AU2004245849B2

AU2004245849B2 - Novel nitrile hydratase

Info

Publication number: AU2004245849B2
Application number: AU2004245849A
Authority: AU
Inventors: Kaoru Furuya; Shin-Ichiro Nagasawa; Ayano Suzuki; Akira Tamaki
Original assignee: Asahi Kasei Corp; Asahi Chemical Industry Co Ltd
Current assignee: Asahi Kasei Corp
Priority date: 2003-06-10
Filing date: 2004-06-10
Publication date: 2008-10-09
Anticipated expiration: 2024-06-10
Also published as: KR100806991B1; AU2004245849A1; WO2004108942A1; TWI321151B; JPWO2004108942A1; JP4108095B2; KR20060016115A; TW200504211A; CN1806047A

Description

DESCRIPTION

NOVEL NITRILE HYDRATASE TECHNICAL FIELD The present invention relates to a technique of producing an amide compound from a nitrile compound by the enzymatic catalysis of a novel nitrile hydratase derived from microorganisms that have been newly isolated from nature.

BACKGROUND ART In a technique of hydrating the nitrile group of a nitrile compound to convert it to an amide group, thereby producing an amide compound corresponding thereto, a method using an enzyme obtained from microorganisms as a catalyst has mainly been applied, instead of the conventional chemical method using a copper catalyst. Such an enzyme has generally been known as nitrile hydratase. Since the initial report regarding the enzyme, a large number of enzymes have been discovered from various types of microorganisms. Examples of such microorganisms include members of: genus Arthrobacter (Agricultural and Biological Chemistry, Vol. 44 pp. 2251-2252, 1980); genus Agrobacterium (Japanese Patent Application Laid-Open No. 05-103681); genus Acinetobacter (Japanese Patent Application Laid-Open No. 61-282089); genus Aeromonas (Japanese Patent Application Laid-Open No. 05-030983); genus Enterobacter (Japanese Patent Application Laid-Open No. 05-236975); genus Erwinia (Japanese Patent Application Laid-Open No. 05-161496); genus Xanthobacter (Japanese Patent Application Laid-Open No. 05-161495); genus Klebsiella (Japanese Patent Application Laid-Open No. 05-030982); genus Corynebacterium (Japanese Patent Application Laid-Open No. 54-129190; it was later turned out to be Rhodococcus genus Pseudomonas (Japanese Patent Application Laid-Open No. 58-86093); genus Citrobacter (Japanese Patent Application Laid-Open No. 05-030984); genus Streptomyces (Japanese Patent Application Laid-Open No. 05-236976); genus Bacillus (Japanese Patent Application Laid-Open Nos. 51-86186 and 7-255494); genus Fusarium (Japanese Patent Application Laid-Open No. 01-086889); genus Rhodococcus (Japanese Patent Application Laid-Open Nos. 63-137688, 02-227069, 2002-369697, and 2-470); genus Rhizobium (Japanese Patent Application Laid-Open No. 05-236977); and genus Pseudonocardia (Japanese Patent Application Laid-Open No. 8-56684). Enzymes obtained from these microorganisms have a variety of amino acid sequences and also a variety of physicochemical properties. Thus, studies regarding such enzymes have been conducted for various purposes. Among such physicochemical properties, stability in the face of heat, amide compounds, nitrile compounds, and the like, is being clarified.

Examples of publications regarding such stability may include: publications regarding the Rhodococcus rhodochrous JI strain (European Journal of Biochemistry, Vol. 196, pp.

581-589, 1991; Applied and Microbiology Biotechnology, Vol. 40, pp. 189-195, 1993); publications regarding the Pseudonocardia thermophila JCM3095 strain (Japanese Patent Application Laid-Open No. 8-187092; Journal of Fermentation and Bioengineering, Vol.

83, pp. 474-477, 1997); publications regarding the genus Bacillus BR449 strain (WO99/55719; Applied Biochemistry and Biotechnology, Vol. 77-79, pp. 671-679, 1999); publications regarding the genus Bacillus RAPc8 strain (Enzyme and Microbial Technology, Vol. 26, pp. 368-373, 2000; Extremophiles, Vol. 2, pp. 347-357, 1998); a publication regarding the Bacillus palidus Dac521 strain (Biochimica et Biophysica Acta, Vol. 1431, pp. 249-260, 1999); and a publication regarding the Bacillus smithii SC-J05-1 strain (Journal of Industrial Microbiology and Biotechnology, Vol. 20, 220-226, 1998).

When an amide compound is industrially produced from a nitrile compound using such unique nitrile hydratase, the production cost of the above enzyme amid the total production cost of an amide compound causes a serious problem. Thus, it is desired that an amide compound be produced using a host, the culture method of which has already been established at an industrial level. Hence, for the purpose of allowing 00 O nitrile hydratase to express in high volumes via genetic engineering, an attempt to c clone the gene has been made. Examples may includes members of genus e( a Pseudomonas (Japanese Patent Application Laid-Open No. 3-251184), genus Rhodococcus (Japanese Patent Application Laid-Open Nos. 2-119778, 4-211379, 09-00973, 07-099980, and 2001-069978), genus Rhizobium (Japanese Patent Application Laid-Open No. 6-25296), genus Klebsiella (Japanese Patent Application Laid-Open No. 6-303971), genus Achromobacter (Japanese Patent 00oo n Application Laid-Open No. 08-266277), genus Pseudonocardia (Japanese Patent Application Laid-Open No. 9-275978), and genus Bacillus (Japanese Patent Application Laid-Open No. 09-248188).

DISCLOSURE OF THE INVENTION It is desired that, as the physicochemical properties, nitrile hydratase have heat stability and maintain high activity even in a high concentration of a nitrile compound used as a substrate, or an amide compound that is a product. Such nitrile hydratase has been reported. However, since the absolute values differ depending on the embodiments of the enzyme used for a reaction or the type of nitrile compound, an enzyme having all the desired properties has not yet been discovered. In addition, it is also desired that a variety of enzymes that are of unknown origin be developed and used as materials for studies regarding the improvement of the physicochemical properties thereof using future evolutionary engineering.

Needless to say, the improvement of physicochemical properties using evolutionary engineering requires the cloning of a gene and the expression thereof. From the viewpoint of the aforementioned restriction of the production cost of an enzyme during the production of an amide compound, it is desired that a genetically modified strain be produced using a host having an established culture method.

That is to say, the present invention provides a method for producing a nitrile hydratase having high stability in the face of heat or a high concentration of compound by isolating the above enzyme from nature; and a method for producing the corresponding amide compound from a nitrile compound using the above enzyme. Moreover, the invention provides the amino acid sequence of 00 O the above enzyme and the gene sequence thereof; a recombinant plasmid c comprising the gene; a transformed strain comprising the above recombinant dU plasmid; a method for producing the above enzyme using the above transformed strain; and a method for producing the corresponding amide compound from a nitrile compound using the above transformed strain. Furthermore there is provided the amino acid sequence of a protein that activates the nitrile hydratase of the above recombinant, and the gene sequence thereof.

00oo Iv The present inventors have found Geobacillus thermoglucosidasius as a Smicroorganism having nitrile hydratase activity in soil located near hot springs in Saitama prefecture. Microorganisms belonging to genus Geobacillus had not been previously known to have nitrile hydratase and to exhibit the nitrile hydratase activity. In addition, the temperature of 65°C that is generally used for the culture of the present microorganisms is higher than the common culture temperature (450C to 600C) used for the conventional thermophilic bacteria having nitrile hydratase.

Moreover, the present inventors have purified nitrile hydratase from the above microorganisms, and have revealed that the nitrile hydratase activity has high stability in the face of heat or high concentrations of nitrile compounds or amide compounds. Furthermore, the present inventors have isolated a nitrile hydratase gene from the chromosomal DNA of the above microorganisms, based on the amino acid sequence at the N-terminus of each subunit of the purified enzyme, and have clarified the amino acid sequence and gene sequence thereof for the first time. As a result, it has been revealed that the nitrile hydratase of the present invention has extremely low homology with existing nitrile hydratases.

Further, the inventors have allowed a gene sequence located downstream of the gene, which is assumed to be an activating protein, to simultaneouslyexpress, so that they have succeeded in producing a genetically modified strain that is capable of expressing the above enzyme in large amount, thereby completing the present invention.

Accordingly in a first aspect of the invention there is provided a purified and/or isolated DNA selected from any one of or wherein: is a purified and/or isolated DNA which comprises a combination of a nucleotide sequence containing a gene which encodes for the amino acid 00 O sequence of subunit a as shown in SEQ ID NO: 1 of the sequence listing, and a Snucleotide sequence containing a gene encoding the amino acid sequence of e( a subunit p as shown in SEQ ID NO: 2 of the sequence listing; is a purified and/or isolated DNA which encodes for a protein which comprises a modified or unmodified subunit a and a modified or unmodified subunit I, wherein said modified subunit comprises a substitution, deletion, o00 addition, or post-translational modification of one or more amino acids, with respect to either one of or both of subunit a, having the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing, and subunit 3 having the amino acid sequence shown in SEQ ID NO: 2; is a purified and/or isolated DNA which comprises a combination of DNA containing a sequence portion 695-1312 of the nucleotide sequence shown in SEQ ID NO: 3 of the sequence listing, and DNA containing a sequence portion 1-681 of the nucleotide sequence shown in SEQ ID NO: 3 of the sequence listing; and is a purified and/or isolated DNA which encodes for a protein, said protein comprising a subunit a which is encoded by either a DNA containing a sequence portion 695-1312 of the nucleotide sequence shown in SEQ ID NO: 3 of the sequence listing, or DNA hybridizing with said DNA under stringent conditions; and a subunit 13 encoded by either a DNA containing a sequence portion 1-681 of the nucleotide sequence shown in SEQ ID NO: 3 of the sequence listing, or DNA hybridizing with said DNA under stringent conditions, (provided that the aforementioned case is excluded); and where the purified and/or isolated DNA of any of or (D) encodes a protein having the following physicochemical properties: 00 O it has nitrile hydratase activity; c substrate specificity; it exhibits its activity to acrylonitrile, adiponitrile, CD acetonitrile, isobutyronitrile, n-valeronitrile, n-butyronitrile, benzonitrile or hexanenitrile as substrates; molecular weight; said protein comprising at least the following two types of subunits, and each subunit has the following molecular weight measured by reduced SDS-polyacrylamide electrophoresis: 00 subunit a molecular weight 25,000 2,000; and subunit 3 molecular weight 28,000 2,000; 0 10 heat stability; after the enzyme in an aqueous solution has been heated at a temperature of 70 0 C for 30 minutes, the activity that is 35% or more of that before heating is maintained; even if 6%-by-weight acrylonitrile solution is used as a substrate, the activity does not decrease, when compared with a case where the substrate concentration is lower than the case of the above substrate; and even in 35%-by-weight acrylamide aqueous solution, it has activity that is based on the existence of acrylonitrile as a substrate.

In a second aspect of the invention there is provided a purified and/or isolated DNA comprising the gene for the subunit a of nitrile hydratase, which gene encodes for the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing.

In a third aspect of the invention there is provided a purified and/or isolated DNA comprising the gene for the subunit p of nitrile hydratase, which DNA encodes for the amino acid sequence shown in SEQ ID NO: 2 of the sequence listing.

In a fourth aspect of the invention there is provided a purified and/or isolated DNA comprising a gene which is associated with the activation of nitrile hydratase, and which gene encodes for the amino acid sequence shown in SEQ ID NO: 4 of the sequence listing.

In a fifth aspect of the invention there is provided a method for producing a protein or a bacterial cell mass-treated product containing said protein, wherein a microorganism belonging to the genus Geobacillus and being capable of 00 O producing a protein having the following physicochemical properties, is cultured in C a medium: S(a) it has nitrile hydratase activity; substrate specificity; it exhibits its activity to acrylonitrile, adiponitrile, acetonitrile, isobutyronitrile, n-valeronitrile, n-butyronitrile, benzonitrile or hexanenitrile as substrates; S(c) molecular weight; it is a protein comprising at least the following two types of subunits, and each subunit has the following molecular weight measured by reduced SDS-polyacrylamide electrophoresis: 10 subunit a molecular weight 25,000 2,000; and subunit p molecular weight 28,000 2,000; heat stability; after the enzyme in an aqueous solution has been heated at a temperature of 70 0 C for 30 minutes, the activity that is 35% or more of that before heating is maintained; even if 6%-by-weight acrylonitrile solution is used as a substrate, the activity does not decrease, when compared with a case where the substrate concentration is lower than the case of the above substrate; and even in 35%-by-weight acrylamide aqueous solution, it has activity that is based on the existence of acrylonitrile as a substrate.

In a sixth aspect of the invention there is provided a purified and/or isolated protein which has the following physicochemical properties: it has nitrile hydratase activity; substrate specificity; it exhibits its activity to acrylonitrile, adiponitrile, acetonitrile, isobutyronitrile, n-valeronitrile, n-butyronitrile, benzonitrile or hexanenitrile as substrates; molecular weight; it is a protein comprising at least the following two types of subunits, and each subunit has the following molecular weight measured by reduced SDS-polyacrylamide electrophoresis: subunit a molecular weight 25,000 2,000; and subunit p molecular weight 28,000 2,000; heat stability; after the enzyme in an aqueous solution has been heated at a temperature of 70 0 C for 30 minutes, the activity that is 35% or more of that before heating is maintained; 00 O even if 6%-by-weight acrylonitrile solution is used as a substrate, the c activity does not decrease, when compared with a case where the substrate l concentration is lower than the case of the above substrate; and even in 35%-by-weight acrylamide aqueous solution, it has activity that is based on the existence of acrylonitrile as a substrate.

In a seventh aspect of the invention there is provided a purified and/or isolated protein of either or wherein: 00O is a protein which comprises a subunit a having the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing and a subunit p having 0 10 the amino acid sequence shown in SEQ ID NO: 2 of the sequence listing; and is a protein which comprises a modified or unmodified subunit a and a modified or unmodified subunit p, wherein said modified subunit comprises a substitution, deletion, addition, or post-translational modification of one or several amino acids, with respect to either one of or both of the subunit a having the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing and the subunit p having the amino acid sequence shown in SEQ ID NO: 2; and which protein has the following physicochemical properties: it has nitrile hydratase activity; substrate specificity; it exhibits its activity to acrylonitrile, adiponitrile, acetonitrile, isobutyronitrile, n-valeronitrile, n-butyronitrile, benzonitrile or hexanenitrile as substrates; molecular weight; said protein comprising at least the following two types of subunits, and each subunit has the following molecular weight measured by reduced SDS-polyacrylamide electrophoresis: subunit a molecular weight 25,000 2,000; and subunit p molecular weight 28,000 2,000; heat stability; after the enzyme in an aqueous solution has been heated at a temperature of 70°C for 30 minutes, the activity that is 35% or more of that before heating is maintained; even if 6%-by-weight acrylonitrile solution is used as a substrate, the activity does not decrease, when compared with a case where the substrate concentration is lower than the case of the above substrate; and 00 O even in 35%-by-weight acrylamide aqueous solution, it has activity that c is based on the existence of acrylonitrile as a substrate.

CD In a eighth aspect of the invention there is provided a purified and/or isolated protein of either or wherein: is a protein which comprises a subunit a encoded by DNA containing a sequence portion 695-1312 of the nucleotide sequence shown in SEQ ID NO: 3 00 of the sequence listing, and a subunit p encoded by DNA containing a sequence 00 portion 1-681 of the nucleotide sequence shown in SEQ ID NO: 3 of the sequence listing; and 0 10 is a protein which comprises a subunit a encoded by either DNA containing a sequence portion 695-1312 of the nucleotide sequence shown in SEQ ID NO: 3 of the sequence listing, or DNA hybridizing with said DNA under stringent conditions, and a subunit 3 encoded by either DNA containing a sequence portion 1-681 of the nucleotide sequence shown in SEQ ID NO: 3 of the sequence listing, or DNA hybridizing with said DNA under stringent conditions (provided that the aforementioned case is excluded), and which protein has the following physicochemical properties: it has nitrile hydratase activity; substrate specificity; it exhibits its activity to acrylonitrile, adiponitrile, acetonitrile, isobutyronitrile, n-valeronitrile, n-butyronitrile, benzonitrile or hexanenitrile as substrates; molecular weight; said protein comprising at least the following two types of subunits, and each subunit has the following molecular weight measured by reduced SDS-polyacrylamide electrophoresis: subunit a molecular weight 25,000 2,000; and subunit 3 molecular weight 28,000 2,000; heat stability; after the enzyme in an aqueous solution has been heated at a temperature of 700C for 30 minutes, the activity that is 35% or more of that before heating is maintained; even if 6%-by-weight acrylonitrile solution is used as a substrate, the activity does not decrease, when compared with a case where the substrate concentration is lower than the case of the above substrate; and 00 O even in 35%-by-weight acrylamide aqueous solution, it has activity that c is based on the existence of acrylonitrile as a substrate.

C

U In a ninth aspect of the invention there is provided a purified and/or isolated protein which comprises at least either one of, or both of, a polypeptide containing a subunit a having the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing, or a post-translationally modified product thereof; and a oo polypeptide containing a subunit p having the amino acid sequence shown in SEQ ID NO: 2 of the sequence listing, or a post-translationally modified product thereof, wherein the protein has the following physicochemical properties: 0 10 it has nitrile hydratase activity; substrate specificity; it exhibits its activity to acrylonitrile, adiponitrile, acetonitrile, isobutyronitrile, n-valeronitrile, n-butyronitrile, benzonitrile or hexanenitrile as substrates; molecular weight; said protein comprising at least the following two types of subunits, and each subunit has the following molecular weight measured by reduced SDS-polyacrylamide electrophoresis: subunit a molecular weight 25,000 2,000; and subunit p molecular weight 28,000 2,000; heat stability; after the enzyme in an aqueous solution has been heated at a temperature of 70°C for 30 minutes, the activity that is 35% or more of that before heating is maintained; even if 6%-by-weight acrylonitrile solution is used as a substrate, the activity does not decrease, when compared with a case where the substrate concentration is lower than the case of the above substrate; and even in 35%-by-weight acrylamide aqueous solution, it has activity that is based on the existence of acrylonitrile as a substrate.

In a tenth aspect of the invention there is provided a method for producing an amide compound, wherein a protein having the following physicochemical properties or a bacterial cell mass-treated product containing said protein, is allowed to act on a nitrile compound, so as to obtain an amide compound induced from the above nitrile compound, said physicochemical properties including: it has nitrile hydratase activity; 00 O substrate specificity; it exhibits its activity to acrylonitrile, adiponitrile, c acetonitrile, isobutyronitrile, n-valeronitrile, n-butyronitrile, benzonitrile or hexanenitrile as substrates; molecular weight; it is a protein comprising at least the following two types of subunits, and each subunit has the following molecular weight measured by reduced SDS-polyacrylamide electrophoresis: 0" subunit a molecular weight 25,000 2,000; and subunit p molecular weight 28,000 2,000; heat stability; after the enzyme in an aqueous solution has been heated 0 10 at a temperature of 70°C for 30 minutes, the activity that is 35% or more of that before heating is maintained; even if 6%-by-weight acrylonitrile solution is used as a substrate, the activity does not decrease, when compared with a case where the substrate concentration is lower than the case of the above substrate; and even in 35%-by-weight acrylamide aqueous solution, it has activity that is based on the existence of acrylonitrile as a substrate.

That is to say, the present invention provides the following: DNA which encodes a protein having the following physicochemical properties: it has nitrile hydratase activity; substrate specificity; it exhibits its activity to acrylonitrile, adiponitrile, acetonitrile, isobutyronitrile, n-valeronitrile, n-butyronitrile, benzonitrile or hexanenitrile as substrates; molecular weight; it is a protein comprising at least the following two types of subunits, and each subunit has the following molecular weight measured by reduced SDS-polyacrylamide electrophoresis: subunit a molecular weight 25,000 2,000; and subunit p molecular weight 28,000 2,000; heat stability; after the enzyme in an aqueous solution has been heated at a temperature of 70°C for 30 minutes, the activity that is 35% or more of that before heating is maintained; 00 O even if 6%-by-weight acrylonitrile solution is used as a substrate, the c activity does not decrease, when compared with a case where the substrate CD concentration is lower than the case of the above substrate; and even in 35%-by-weight acrylamide aqueous solution, it has activity that is based on the existence of acrylonitrile as a substrate.

DNA of any of the following or 0 DNA which comprises a combination of a nucleotide sequence oo containing a subunit a gene encoding the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing, and a nucleotide sequence containing a subunit p 0 10 gene encoding the amino acid sequence shown in SEQ ID NO: 2 of the sequence listing; and DNA encoding a protein which comprises a modified or unmodified subunit a and a modified or unmodified subunit 3 comprising a substitution, deletion, addition, or post-translational modification of one or several amino acids, with respect to either one of or both of the subunit a having the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing and the subunit 1 having the amino acid sequence shown in SEQ ID NO: 2; and which has nitrile hydratase activity.

DNA of any of the following or DNA which comprises a combination of DNA containing a sequence portion 695-1312 of the nucleotide sequence shown in SEQ ID NO: 3 of the sequence listing, and DNA containing a sequence portion 1-681 of the nucleotide sequence shown in SEQ ID NO: 3 of the sequence listing; and DNA encoding a protein, which comprises: a subunit a encoded by either DNA containing a sequence portion 695-1312 of the nucleotide sequence shown in SEQ ID NO: 3 of the sequence listing, or DNA hybridizing with said DNA under stringent conditions; and a subunit 3 encoded by either DNA containing a sequence portion 1-681 of the nucleotide sequence shown in SEQ ID NO: 3 of the sequence listing, or DNA hybridizing with said DNA under stringent conditions, and which has nitrile hydratase activity, provided that the aforementioned case is excluded.

The DNA according to any of to above, which further comprises either DNA having a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 4 of the sequence listing, or DNA encoding a protein which comprises a substitution, deletion, addition, or post-translational modification of one or several amino acids with respect to said amino acid sequence, and is associated with activation of nitrile hydratase.

The DNA according to any of to above, which further comprises either DNA containing a sequence portion 1325-1663 of the nucleotide sequence shown in SEQ ID NO: 3 of the sequence listing, or DNA hybridizing with said DNA under stringent conditions and encoding a protein that is associated with activation of nitrile hydratase.

DNA comprising the subunit a gene of nitrile hydratase encoding the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing.

DNA comprising the subunit P gene of nitrile hydratase encoding the amino acid sequence shown in SEQ ID NO: 2 of the sequence listing.

DNA comprising a gene which is associated with activation of nitrile hydratase encoding the amino acid sequence shown in SEQ ID NO: 4 of the sequence listing.

The DNA according to any of to above, which is derived from genus Geobacillus.

The DNA according to any of to above, which is derived from Geobacillus thermoglucosidasius.

(11) The DNA according to any of to above, which is derived from the Geobacillus thermoglucosidasius Q-6 strain (FERM BP-08658).

(12) A recombinant vector, into which the DNA according to any of to (11) has been incorporated.

(13) A microorganism selected from a microorganism transformed with the DNA according to any of to (11) above, or a Geobacillus thermoglucosidasius Q-6 strain (FERM BP-08658) and a mutant thereof.

(14) A method for producing said protein or a bacterial cell mass-treated product containing said protein, wherein a microorganism transformed with the DNA according to any of to (11) above is cultured in a medium.

A method for producing said protein or a bacterial cell mass-treated product containing said protein, wherein a microorganism belonging to genus Geobacillus and being capable of producing a protein having the following physicochemical properties is cultured in a medium; it has nitrile hydratase activity; substrate specificity; it exhibits its activity to acrylonitrile, adiponitrile, acetonitrile, isobutyronitrile, n-valeronitrile, n-butyronitrile, benzonitrile or hexanenitrile as substrates; molecular weight; it is a protein comprising at least the following two types of subunits, and each subunit has the following molecular weight measured by reduced SDS-polyacrylamide electrophoresis: subunit a molecular weight 25,000 2,000; and subunit p molecular weight 28,000 2,000; heat stability; after the enzyme in an aqueous solution has been heated at a temperature of 70 0 C for 30 minutes, the activity that is 35% or more of that before heating is maintained; even if 6%-by-weight acrylonitrile solution is used as a substrate, the activity does not decrease, when compared with a case where the substrate concentration is lower than the case of the above substrate; and even in 35%-by-weight acrylamide aqueous solution, it has activity that is based on the existence of acrylonitrile as a substrate.

(16) Said protein or a bacterial cell mass-treated product containing said protein, which is obtained from a microorganism cultured by the production method according to either (14) or (15) above.

(17) A protein which has the following physicochemical properties: it has nitrile hydratase activity; substrate specificity; it exhibits its activity to acrylonitrile, adiponitrile, acetonitrile, isobutyronitrile, n-valeronitrile, n-butyronitrile, benzonitrile or hexanenitrile as substrates; molecular weight; it is a protein comprising at least the following two types of subunits, and each subunit has the following molecular weight measured by reduced SDS-polyacrylamide electrophoresis: subunit a molecular weight 25,000 2,000; and subunit P molecular weight 28,000 2,000; heat stability; after the enzyme in an aqueous solution has been heated at a temperature of 70°C for 30 minutes, the activity that is 35% or more of that before heating is maintained; even if 6%-by-weight acrylonitrile solution is used as a substrate, the activity does not decrease, when compared with a case where the substrate concentration is lower than the case of the above substrate; and even in 35%-by-weight acrylamide aqueous solution, it has activity that is based on the existence of acrylonitrile as a substrate.

(18) A protein of any of the following or a protein which comprises a subunit ax having the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing and a subunit P having the amino acid sequence shown in SEQ ID NO: 2 of the sequence listing; and a protein which comprises a modified or unmodified subunit ax and a modified or unmodified subunit P, comprising a substitution, deletion, addition, or post-translational modification of one or several amino acids, with respect to either one of or both of the subunit c having the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing and the subunit P having the amino acid sequence shown in SEQ ID NO: 2; and which has nitrile hydratase activity.

(19) A protein of any of the following or a protein which comprises a subunit cx encoded by DNA containing a sequence portion 695-1312 of the nucleotide sequence shown in SEQ ID NO: 3 of the sequence listing, and a subunit P encoded by DNA containing a sequence portion 1-681 of the nucleotide sequence shown in SEQ ID NO: 3 of the sequence listing; and a protein which comprises: a subunit ca encoded by either DNA containing a sequence portion 695-1312 of the nucleotide sequence shown in SEQ ID NO: 3 of the sequence listing, or DNA hybridizing with said DNA under stringent conditions; and a subunit P encoded by either DNA containing a sequence portion 1-681 of the nucleotide sequence shown in SEQ ID NO: 3 of the sequence listing, or DNA hybridizing with said DNA under stringent conditions, and which has nitrile hydratase activity, provided that the aforementioned case is excluded.

A protein which comprises at least either one of, or both of: a polypeptide containing a subunit ca having the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing, or a post-translationally modified product thereof; and a polypeptide containing a subunit p having the amino acid sequence shown in SEQ ID NO: 2 of the sequence listing, or a post-translationally modified product thereof.

(21) A method for producing an amide compound, wherein a protein having the following physicochemical properties or a bacterial cell mass-treated product containing said protein is allowed to act on a nitrile compound, so as to obtain an amide compound induced from the above nitrile compound: it has nitrile hydratase activity; substrate specificity; it exhibits its activity to acrylonitrile, adiponitrile, acetonitrile, isobutyronitrile, n-valeronitrile, n-butyronitrile, benzonitrile or hexanenitrile as substrates; molecular weight; it is a protein comprising at least the following two types of subunits, and each subunit has the following molecular weight measured by reduced SDS-polyacrylamide electrophoresis: subunit a molecular weight 25,000 2,000; and subunit p molecular weight 28,000 2,000; heat stability; after the enzyme in an aqueous solution has been heated at a temperature of 70 0 C for 30 minutes, the activity that is 35% or more of that before heating is maintained; even if 6%-by-weight acrylonitrile solution is used as a substrate, the activity does not decrease, when compared with a case where the substrate concentration is lower than the case of the above substrate; and even in 35%-by-weight acrylamide aqueous solution, it has activity that is based on the existence of acrylonitrile as a substrate.

(22) A method for producing an amide compound, wherein a protein obtained by culturing in a medium either a microorganism transformed with the DNA according to any of to (11) or a microorganism belonging to genus Geobacillus and being capable of producing a protein having the following physicochemical properties, or a bacterial cell mass-treated product containing said protein, is used: it has nitrile hydratase activity; substrate specificity; it exhibits its activity to acrylonitrile, adiponitrile, acetonitrile, isobutyronitrile, n-valeronitrile, n-butyronitrile, benzonitrile or hexanenitrile as substrates; molecular weight; it is a protein comprising at least the following two types of subunits, and each subunit has the following molecular weight measured by reduced SDS-polyacrylamide electrophoresis: subunit a molecular weight 25,000 2,000; and subunit P molecular weight 28,000 2,000; heat stability; after the enzyme in an aqueous solution has been heated at a temperature of 70 0 C for 30 minutes, the activity that is 35% or more of that before heating is maintained; even if 6%-by-weight acrylonitrile solution is used as a substrate, the activity does not decrease, when compared with a case where the substrate concentration is lower than the case of the above substrate; and even in 35%-by-weight acrylamide aqueous solution, it has activity that is based on the existence of acrylonitrile as a substrate.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the gene structures of the nitrile hydratase subunit P, subunit a, and a group of downstream genes of the Geobacillus thermoglucosidasius Q-6 strain, and it also shows the restriction map thereof. The figure indicates the position of a fragment (Hin2.3) obtained by colony hybridization and the positions of fragments (pa, pal, and pal2) used for expression in Escherichia coli.

BEST MODE FOR CARRYING OUT THE INVENTION First, the nitrile hydratase of the present invention will be described. In the present invention, the expression "have nitrile hydratase activity" means that it has activity of adding a water molecule to a nitrile compound, so as to convert it to an amide compound, such as conversion of acetonitrile to acetamide, conversion of n-propionitrile to n-propioamide, or conversion of acrylonitrile to acrylamide. The generated compound is fractionated by liquid chromatography, and it is then identified using gas chromatography/mass spectrometry (GC/MS), infrared absorption spectrometry (IR), nuclear magnetic resonance spectrometry (NMR), and the like.

When nitrile hydratase activity is measured in the present invention, for example, ld of a nitrile hydratase enzyme solution is added to 1 ml of 0.1%-by-weight nitrile compound solution (0.05 M phosphate buffer; pH and the obtained mixture is incubated at a reaction temperature between 27 0 C and 60 0 C for 1 to 60 minutes.

Thereafter, the reaction is terminated by addition of 0.1 ml of 1 N HC1, and an aliquot of the reaction solution is analyzed by liquid chromatography, so as to examine the presence or absence of the generation of an amide compound.

Examples of a nitrile compound used as a substrate in the present invention may include: aliphatic nitrile compounds such as acetonitrile, n-propionitrile, n-butyronitrile, isobutyronitrile, n-valeronitrile, or n-hexanenitrile; nitrile compounds containing a halogen atom, such as 2-chloropropionitrile; aliphatic nitrile compounds having an unsaturated bond, such as acrylonitrile, crotononitrile, or methacrylonitrile; hydroxynitrile compounds such as lactonitrile or mandelonitrile; aminonitrile compounds such as 2-phenylglycinonitrile; aromatic nitrile compounds such as benzonitrile or cyanopyridine; dinitrile compounds such as malononitrile, succinonitrile, or adiponitrile; and trinitrile compounds.

The substrate specificity of the nitrile hydratase of the present invention can be determined by using various substrates under the aforementioned measurement conditions and then measuring whether or not the nitrile hydratase of the present invention has its activity on each substrate. If nitrile hydratase has wide substrate specificity, the number of types of amide compounds corresponding thereto, which can be produced, also favorably increases. The present enzyme has, as its substrates, at least acrylonitrile, adiponitrile, acetonitrile, isobutyronitrile, n-valeronitrile, n-butyronitrile, benzonitrile, and hexanenitrile.

When the nitrile hydratase of the present invention is subjected to reduced SDS (sodium dodecyl sulfate)-polyacrylamide electrophoresis, two subunits, one having a molecular weight of 25,000 2,000 and the other having a molecular weight of 28,000 2,000, are detected by staining with Coomassie brilliant blue. The former is called a subunit ca, and the latter is called a subunit P.

Even after the nitrile hydratase of the present invention has been subjected to a heat treatment at 70 0 C for 30 minutes in an aqueous solution containing no stabilizers such as organic acid prior to the measurement of its activity, it can maintain 35% of the activity that it exhibited before heating.

It has been reported that a high concentration of nitrile substrate chemically inactivates enzymes. However, in the case of the nitrile hydratase of the present invention, even if 6%-by-weight acrylonitrile solution is used as a substrate, such a phenomenon does not occur.

It has also been reported that a high concentration of amide compound as a reaction product chemically inhibits the reaction. This phenomenon is considered to become seriously problematic when a high concentration of reaction product is obtained.

However, in the case of the nitrile hydratase of the present invention, even if 35% by weight of acrylamide is added to a solution used for the measurement of the activity, the concentration of acrylonitrile used as a substrate significantly decreases, and thus the activity is maintained.

The present invention is exemplified by those described in (18) above. That is to say, a preferred example of the nitrile hydratase of the present invention is a nitrile hydratase, which comprises a subunit a consisting of 205 amino acids shown in SEQ ID NO: 1 of the sequence listing and a subunit P consisting of 226 amino acids shown in SEQ ID NO: 2 of the sequence listing. The nitrile hydratase of the present invention may also comprise metals, other peptides, and the like, as well as such two subunits. As such metals, the nitrile hydratase of the present invention may often particularly comprise iron or cobalt. Moreover, it may also be a protein containing either one of such subunits. The amino acid sequence of each subunit may comprise a substitution, deletion, or insertion of one or several amino acids with respect to the aforementioned amino acid sequence, as long as it forms a complex with another subunit and has nitrile hydratase activity. Also, the nitrile hydratase of the present invention is naturally anticipated to undergo post-translational modification depending on the type of host. In particular, in the subunit a of nitrile hydratase, a cysteine residue may often be modified to cysteinesulfinic acid or cysteinesulfenic acid after translation. An amino acid sequence comprising a substitution, deletion, insertion, or post-translational modification of 1 to 30 amino acids with respect to the above amino acid sequence is also a preferred example. The number of amino acids that are substituted, deleted, inserted, or post-translationally modified, is more preferably 1 to 10, further more preferably 1 to and most preferably 1 to 3. Such a nitrile hydratase enzyme having an amino acid sequence comprising a substitution, deletion, or insertion, can be obtained by using DNA comprising such a substitution, deletion, or insertion in the corresponding site(s) of its nucleotide sequence according to a known site-directed mutagenesis such as the method described in Molecular Cloning 2nd Edition, Cold Spring Harbor Laboratory Press (1989), and by introducing the DNA into host microorganisms and allowing it to express therein as described later. It is also possible to try to produce a mutant enzyme, which achieves industrially desired properties such as heat stability, improved resistance to organic solvents, and change in substrate specificity. Taking into consideration such technical level, when such mutant enzymes have nitrile hydratase activity, they are included in the present invention.

Moreover, the present invention is also exemplified by those described in (19) above. These proteins will be described in detail later in the section describing the DNA of the present invention.

The nitrile hydratase having the aforementioned physicochemical properties can be obtained by culturing microorganisms belonging to genus Geobacillus, for example.

The type of microorganisms used in the present invention is not particularly limited, as long as they belong to genus Geobacillus and have hydration activity of converting a nitrile compound to an amide compound. Examples of microorganisms belonging to genus Geobacillus may include Geobacillus caldoxylosilyticus, Geobacillus kaustophilus, Geobacillus lituanicus, Geobacillus stearothermophilus, Geobacillus subterraneus, Geobacillus thermocatenulatus, Geobacillus thermodenitrificans, Geobacillus thermoglucosidasius, Geobacillus thermoleovorans, Geobacillus toebii, and Geobacillus uzenensis. In addition, microorganisms used in the present invention are not particularly limited to those belonging to genus Geobacillus, and nitrile hydratase genes derived from other microorganism strains are also included. Examples of such a microorganism strain may include members of genus Agrobacterium, genus Achromobacter, genus Acinetobacter, genus Aeromonas, genus Enterobacter, genus Erwinia, genus Xanthobacter, genus Klebsiella, genus Corynebacterium, genus Sinorhizobium, genus Pseudomonas, genus Streptomyces, genus Nocardia, genus Bacillus, genus Micrococcus, genus Rhodococcus, genus Rhodopseudomonas, genus Rhizobium, and genus Pseudonocardia. Specifically, screening was carried out by the following method in the present invention. First, a small amount of soil collected from various places is placed in a test tube filled with water or a normal saline solution, and it is then subjected to a shaking culture for 2 to 14 days in a shaking incubator at 65 0

C.

Thereafter, an aliquot of the obtained culture solution is placed in a general-purpose medium used for the growth of microorganisms, such as a liquid medium containing, as main ingredients, glycerol, polypeptone, yeast extract, or the like. The obtained mixture is then cultured at a culture temperature of 65 0 C for 1 to 7 days. Thereafter, an aliquot of the obtained culture solution is spread on an agar plate medium containing the aforementioned ingredients for a medium for the growth of microorganisms, and the resultant is further cultured at 65 0 C, so as to form colonies, thereby isolating microorganisms. The thus obtained microorganisms are subjected to a shaking culture at a culture temperature of 65 0 C for an appropriate period of time, such as for approximately 12 hours to 7 days, in a test tube or flask that is filled with a liquid medium prepared by further adding a nitrile compound such as n-valeronitrile or an amide compound such as methacrylamide to the aforementioned medium, so as to allow the microorganisms to grow. Thereafter, based on the aforementioned common method for measuring nitrile hydratase activity, microorganisms of interest are selected. A typical microorganism strain was identified based on 16SrRNA and the biochemical properties mentioned below. As a result, it was found that such a typical strain is Geobacillus thermoglucosidasius. This strain was named as Geobacillus thermoglucosidasius Q-6, and it was deposited with the National Institute of Advanced Industrial Science and Technology, an Independent Administrative Institution under the Ministry of Economy, Trade and Industry (Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan), under accession No. FERM P-19351 (date of receipt: May 16, 2003).

This strain was then transferred to an international depository under the provisions of the Budapest Treaty (receiving date: March 11, 2004) under FERM BP-08658. Although various patents or publications were examined, there were no descriptions regarding the fact that microorganisms belonging to Geobacillus thermoglucosidasius have nitrile hydratase activity. From these results, it is considered that Geobacillus thermoglucosidasius Q-6 is a novel strain. It is also possible to produce an amide compound by using mutants of this new strain, namely, using mutants induced from the Geobacillus thermoglucosidasius Q-6 strain, cell fusion strains, and genetically modified strains. The Geobacillus thermoglucosidasius Q-6 strain has the following properties: Morphological characteristics Culture conditions: Nutrient Agar (Oxoid, England, UK) medium, 1. Shape and size of cell Shape: coryneform Size: 0.8 x 2.0 to 3.0 uLm 2. Presence or absence of cell polymorphism: 3. Presence or absence of motility: Epiphytic state of flagella: peritrichous 4. Presence or absence of spore: Site of spore: boundary Culture characteristics Culture conditions: Nutrient Agar (Oxoid, England, UK) medium, 1. Color: cream color 2. Luster: 3. Pigment production: Culture conditions: Nutrient broth (Oxoid, England, UK) medium, 1. Presence or absence of growth on surface: 2. Presence or absence of opacity of medium: Culture conditions: gelatin stab culture, 1. Growth state: 2. Gelatin liquefaction: Culture conditions: litmus milk, 1. Coagulation: 2. Liquefaction: Physiological characteristics 1. Gram staining: inconstant 2. Reduction of nitrate: 2. Denitrification: 3. MR test: 4. VP test: Generation of indole: 6. Generation of hydrogen sulfide: 7. Hydrolysis of starch: 8. Use of citric acid Koser: Christensen: 9. Use of inorganic nitrogen source Nitrate: Ammonium salt: Generation of pigment: 11. Urease activity: 12. Oxidase: 13. Catalase: 14. Growth range pH: 5.5 to Temperature: 45 0 C to 72 0

C

Attitude to oxygen: facultatively anaerobic 16. O-F test: Generation of acid/gas from saccharides 1. L-arabinose: 2. D-xylose: 3. D-glucose: 4. D-mannose: D-fructose: 6. D-galactose: 7. Maltose: 8. Sucrose: 9. Lactose: Trehalose: 11. D-sorbitol: 12. D-mannitol: 13. Inositol: 14. Glycerine: Other characteristics 1. P-galactosidase activity: 2. Arginine dihydrolase activity: 3. Lysine decarboxylase activity: 4. Tryptophan deaminase activity: Gelatinase activity: Microorganisms used in the method of the present invention are cultured in accordance with a common microorganism culture method. Either solid culture or liquid culture may be applied. Since Geobacillus thermoglucosidasius is a facultative anaerobic microorganism, it can be cultured under the same culture conditions as those for common facultative anaerobic microorganisms. The culture temperature may be appropriately changed within a range where microorganisms grow. For example, the culture temperature is between 40'C and 75°C. The pH of a medium is between pH 4 and pH 9, for example. In particular, in the case of Geobacillus thermoglucosidasius Q-6, the culture temperature is between 45°C and 72°C, and preferably between and 70C, and the pH of a medium is between pH 5 and pH 8. The culture time differs depending on various conditions, but it is preferably between approximately 1 and 7 days.

As a medium used herein, various types of mediums, which are generally used for microorganisms and appropriately contain a common carbon source, nitrogen source, organic or inorganic salts, and the like, are used. Examples of a carbon source used herein may include glycerol, glucose, sucrose, molasses, organic acid, and animal or vegetable oil. Examples of a nitrogen source used herein may include yeast extract, peptone, malt extract, meat extract, urea, and sodium nitrate. Examples of organic or inorganic salts used herein may include sodium chloride, magnesium sulfate, potassium chloride, ferrous sulfate, manganese sulfate, cobalt chloride, zinc sulfate, copper sulfate, sodium acetate, calcium carbonate, potassium dihydrogen phosphate, and dibasic potassium phosphate. In the method of the present invention, in order to enhance the nitrile hydratase activity of microorganisms used, it is preferable to add a nitrile compound such as n-valeronitrile, isovaleronitrile or crotononitrile, or an amide compound such as methacrylamide, to a medium. As an additive amount, such a compound is added to 1 L of medium, at a suitable amount between 0.01 g and 10 g. In addition, 0.1 [tg/ml or more Fe ions or Co ions are preferably allowed to exist.

In order to obtain information regarding the amino acid sequence of the enzyme of the present invention, after the enzyme of the present invention has been purified, each subunit is separated by reduced SDS-polyacrylamide electrophoresis, and each band is then cut out of the gel. Thereafter, a part of the amino acid sequence can be determined using a protein sequencer.

Moreover, the present invention relates to DNA encoding nitrile hydratase.

Specific examples of such DNA may include: DNA containing a sequence portion 695-1312 of the nucleotide sequence shown in SEQ ID NO: 3 of the sequence listing; and DNA containing a sequence portion 1-681 of the nucleotide sequence shown in SEQ ID NO: 3 of the sequence listing. Each DNA encodes a subunit cx and a subunit 3.

However, examples of such DNA are not limited thereto, and any DNA containing such a nucleotide sequence can be applied herein. Moreover, DNA which hybridizes with DNA having a nucleotide sequence complementary to the aforementioned sequences under stringent conditions, may also be included in the present invention, as long as it has nitrile hydratase activity. In other words, the nitrile hydratase of the present invention is allowed to express, using these DNAs. Examples of stringent conditions may include the conditions described in manual included with an ECL direct nucleic acid labeling and detection system (manufactured by Amersham Pharmacia Biotech) (wash: 42°C; a primary wash buffer containing 0.5 x SSC). An example of DNA capable of hybridizing with the aforementioned DNA under such stringent conditions may be DNA, which hybridizes with DNA containing a sequence portion 695-1312 of the nucleotide sequence shown in SEQ ID NO: 3 of the sequence listing, or DNA, which hybridizes with a detection sample that is a nucleotide sequence portion consisting of any number of, generally at least 20, preferably at least 50, and particularly preferably at least 100 contiguous nucleotides of a nucleotide sequence complementary to DNA containing a sequence portion 1-681 of the nucleotide sequence shown in SEQ ID NO: 3 of the sequence listing, under the aforementioned stringent conditions.

DNA encoding the nitrile hydratase of the present invention can be obtained by the method mentioned below. In the present specification, a genetic recombination technique, a technique of producing a recombinant protein, and an analysis method, which are known in the present field, can be adopted, unless otherwise specified.

DNA encoding the nitrile hydratase of the present invention can be obtained from microorganisms containing the nitrile hydratase of the present invention, such as the Geobacillus thermoglucosidasius Q-6 strain, based on the nucleotide sequences or amino acid sequences disclosed in the specification of the present application, or in some cases, based on sequence information such as an amino acid sequence determined from the aforementioned purified enzyme. Chromosomal DNA of microorganisms containing nitrile hydratase is digested with restriction enzymes, so as to obtain a DNA fragment.

Thereafter, the DNA fragment is introduced into a phage or plasmid, and a host is then transformed with such a phage or plasmid, so as to obtain a library. Thereafter, DNA encoding the nitrile hydratase of the present invention can be obtained from the library by plaque hybridization or colony hybridization using oligonucleotide synthesized based on the amino acid sequence as a probe. Alternatively, oligonucleotide is not used as a probe, but primers are produced based on information regarding the amino acid sequences at the N-termini of both subunits determined from the aforementioned purified enzyme. Thereafter, a part of a nitrile hydratase gene is amplified using such primers by polymerase chain reaction (PCR), and the thus amplified product is used as a probe.

Using the probe, DNA encoding the nitrile hydratase of the present invention can be obtained by the same above process. The obtained DNA is inserted into a plasmid vector such as pUC118, followed by cloning. Thereafter, the nucleotide sequence thereof can be determined by a publicly known method such as the dideoxy terminator method (Proceedings of the National Academy of Sciences. 74: 5463-5467, 1977). By applying the aforementioned common method for measuring the activity of an expression product that is contained in Escherichia coli used as a host and transformed with the thus prepared gene, it can be confirmed that the above gene is DNA encoding nitrile hydratase.

Furthermore, the present invention provides a recombinant vector, wherein the aforementioned DNA is ligated to a vector.

The recombinant vector of the present invention can be produced by ligating the DNA obtained by the aforementioned method downstream of a promoter region suitable for host microorganisms, such that the 5'-terminal side of the above DNA is able to function, and as necessary, by inserting a transcription termination sequence downstream thereof, and then incorporating the resultant product into a suitable expression vector.

The type of a suitable expression vector is not particularly limited, as long as it is able to replicate and grow in host microorganisms. Moreover, if a host has capable system for chromosomal integration, the vector does not need to have a region capable of autonomous replication. When Escherichia coli is used as a host for example, an expression vector can be selected from among any given vectors generally used in Escherichia coli, such as pUS, pGEX, pET, pT7, pBluescript, pKK, pBS, pBC, and pCAL, which include strong promoters such as lac, trp, tac, trc, T7, or PL, and a pyruvate oxidase gene promoter (Japanese Patent Publication No. 2579506). Furthermore, a subunit a gene and a subunit 3 gene may be expressed from different promoters as independent cistrons. Otherwise, these genes may also be expressed from a single promoter as a polycistron. Further, when these genes are expressed as independent cistrons, each subunit gene may be on a different vector.

Sill further, the present inventors have found that nitrile hydratase activity is further increased by incorporating a gene located downstream of the nitrile hydratase gene of the present invention into the aforementioned nitrile hydratase recombinant vector. Thus, the present invention also provides such a vector. Specifically, a plasmid vector containing a promoter, a transcription termination factor, or the like, which are necessary for expression as described above, is used. Genes encoding proteins associated with activation of nitrile hydratase, that are, a subunit a gene and a subunit P gene of nitrile hydratase, may be expressed as independent cistrons, or they may also be expressed from a single control region as a polycistron. Likewise, each gene may be on a different vector.

Thus, the present invention provides DNA encoding a protein associated with activation of nitrile hydratase. A specific example may be a portion 1,325-1663 of the nucleotide sequence shown in SEQ ID NO: 3 of the sequence listing. However, examples are not limited thereto, and any DNA containing this nucleotide sequence may be used. In addition, DNA which hybridizes with DNA having a nucleotide sequence complementary to the above sequence under stringent conditions, may also included in the present invention, as long as it is associated with activation of nitrile hydratase.

That is to say, the nitrile hydratase of the present invention can be further activated using these DNAs. Examples of stringent conditions may include the conditions described in manual included with an ECL direct nucleic acid labeling and detection system (manufactured by Amersham Pharmacia Biotech) (wash: 42"C; a primary wash buffer containing 0.5 x SSC). An example of DNA capable of hybridizing with the aforementioned DNA under stringent conditions may be DNA, which hybridizes with a detection sample that is a nucleotide sequence portion consisting of any number of, generally at least 20, preferably at least 50, and particularly preferably at least 100 contiguous nucleotides of a nucleotide sequence complementary to DNA containing a portion 1,325-1663 of the nucleotide sequence shown in SEQ ID NO: 3 of the sequence listing under the aforementioned stringent conditions. In addition, the protein associated with activation of the nitrile hydratase of the present invention is a protein having the amino acid sequence shown in SEQ ID NO: 4 of the sequence listing, which consists of 112 amino acids. However, such a protein may comprise a substitution, deletion, or insertion of one or several amino acids with respect to the above amino acid sequence, as long as it has ability to get involved in activation of nitrile hydratase. Also, the protein of the present invention is naturally anticipated to undergo post-translational modification depending on the type of host. A preferred example may be an amino acid sequence comprising a substitution, deletion, insertion, or post-translational modification of 1 to 25 amino acids with respect to the above amino acid sequence. The number of amino acids that are substituted, deleted, inserted, or post-translationally modified, is more preferably 1 to 10, further more preferably 1 to 5, and most preferably 1 to 3.

Moreover, the present invention provides a transformant, which is obtained by introducing the aforementioned DNA into host cells for transformation.

Such a transformant can be obtained by transforming host cells with the expression vector produced by the aforementioned method. Examples of host cells may include microorganisms, mammalian cells, and plant cells. Of these, microorganisms are preferably used. An example of such microorganisms may be Escherichia coli, as described later in the example section, but examples are not particularly limited thereto.

Other examples may include microorganisms belonging to genus Bacillus, genus Pseudomonas, genus Corynebacterium, genus Brevibacterium, genus Streptococcus and genus Rhodococcus, Actinomycetes, and yeasts.

A gene can be introduced into preferred host microorganisms by any given method publicly known in the present technical field, such as genetic transformation, genetic transduction, conjugal transfer, or electroporation.

As a method for producing the nitrile hydratase of the present invention or a bacterial cell mass-treated product containing the same, as described above, microorganisms capable of producing the above nitrile hydratase, such as those belonging to genus Geobacillus, and particularly preferably Geobacillus thermoglucosidasius Q-6 strain, are cultured, and thereafter, the above enzyme can be obtained from the culture product by appropriately combining known purification methods. In addition, as described above, the enzyme can also be obtained from a transformant transformed with a nitrile hydratase gene.

A method for culturing microorganisms belonging to genus Geobacillus, so as to obtain the above enzyme, is the same as that described above. In general, the aforementioned transformant is preferably cultured in a medium containing nutrients that can be assimilated by such microorganisms. For example, the transformant can be cultured by a common method for producing enzymes or antibiotics. In general, either solid culture or liquid culture may be applied. For example, the following nutrients may appropriately be mixed into a medium used: carbohydrates such as glucose or sucrose; alcohols such as sorbitol or glycerol; organic acids such as citric acid or acetic acid; carbon sources such as soybean oil or a mixture thereof; nitrogen-containing inorganic or organic nitrogen sources such as yeast extract, meat extract, ammonium sulfate, or ammonia; inorganic nutrients such as phfiosphate, magnesium, iron, cobalt, manganese, or potassium; and vitamins such as biotin or thiamine. More preferably, 0.1 tg/ml or more Fe ions or Co ions may be added to such medium composition. The culture is preferably carried out under aerobic conditions. The culture temperature is not particularly limited as long as it enables the growth of host microorganisms. It is generally between 5°C and 80'C, preferably between 20 0 C and 70'C, and more preferably between 25°C and 42C. The pH applied during the culture is not particularly limited, as long as it enables the growth of host microorganisms. It is generally between pH 3 and pH 9, preferably between pH 5 and pH 8, and more preferably between pH 6 and 7.

Furthermore, in the present invention, a nitrile compound can be produced using the enzyme of the present invention. When the aforementioned enzyme is used in this invention, the purification degree thereof is not limited, unless the action of the enzyme of the present invention is not inhibited. Not only a purified enzyme of the present invention, but also a product containing the above enzyme may be used. Further, a microorganism producing the above enzyme, or a transformant obtained by introducing the enzyme gene, may also be used. When such a microorganism or a transformant is used, a bacterial cell mass may be used. Examples of such a bacterial cell mass used herein may include a living bacterial cell mass and a bacterial cell mass with increased permeability of compounds that is obtained by treating with a solvent such as acetone or toluene, or by freeze-drying. In some cases, products containing the enzyme, such as a product obtained by disintegration of a bacterial cell mass or a bacterial cell mass extract, may also be used. In order to produce a bacterial cell mass-treated product containing the above enzyme, for example, a culture product is first subjected to solid-liquid separation, the obtained wet bacterial cell mass is then suspended in a buffer solution such as a phosphate buffer or a Tris-HC1 buffer, as necessary. Thereafter, the suspension is appropriately subjected to the combined use of several bacterial cell mass disintegration treatments such as ultrasonication, French press treatment, a grinding treatment using glass beads, or a treatment using a cell wall digesting enzyme such as lysozyme or protease, so as to extract the enzyme from the bacterial cell mass, thereby obtaining a nitrile hydratase-containing crude solution. This enzyme-containing crude solution is subjected to known means for isolating and purifying proteins or enzymes, as necessary, so as to further purify it. For example, a method comprising: adding an organic solvent such as acetone or ethanol to such an enzyme-containing crude solution, so as to separate and precipitate the enzyme, or adding ammonium sulfate or the like thereto, so as to salting-out the enzyme; and then precipitating a fraction containing nitrile hydratase from the aqueous solution and recovering it, is applied. Moreover, the enzyme can be purified by appropriately combining anion exchange, cation exchange, gel filtration, affinity chromatography using an antibody, chelate, or the like. As a matter of course, such an enzyme, bacterial cell mass, bacterial cell mass-treated product containing the enzyme, or the like, may be filled into a column by a known method, or may be immobilized on a carrier. In particular, in the case of a bacterial cell mass, it may be embedded in a polymer such as polyacrylamide gel. Such a bacterial cell mass or a bacterial cell mass-treated product is suspended in water or an aqueous solution such as a phosphate buffer, and a nitrile compound is then added to the suspension, so as to promote the reaction. The concentration of a bacterial cell mass or a bacterial cell mass-treated product used is between 0.01% and 20% by weight, and preferably between 0.1% and 10% by weight. The upper limit of the reaction temperature is preferably more preferably 85°C, and further more preferably 70 0 C. The lower limit of the 00 O reaction temperature is for example 1 0 C, preferably 40C, and more preferably 100C. The pH for the reaction is for example between pH 5 and pH 10, and e( a preferably between pH 6 and pH8. The reaction time is for example for 1 minute to 72 hours. Moreover, a high concentration of amide compound can be generated and accumulated by gradually adding a nitrile compound dropwise to o the suspension. In order to recover the generated amide compound from the 00 reaction solution, a method comprising eliminating a bacterial cell mass, a

I€'

bacterial cell mass-treated product, or the like by filtration, centrifugation, or the like, and then collecting the resultant by crystallization or the like, can be applied, C 10 for example.

(N

Comprises/comprising and grammatical variations thereof when used in this specification are to be taken to specify the presence of stated features, integers, steps or components or groups thereof, but do not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

EXAMPLES

The present invention will be described in detail in the following examples.

These examples are not intended to limit the scope of the present invention.

Example 1: Separation of bacterial cell mass A small amount (approximately 1 g) of soil collected from the place located near hot springs in Saitama prefecture was placed in a test tube containing 5 ml of a normal saline solution. The obtained mixture was subjected to a shaking culture for 3 days in a shaking incubator at 650C. An aliquot (0.5 ml) of the culture solution was added to a medium (pH 7.0) of 1.0% by weight of glucose, by weight of polypeptone, and 0.3% by weight of yeast extract, and the obtained mixture was then subjected to a reciprocal shaking culture at 650C for 2 days. An aliquot (0.1 ml) of the culture solution obtained by this culture was spread on an agar plate medium containing the aforementioned medium composition, and it was further cultured at 650C for 2 days, so as to form 28a 00 O colonies, thereby isolating microorganisms. The isolated microorganisms were C inoculated into a liquid medium obtained by adding 0.1% by weight of n- D valeronitrile to a medium with the same above composition, and the obtained mixture 00 was cultured at 65 0 C for 24 hours, so as to obtain a culture solution containing microorganisms having high nitrile-assimilating ability. 1 ml of this culture solution was added to 9 ml of a solution containing 1.1% by weight of acrylonitrile (0.05 M phosphate buffer; pH and the reaction was then initiated at 27 0 C. Ten minutes later, the reaction was terminated by addition of 1 ml of 1 N HC1. An aliquot of the reaction solution was analyzed by liquid chromatography (HPLC), and the presence or absence of the generation of acrylamide was examined, so as to screen microorganisms having nitrile hydratase activity. Thus, a microorganism having hydration activity of converting a nitrile compound to an amide compound, Geobacillus thermoglucosidasius Q-6 strain, was obtained.

(Conditions for liquid chromatography analysis) Main body: HITACHI D-7000 (manufactured by Hitachi, Ltd.) Column: Inertsil ODS-3 (manufactured by GL Sciences Inc.) Length: 200 mm Column temperature: Flow rate: 1 ml/min Amount of sample injected: 10 pl Solution: 0.1%-by-weight aqueous phosphate solution Example 2: Upper limit of temperature for growth of bacterial cell mass The Geobacillus thermoglucosidasius Q-6 strain obtained in Example 1 was applied to an agar plate medium containing the medium composition used in Example 1, and the obtained mixture was cultured at a plurality of different temperatures, so as to examine the growth state of the bacterial cell mass. The results are shown in Table 1.

The Geobacillus thermoglucosidasius Q-6 strain normally proliferated up to a temperature of 70 0 C, and the strain was able to grow even at 72 0

C.

Table 1 Evaluation standard: not proliferated; proliferated; significantly proliferated Culture Proliferation level of Geobacillus temperature thermoglucosidasius Q-6 72 Example 3: Measurement of nitrile hydratase activity in bacterial cell mass of Geobacillus thermoglucosidasius Q-6 strain and temperature dependence thereof 100 ml of a sterilized medium (pH 7.0) containing 0.2% by weight of glycerol, 0.2% by weight of trisodium citrate dehydrate, 0.1% by weight of potassium dihydrogen phosphate, 0.1% by weight of dibasic potassium phosphate, 0.1% by weight of polypeptone, 0.1% by weight of yeast extract, 0.1% by weight of sodium chloride, 0.1% by weight of n-valeronitrile, 0.02% by weight of magnesium sulfate heptahydrate, 0.003% by weight of iron (II) sulfate heptahydrate, and 0.0002% by weight cobalt chloride hexahydrate, was placed in 500-ml Erlenmeyer flask. Thereafter, 1 ml of a culture solution containing the Geobacillus thermoglucosidasius Q-6 strain, which had previously been cultured in the same type of medium, was inoculated in the above-sterilized medium. The obtained mixture was subjected to a roll-tube shaking culture at 65 0 C for 1 day at 200 strokes/min, so as to obtain a bacterial cell mass culture solution. 300 ml of this bacterial cell mass culture solution of the Geobacillus thermoglucosidasius Q-6 strain was centrifuged (10,000 x g; 15 minutes), so as to collect a bacterial cell mass. The collected bacterial cell mass was washed with a 0.05 M phosphate buffer (pH 7.5) and was then suspended in 50 ml of the same buffer. The thus prepared bacterial cell mass suspension was allowed to react for 5 minutes by the aforementioned method, and the hydration activity of converting a nitrile compound to an amide compound was measured. With regard to the unit of enzyme activity, activity necessary for converting 1 pmol acrylonitrile to acrylamide for 1 minute was defined as 1 unit (hereinafter referred to as The nitrile hydratase activity (U/mg) per wet bacterial cell mass weight at 27 0 C was found to be 9.37 U/mg. Moreover, a bacterial cell mass suspension containing 0.5% by weight of acrylonitrile was prepared to result in a nitrile hydratase activity of 5 U/ml at 10 0 C. Using this bacterial cell mass suspension, the nitrile hydratase activity was obtained in the same above manner under temperature conditions of 30 0 C, 40°C, 50 0 C, 60 0 C, and 70 0 C. The results are shown in Table 2.

As a result, it was found that when a bacterial cell mass is used for the reaction, the optimal temperature was approximately 60°C, and that high activity was exhibited particularly in a high temperature range.

Table 2 Reaction temperature Nitrile hydratase activity (U/ml) 19.2 39.4 49.2 50.6 42.4 Example 4: Heat stability of nitrile hydratase in bacterial cell mass of Geobacillus thermoglucosidasius Q-6 strain In order to examine the heat stability of nitrile hydratase activity in the bacterial cell mass of the Geobacillus thermoglucosidasius Q-6 strain, the bacterial cell mass obtained by the culture method in Example 3 was suspended in distilled water, resulting in 10 U/ml. The suspension was subjected to a heat retention treatment at a certain temperature for 30 minutes, and the remaining activity was then measured. 0.5 ml of the bacterial cell mass solution obtained after the heat retention treatment was added to ml of l%-by-weight acrylonitrile solution (0.05 M potassium phosphate buffer; pH The mixture was stirred at 27 0 C, and thus the reaction was initiated. Five minutes later, 100 pl of 1 N hydrochloric acid was added to the reaction solution, so as to terminate the reaction. The activity after the preservation treatment was calculated.

The activity before the preservation treatment was defined as a standard (100), and the activity after the preservation treatment was indicated relative to such a standard. The results are shown in Table 3. From these results, it can be said that the enzyme activity of nitrile hydratase in the bacterial cell mass of the Geobacillus thermoglucosidasius Q-6 strain is stably maintained at a high temperature, that 80% or more of the activity can be maintained at a high temperature of 70 0 C, and that 30% or more of the activity can be maintained even at a high temperature of 80 0

C.

Table 3 Treatment temperature (oC) Activity (U/ml) Remaining activity 5 100 4.8 96 4.5 4.4 88 4.2 84 1.6 32 Example 5: Reaction using various nitrile compounds as substrates The nitrile hydratase activity necessary for converting various nitrile compounds described in Table 4 below to amide compounds corresponding thereto was examined.

1 ml of a bacterial cell mass suspension was added to 9 ml of 1.1% nitrile solution (0.05 M potassium phosphate buffer; pH and the reaction was initiated at a reaction temperature of 30 0 C. Ten minutes later, 1 ml of 1 N HC1 was added to the reaction solution, so as to terminate the reaction. Conditions for HPLC analysis were the same as those applied in Example 1. However, as a solution, distilled water containing by weight of acetonitrile was used. As a result, nitrile hydratase activity was observed for all of the nitrile compounds described in Table 4 as substrates.

Table 4 Nitrile compounds used Adiponitrile n-butyronitrile Acetonitrile Hexanenitrile Isobutyronitrile Benzonitrile n-valeronitrile The details of the present invention about clarification of the amino acid sequences and nucleotide sequences of the nitrile hydratase subunit a (ORF2), subunit P (ORF1), and nitrile hydratase activator (ORF3) derived from the Geobacillus thermoglucosidasius Q-6 strain described in Example 6 and in examples following it, will be summarized below.

A bacterial cell mass obtained by the culture of the Geobacillus thermoglucosidasius Q-6 strain was disintegrated. The disintegrated product was then subjected to precipitation with ammonium sulfate, anion exchange column chromatography, DEAE column, and hydroxy apatite column, and it was then subjected to gel filtration chromatography and dialysis, so as to purify nitrile hydratase.

The amino acid sequence of approximately 30 residues at the N-terminus of each of the subunit cx and subunit P of the purified nitrile hydratase was determined. Taking into consideration the use of amino acid codon based on the genus to which the above strain belongs, degenerate oligonucleotide primers used for gene amplification were produced. Degenerate PCR was carried out using chromosomal DNA extracted from the above bacterial cell mass as a template, so as to obtain an amplified DNA fragment.

The amplified DNA fragment was cloned, and the nucleotide sequence of an insert fragment was determined. An amino acid sequence deduced from the above nucleotide sequence was compared with the N-terminal amino acid sequence of each of the nitrile hydratase subunit c and subunit P purified from the Geobacillus thermoglucosidasius Q-6 strain, and it was confirmed that the cloned sequence encodes nitrile hydratase.

As a result, it was revealed that nitrile hydratase genes are located adjacent to each other in the order of the subunit p and the subunit a from upstream of the side in the Geobacillus thermoglucosidasius Q-6 strain.

Degenerate oligonucleotide primers used for gene amplification were produced using sequences with high homology from among downstream genes of various types of known nitrile hydratase subunit a. Degenerate PCR was carried out using chromosomal DNA extracted from the above bacterial cell mass as a template, so as to obtain an amplified DNA fragment. The amplified DNA fragment of the subunit a portion of the above-obtained strain was cloned, and the nucleotide sequence thereof was determined.

The thus obtained nitrile hydratase subunit a and subunit P of the Geobacillus thermoglucosidasius Q-6 strain were introduced into a suitable expression vector.

Using the thus constructed expression plasmid, a suitable host strain was transformed.

Examples of a host used herein may include members of genus Rhodococcus or genus Corynebacterium, and Escherichia coli. A host that does not have amidase is preferably used. The obtained transformant was cultured, and the obtained bacterial cell mass was allowed to come into contact with acrylonitrile in an aqueous vehicle, so as to confirm the generation of acrylamide. Thereafter, a comparison was made in terms of generation efficiency and nitrile hydratase activity.

Subsequently, colony hybridization was carried out using the above obtained DNA fragment as a probe, peripheral genes including genes downstream of the nitrile hydratase subunit a and subunit P of the Geobacillus thermoglucosidasius Q-6 strain were cloned.

Such downstream genes were allowed to expressed together with the nitrile hydratase subunit a and subunit P, and nitrile hydratase activities thereof were compared.

As a result, it was found that such downstream genes are associated with activation for significantly increasing nitrile hydratase activity.

Example 6: Purification of nitrile hydratase enzyme derived from Geobacillus thermoglucosidasius Q-6 strain The Geobacillus thermoglucosidasius Q-6 strain was cultured and then subjected to various columns, so as to purify a nitrile hydratase active fraction.

The nitrile hydratase active fraction was measured by chromatography as follows. 1% by weight of acrylonitrile was added to an eluant of each fraction diluted with an HEPES buffer (100 mM; pH and the mixture was then reacted at 27 0 C for 1 minute. 1 N HC1 was added to the reaction solution at an amount of 10% by fluid weight, so as to terminate the reaction. The concentration of the generated acrylamide was measured by the HPLC analysis method described in Example 1.

In order to purify nitrile hydratase derived from the Geobacillus thermoglucosidasius Q-6 strain, the above strain was first inoculated into a V/F medium containing 0.1% by weight of n-valeronitrile by weight of glycerol, 0.2% by weight of trisodium citrate dehydrate, 0.1% by weight of potassium dihydrogen phosphate, 0.1% by weight of dibasic potassium phosphate, 0.1% by weight of polypeptone, 0.1% by weight of yeast extract, 0.1% by weight of sodium chloride, 0.1% by weight of n-valeronitrile, 0.02% by weight of magnesium sulfate heptahydrate, 0.003% by weight of iron (II) sulfate heptahydrate, and 0.0002% by weight cobalt chloride hexahydrate), and the obtained mixture was then cultured at 65 0 C for 24 hours.

For the culture, a 2-ml deep plate with 96 wells (COSTAR) was used. After completion of the culture, cells were collected by centrifugation at 8,000 g for 10 minutes.

Thereafter, 3 g of the obtained wet bacterial cell mass was resuspended in 20 ml of an HEPES buffer (100 mM; pH The bacterial cell mass was disintegrated with an ultrasonic disintegrator under cooling, and ammonium sulfate (30% saturated concentration) was then added to the solution containing the disintegrated bacterial cell mass. The mixture was gently stirred at 4 0 C for 30 minutes, and it was then centrifuged at 20,000 g for 10 minutes, so as to obtain a supernatant. Thereafter, ammonium sulfate saturated concentration) was added to the centrifuge supernatant, and the obtained mixture was then gently stirred at 4 0 C for 30 minutes. Thereafter, the mixture was centrifuged at 20,000 g for 10 minutes, so as to obtain a precipitate. The obtained precipitate was redissolved in 9 ml of an HEPES buffer (100 mM; pH Dialysis was carried out at 4 0 C for 24 hours in 1 L of the same solution, and the resultant was then subjected to anion exchange chromatography (Amersham Biosciences; HiTrap DEAE FF (column volume: 5 ml x 5 columns). An HEPES buffer (100 mM; pH 7.2) was used as a developing solution, and a fraction was eluted by linearly increasing the potassium chloride concentration from 0.0 M to 0.5 M, thereby obtaining a fraction having nitrile hydratase activity. The obtained fraction was subjected to apatite column chromatography (manufactured by BIO-RAD; CHT2-I (column volume: 2 Using an aqueous 0.01 M potassium phosphate solution (pH 7.2) as a developing solution, and a fraction was eluted by linearly increasing the potassium chloride concentration from 0.01 M to 0.3 M, thereby obtaining a fraction having nitrile hydratase activity. The obtained fraction was subjected to gel filtration chromatography (Amersham Biosciences; Superdex 200 HR 10/30), using a 0.05 M aqueous sodium phosphate solution (pH 7.2) containing 0.15 M NaC1 as a developing solution, thereby obtaining a nitrile hydratase active fraction. The nitrile hydratase active fraction obtained by the gel filtration chromatography was used in the following examples.

Example 7: Reaction temperature dependence of nitrile hydratase in nitrile hydratase active fraction purified from Geobacillus thermoglucosidasius Q-6 strain With regard to a nitrile hydratase active fraction solution (3.2 mg/ml; 0.05 M phosphate buffer (pH derived from the Geobacillus thermoglucosidasius Q-6 strain, the nitrile hydratase activity of converting a nitrile compound to an amide compound at each reaction temperature shown in Table 5 was measured. The nitrile hydratase active fraction solution was added to 1 ml of 0.5%-by-weight acrylonitrile solution (a 0.05 M potassium phosphate buffer; pH and the obtained mixture was stirred at each temperature, so as to initiate the reaction. Two minutes later, 100 tl1 of 1 N hydrochloric acid was added to the reaction solution, so as to terminate the reaction.

With regard to the unit of enzyme activity, activity necessary for converting 1 p.mol acrylonitrile to acrylamide for 1 minute was defined as 1 unit (hereinafter referred to as The hydration activity (U/mg) per weight of enzyme was shown in Table 5. From these results, it was found that the nitrile hydratase activity in the nitrile hydratase active fraction purified from the Geobacillus thermoglucosidasius Q-6 strain increased up to a high temperature of 60° C, as the reaction temperature increased. Thus, it is considered that the optimal temperature is approximately 600 C, as in the case of using a bacterial cell mass for the reaction. A significantly high nitrile hydratase activity was exhibited even at a high temperature of 70 0

C.

Table Reaction temperature (0 C) Activity (U/mg) 210.4 27 550.7 1135.3 2228.8 2823.3 2781.1 Example 8: Heat stability of nitrile hydratase purified from Geobacillus thermoglucosidasius Q-6 strain In order to examine the heat stability of activity of nitrile hydratase purified from the Geobacillus thermoglucosidasius Q-6 strain, a nitrile hydratase active fraction 38 solution (3.2 mg/ml; 0.05 M phosphate buffer (pH was subjected to a heat retention treatment at a certain temperature for 30 minutes, and the remaining activity was then measured. 5 pl of the nitrile hydratase solution obtained after the heat retention treatment was added to 1 ml of 0.5%-by-weight acrylonitrile solution (0.05 M potassium phosphate buffer; pH The mixture was stirred at 27 0 C, and thus the reaction was initiated. Two minutes later, 100 ul of 1 N HC1 was added to the reaction solution, so as to terminate the reaction. The activity after the preservation treatment (remaining activity) was calculated. The activity before the preservation treatment was defined as a standard (100), and the activity after the preservation treatment was indicated relative to such a standard. The results are shown in Table 6. From these results, it can be said that the enzyme activity of nitrile hydratase in an aqueous solution containing a nitrile hydratase active fraction purified from the Geobacillus thermoglucosidasius Q-6 strain is stably maintained at a high temperature, that 60% or more of the activity can be maintained at a high temperature of 60 0 C, and that 35% or more of the activity can be maintained even at a high temperature of 70 0

C.

Table 6 Treatment temperature Remaining activity 89.2 27 85.6 82.3 78.9 66.9 38.8 Example 9: Acrylonitrile concentration dependence and concentration resistance of nitrile hydratase purified from Geobacillus thermoglucosidasius Q-6 strain With regard to nitrile hydratase purified from the Geobacillus thermoglucosidasius Q-6 strain, in order to examine its dependence on the concentration of acrylonitrile acting as a substrate and its resistance to the concentration, 4 .1 of a nitrile hydratase active fraction solution (3.2 mg/ml; 0.05 M phosphate buffer (pH was added to 5 ml of a solution (0.05 M potassium phosphate buffer; pH 7.5) containing various weight-% acrylonitrile, and the obtained mixture was stirred at 27 0 C, so as to initiate the reaction. 5 minutes, 10 minutes, 20 minutes, and 40 minutes later, 1 ml each of the reaction solution was taken out, and 100 pl of 1 N HCI was added thereto, so as to terminate the reaction. The concentration of the generated acrylamide was quantified by HPLC. The results were shown in Table 7. From these results, it can be said that the enzyme activity of nitrile hydratase in an aqueous solution containing a nitrile hydratase active fraction purified from the Geobacillus thermoglucosidasius Q-6 strain is stably maintained in a high concentration of acrylonitrile. Even after the reaction was carried out for 40 minutes in an acrylonitrile solution with a high concentration of 6%, the activity did not decrease when compared with the case of a lower acrylonitrile concentration, and rather, the activity increased, as the concentration of the substrate increased.

Table 7 Acrylonitrile concentration (weight at the time of initiation of reaction Acrylamide concentration (weight 5 minutes after initiation of reaction Acrylamide concentration (weight 10 minutes after initiation of reaction Acrylamide concentration (weight 20 minutes after initiation of reaction Acrylamide concentration (weight minutes after initiation of reaction 0.018 0.032 0.052 0.087 0.023 0.042 0.070 0.107 0.026 0.046 0.077 0.137 0.030 0.051 0.082 0.169 Example 10: Acrylamide concentration resistance of nitrile hydratase purified from Geobacillus thermoglucosidasius Q-6 strain In order to examine inhibition of acrylamide as a product against nitrile hydratase purified from the Geobacillus thermoglucosidasius Q-6 strain, 10 pl of a nitrile hydratase active fraction solution (3.2 mg/ml; 0.05 M phosphate buffer (pH was added to 1 ml of a solution (0.05 M potassium phosphate buffer; pH 7.5) containing by weight of acrylonitrile and 35% by weight of acrylamide. The obtained mixture was stirred at 27 0 C, so as to conduct the reaction for 10 minutes, and thereafter, the concentration of acrylonitrile in the solution was assayed by HPLC after completion of the reaction. As a result, it was found that all acrylonitrile was converted to acrylamide.

From this result, it can be said that the enzyme activity of nitrile hydratase in an aqueous solution containing a nitrile hydratase active fraction purified from the Geobacillus thermoglucosidasius Q-6 strain is maintained even in a high concentration of acrylamide such as a concentration of Example 11: Cloning of genes of nitrile hydratase subunit P and subunit a portions derived from Geobacillus thermoglucosidasius Q-6 strain Confirmation of nitrile hydratase enzyme derived from Geobacillus thermoglucosidasius Q-6 strain and determination of N-terminal amino acid sequence A nitrile hydratase active fraction eluant obtained by gel filtration chromatography in Example 6 was subjected to reduced SDS-polyacrylamide electrophoresis under reduction conditions. After completion of the electrophoresis, the resultant protein was stained with Coomassie brilliant blue (CBB), followed by decolorization. As a result, two main bands, one having a molecular weight of approximately 25 K dalton and the other having a molecular weight of approximately 28 K Dalton, were confirmed. These two main purified proteins were transcribed on a PVDF membrane (manufactured by MILLIPORE), using a blotting device (BIO-RAD), and then stained with CBB. Thereafter, portions on which the two bands of interests adsorbed were cut out of the PVDF membrane. Subsequently, the N-terminal amino acid sequences of the two types of proteins were decoded using a full automatic protein primary structure analyzer PPSQ-23A (Shimadzu Corp.). As a result, it was found that the N-terminal amino acid sequence of the protein with a molecular weight of 25 K dalton is SEQ ID NO: 23 of the sequence listing, and that the N-terminal amino acid sequence of the protein with a molecular weight of 28 K dalton is SEQ ID NO: 24 of the sequence listing.

When the above sequences were compared with the amino acid sequences of known nitrile hydratases, the polypeptide chain of 25 K Dalton showed low homology with the nitrile hydratase subunit a, and the polypeptide chain of 28 K Dalton showed low homology with the nitrile hydratase subunit P. Thus, it was suggested that these amino acid sequences encode the above proteins.

Synthesis of oligonucleotide primers corresponding to N-terminal amino acid sequences From the N-terminal amino acid sequences of the two types of proteins as decoded above, the following 12 types of oligonucleotide primers used for degenerate PCR were synthesized, taking into consideration the use of codon based on the above genus: primer 1 (aFl) shown in SEQ ID NO: 5 of sequence listing; primer 2 (aF2) shown in SEQ ID NO: 6 thereof; primer 3 (aF3) shown in SEQ ID NO: 7 thereof; primer 4 (aRl) shown in SEQ ID NO: 8 thereof; primer 5 (aR2) shown in SEQ ID NO: 9 thereof; primer 6 (aR3) shown in SEQ ID NO: 10 thereof; primer 7 (pF1) shown in SEQ ID NO: 11 thereof; primer 8 (3F2) shown in SEQ ID NO: 12 thereof; primer 9 (pF3) shown in SEQ ID NO: 13 thereof; primer 10 (pR1) shown in SEQ ID NO: 14 thereof; primer 11 (3R2) shown in SEQ ID NO: 15 thereof; and primer 12 (pR3) shown in SEQ ID NO: 16 thereof. It is to be noted that y represents c or t; r represents a or g; m represents a or c; k represents g or t; s represents c or g; w represents a or t; d represents a, g, or t; and n represents a, c, g, or t. Moreover, taking into consideration the positions of genes encoding the subunit a and the subunit P on a chromosome, the primers were produced.

Extraction of chromosomal DNA from Geobacillus thermoglucosidasius Q-6 strain and degenerate PCR The Geobacillus thermoglucosidasius Q-6 strain was cultured by the same method as that in Example 6, and a bacterial cell mass was recovered. Thereafter, chromosomal DNA was extracted from the bacterial cell mass, using Genomic-tip System (500/G) kit of QIAGEN. Using, as a template, 0.1 tg of the chromosomal DNA of the Geobacillus thermoglucosidasius Q-6 strain dissolved in a TE solution, degenerate PCR was carried out. The generate PCR was carried out 36 times, using the primers 1 to 6 shown in SEQ ID NOS: 5 to 10 of the sequence listing with the combination of the primers 7 to 12 shown in SEQ ID NOS: 11 to 16 thereof. Using two types of 100-pmol primers, the degenerate PCR was carried out with a reaction solution of a total amount of 100 .1 containing 5 U Takara Ex Taq DNA polymerase and a buffer, so as to amplify a DNA fragment. The following reaction conditions were applied. That is, after heat denaturation at 96 0 C for 3 minutes, a cycle consisting of heat denaturation at 96 0 C for seconds, annealing at 42 0 C for 30 seconds, and elongation at 72 0 C for 1.5 minutes was repeated 35 times, and an elongation reaction was then carried out at 72 0 C for 5 minutes, followed by cold insulation at 4 0 C. Each PCR product was subjected to l%-by-weight agarose electrophoresis, and amplification of DNA was then confirmed. As a result, it was confirmed that an approximately 700-bp DNA fragment was amplified only in the two cases where PCR was carried out, using the primer 5 (aR2) shown in SEQ ID NO: 9 of the sequence listing with the combination of the primer 7 (3F1) shown in SEQ ID NO: 11 thereof, and using the primer 5 (aR2) shown in SEQ ID NO: 9 of the sequence listing with the combination of the primer 8 (pF2) shown in SEQ ID NO: 12 thereof.

Cloning of degenerate PCR product and decoding of nucleotide sequence of amplified DNA fragment The amplified DNA fragment was cut out of the gel, and it was then extracted using QIAquick Gel Extraction Kit (QIAGEN). The extract was ligated to pGEM-T Vector (Promega), using T4 DNA Ligase (Takara). As a result of PCR using Ex Taq, the characteristic whereby a nucleotide A is added to the 3'-terminus was utilized. After completion of the ligation reaction, an Escherichia coli JM109 strain was transformed with the ligate, and it was then cultured in an LB agar medium (50 p.g/ml ampicillin, by weight of bacto yeast extract, 1% by weight of bacto trypton, 0.5% by weight of NaC1, and 2.0% by weight of Bacto Agar (pH at 37 0 C overnight. Thereafter, a transformant was selected using amplicillin. Plasmid DNA was extracted from the transformant that had been cultured in an LB medium containing ampicillin by common methods. The nucleotide sequence of an approximately 700-bp insert portion was decoded, using, as primers, the sequences of SP6 and T7 promoters existing on the vector.

As a result, a 681-bp open reading frame (hereinafter referred to as ORFI) was confirmed in the amplified DNA fragment. The space between the translational stop codon of ORF1 and the translational start codon of the next open reading frame (hereinafter referred to as ORF2) was found to be 13 bp. An amino acid sequence consisting of 25 amino acids on the N-terminal side that is estimated from the nucleotide sequence of ORF1 was completely identical to an amino acid sequence consisting of amino acids on the N-terminal side of the polypeptide chain with 28 K dalton that had been purified as described above. It corresponds to a sequence portion from positions 1 to 25 of the amino acid sequence shown in SEQ ID NO: 2 of the sequence listing. The amino acid sequence of ORFI shows low homology with the amino acid sequence of the subunit P of a known nitrile hydratase. Thus, it was suggested that the above amino acid sequence encodes the above protein.

The nitrile hydratase subunit P of the Geobacillus thermoglucosidasius Q-6 strain encodes 226 amino acids. With regard to the coincidence between the amino acid sequence of the above nitrile hydratase subunit P and those of proteins having homology therewith in known database, the above subunit 1 has identity of 43% with nitrile hydratase subunit 1 of genus Klebsiella MC12609 strain, has identity of 42% with that of genus Agrobacterium, and has identity of 40% with that of genus Rhodopseudomonas CGA009 strain. Thus, the identity of the nitrile hydratase subunit P of the Geobacillus thermoglucosidasius Q-6 strain with those of other strains is extremely low. In addition, with regard to the coincidence between the amino acids of the Geobacillus thermoglucosidasius Q-6 strain and those of proteins derived from genus Bacillus, which are closely related to genus Geogacillus, the above subunit P has identity of 35.0% with the nitrile hydratase subunit P of thermophilic bacterium Bacillus BR449 strain, and has identity of 34.5% with that of thermophilic bacterium Bacillus smithii SC-J05-1 strain.

Thus, the identity is extremely low. In contrast, the nitrile hydratase subunit 0 of the thermophilic bacterium Bacillus BR449 strain has high identity of 85.6% with that of the thermophilic bacterium Bacillus smithii SC-J05-1 strain. Moreover, An N-terminal amino acid sequence that is deduced from the nucleotide sequence of ORF2 was completely identical to the N-terminal amino acid sequence of the polypeptide chain with K dalton purified as described above.

From these result, it was revealed that in the Geobacillus thermoglucosidasius Q-6 strain, a gene encoding the nitrile hydratase subunit P with 28 K dalton and a gene encoding the nitrile hydratase subunit a with 25 K dalton are adjacent to each other in this order from upstream of the 5'-terminal side.

Cloning of gene of nitrile hydratase subunit a portion of Geobacillus thermoglucosidasius Q-6 strain In order to clone peripheral genes of the gene of the nitrile hydratase subunit P portion and the gene of the subunit a portion of the Geobacillus thermoglucosidasius Q-6 strain as obtained above, degenerate PCR was carried out. With reference to known genes located downstream of the nitrile hydratase subunit a, the following two types of oligonucleotide primers used for degenerate PCR were produced: primer 13 (pR1) shown in SEQ ID NO: 17 of the sequence listing; and primer 14 (pR2) shown in SEQ ID NO: 18 thereof.

Moreover, the following two types of oligonucleotide primers used for PCR amplification were produced in the nitrile hydratase subunit P of the Geobacillus thermoglucosidasius Q-6 strain, the nucleotide sequence of which had previously been decoded: primer 15 (Q6AposF) shown in SEQ ID NO: 19 of the sequence listing; and primer 16 (Q6abFl) shown in SEQ ID NO: 20 thereof. Using 0.1 pg of the chromosomal DNA of the Geobacillus thermoglucosidasius Q-6 strain as a template, degenerate PCR was carried out. The degenerate PCR was carried out at an annealing temperature of 50°C, 4 times, using the primers 13 and 14 shown in SEQ ID NOS: 17 and 18 of the sequence listing with the combination of the primers 15 and 16 shown in SEQ ID NOS: 18 and 19. As a result, the presence of an approximately 0.8-kb amplified DNA product was confirmed by PCR using the primer 13 (pRI) shown in SEQ ID NO: 17 of the sequence listing with the combination of the primer 15 (Q6AposF) shown in SEQ ID NO: 18 thereof. Furthermore, the presence of an approximately amplified DNA product was confirmed by PCR using the primer 13 (pRI) shown in SEQ ID NO: 16 of the sequence listing with the combination of the primer 16 (Q6abF1) shown in SEQ ID NO: 19 thereof. When PCR was carried out using the primers in other combinations, the existence of such an amplified DNA product could not be confirmed.

The 0.8-kb DNA fragment, which had been amplified by degenerate PCR using the primer 13 (pR1) shown in SEQ ID NO: 16 of the sequence listing with the combination of the primer 15 (Q6AposF) shown in SEQ ID NO: 19 thereof, was cut out of the agarose gel. DNA was then extracted from the DNA fragment by a common method, and it was then incorporated into pGEM-V Vector (Promega). The Escherichia coli JM109 strain was transformed with the vector, and a recombinant was then selected using 50 p.g/ml ampicillin. The transformant was cultured in an LB medium containing ampicillin, and plasmid DNA was then extracted by a common method. Thereafter, the nucleotide sequence of an approximately 0.8-kb insert portion was decoded. As a result, a 618-bp open reading frame (hereinafter referred to as ORF2) was confirmed. An amino acid sequence consisting of 29 amino acids on the N-terminal side that is deduced from the nucleotide sequence of ORF2 was completely identical to the amino acid sequence consisting of 29 amino acids on the N-terminal side of the polypeptide chain with 25 K dalton that had been purified as described above. It corresponds to a sequence portion from positions 1 to 29 of the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing. The amino acid sequence of ORF2 shows low homology with the amino acid sequence of the subunit a of a known nitrile hydratase. Thus, it was suggested that the above amino acid sequence encodes the above protein.

The nitrile hydratase subunit a of the Geobacillus' thermoglucosidasius Q-6 strain encodes 205 amino acids. With regard to the coincidence between the amino acid sequence of the above nitrile hydratase subunit a and those of proteins having homology therewith in known database, the above subunit a has identity of 66.3% with the nitrile hydratase subunit P of thermophilic bacterium Bacillus BR449 strain, and has identity of 63.9% with the nitrile hydratase subunit P of thermophilic bacterium Bacillus smithii SC-J05-1 strain. Thus, the identity of the nitrile hydratase subunit a of the Geobacillus thermoglucosidasius Q-6 strain with those of other strains is low. In contrast, the nitrile hydratase subunit P of the thermophilic bacterium Bacillus BR449 strain has identity of 88.8% with that of the thermophilic bacterium Bacillus smithii SC-J05-1 strain. Thus, these exhibit high homology with each other.

Example 12: Incorporation of nitrile hydratase subunit a and P portions of Geobacillus thermoglucosidasius Q-6 strain into expression vector, and expression in Escherichia coli Incorporation of nitrile hydratase subunit a and P portions of Geobacillus thermoglucosidasius Q-6 strain into expression vector Based on the nucleotide sequences as decoded above, the following two types of oligonucleotide primers were produced for amplification of the nitrile hydratase subunit a and P portions by PCR: primer 17 (Q6ab-Fl-T) shown in SEQ ID NO: 21 of the sequence listing; and primer 18 (Q6ABall-R1-BglII-T) shown in SEQ ID NO: 22 thereof.

In the primer 17 shown in SEQ ID NO: 21 of the sequence listing, the translational start codon of the nitrile hydratase subunit P of the Geobacillus thermoglucosidasius Q-6 strain was designed in the restriction site NdeI. In the primer 18 shown in SEQ ID NO: 22 of the sequence listing, the restriction enzyme BglII site was introduced into directly below the translational stop codon of the nitrile hydratase subunit a of the Geobacillus thermoglucosidasius Q-6 strain. Using the chromosomal DNA of the Geobacillus thermoglucosidasius Q-6 strain as a template, PCR was carried out with 100 pmol each of the primers 17 and 18 shown in SEQ ID NOS: 21 and 22 of the sequence listing, respectively. Using Ex Taq DNA polymerase, the PCR was carried out on a total amount of 100 tl of reaction solution. The following PCR reaction conditions were applied. That is, after heat denaturation at 96 0 C for 3 minutes, a cycle consisting of heat denaturation at 96 0 C for 30 seconds, annealing at 60°C for 30 seconds, and elongation at 72 0 C for 1.5 minutes was repeated 30 times, and an elongation reaction was then carried out at 72 0 C for 5 minutes, followed by cooling to 4 0 C. The solution obtained after PCR was subjected to 1.5%-by-weight agarose electrophoresis. As a result, it was confirmed that an approximately 1.3-kb DNA fragment was amplified.

This amplified DNA product was extracted from the agarose gel by a common method, and it was then ligated to pGEM-T easy Vector of Promega. Thereafter, the Escherichia coli JM109 strain was transformed with the ligate. From the obtained transformant, plasmid DNA was extracted, and the nucleotide sequence of an insert portion was then decoded, so that it was confirmed that the above amplification by PCR did not include any errors.

Subsequently, this plasmid was digested with the restriction enzymes NdeI and EcoRI, and the resultant was then subjected to 1.5%-by-weight agarose electrophoresis.

Approximately 1.3-kb insert DNA was cut out of the agarose gel, and it was then extracted by a common method. As expression vectors, pET-26b(+) Vector and pET-28a(+) Vector manufactured by Novagene were used. Each of such two types of vector DNA was digested with the restriction enzymes NdeI and EcoRI, and the resultant was subjected to 1%-by-weight agarose electrophoresis. Thereafter, an approximately 5.3-kb DNA fragment was extracted by a common method. These insert portions and vectors were subjected to a ligation reaction according to a common method, and the Escherichia coli JM109 strain was transformed with the ligate. From a transformant selected in terms of kanamycin resistance, plasmid DNA was extracted, and a plasmid into which an insert had been introduced was selected. As stated above, expression plasmids, into which the nitrile hydratase subunit a or P portion of the Geobacillus thermoglucosidasius Q-6 strain had been introduced as an insert, were obtained.

Hereinafter, the thus completed plasmids are referred to as pET-26b(+)-pa and pET-28a(+)-pa.

Nitrile hydratase activity of Geobacillus thermoglucosidasius Q-6 strain expressed in Escherichia coli Using expression plasmids pET-26b(+)-pa and pET-28a(+)-3a, the Escherichia coli BL21(DE3)LysE strain of Novagene was transformed. The nitrile hydratase subunit p protein and subunit a protein of the Geobacillus thermoglucosidasius Q-6 strain were allowed to coexpress as a polycistron, using a T7 promoter.

Using each expression plasmid, the competent cells of the Escherichia coli BL21(DE3)LysE strain of Novagene were transformed, and the transformed cell solution was dispersed on an LB agar medium by weight of bacto yeast extract, 1% by weight of bacto tryptone, 0.5% by weight of NaC1, and 2.0% by weight of agar; pH containing 30 ig/ml kanamycin, and the obtained mixture was cultured at 30 0

C

overnight, so as to conduct selection with kanamycin. This transformant was inoculated into 2 ml of an LB medium containing 30 ig/ml kanamycin, and the obtained mixture was then subjected to a shaking culture at 30 0 C at 200 rpm overnight. Thereafter, 2% by weight of the culture solution was inoculated into 10 ml of an LB medium containing jtg/ml cobalt chloride hexahydrate (CoC12-6H20) and 30 pg/ml kanamycin, and the obtained mixture was then subjected to a shaking culture at 30 0 C at 200 rpm for about 3 hours until it resulted in OD600 0.5. Thereafter, 0.1 mM IPTG was added thereto to induce expression from the T7 promoter, and the resultant was then subjected to a shaking culture at 200 rpm for 4 hours. Thereafter, a bacterial cell mass was recovered.

Using the obtained bacterial cell mass, the nitrile hydratase hydration activity thereof of converting a nitrile compound to an amide compound at 27 0 C was measured.

The bacterial cell mass of Escherichia coli, in which nitrile hydratase derived from the Geobacillus thermoglucosidasius Q-6 strain had been allowed to express, was redissolved in a TBS buffer (pH 7.5) containing 20 mM Tris-HC1 and 15 mM NaC1.

The obtained solution was diluted to result in OD=0.2, so as to produce a reaction solution containing acrylonitrile having a final concentration of 0.2% by weight. The reaction solution was stirred at 27°C, so as to conduct the reaction. Thirty minutes later, the reaction was terminated by addition of 100 p1 of 1 N hydrochloric acid. With regard to the unit of enzyme activity, activity necessary for converting 1 p.mol acrylonitrile to acrylamide for 1 minute was defined as 1 unit (hereinafter referred to as The hydration activity (U/mg) per wet bacterial cell mass weight was shown in Table 8.

Table 8 Expression plasmid Activity (U/mg) pET-26b(+) vector 0 pET-26b(+)-pa 0.200 pET-28a(+) vector 0 pET-28a(+)-pa 0.212 From these results, it was revealed that when the nitrile hydratase subunit a and subunit p of the Geobacillus thermoglucosidasius Q-6 strain were allowed to express in Escherichia coli, they have nitrile hydratase activity of converting acrylonitrile to acrylamide.

Example 13: Obtainment of nitrile hydratase peripheral genes derived from Geobacillus thermoglucosidasius Q-6 strain by colony hybridization Production of fluorescently labeled DIG probe Using the DNA of the nitrile hydratase subunit a portion of the Geobacillus thermoglucosidasius Q-6 strain shown in SEQ ID NO: 25 of the sequence listing as a template, a fluorescently-labeled probe was produced using DIG-DNA labeling kit manufactured by Roche. Such a probe was produced in accordance with the DIG manual of Roche.

Chromosome Southern hybridization The chromosomal DNA of the Geobacillus thermoglucosidasius Q-6 strain prepared in Example 11 was digested with various restriction enzymes, and the digest was then subjected to 1%-by-weight agarose gel electrophoresis. DNA in the agarose gel was transcribed on a nylon membrane Hybond-N+ (Amersham). Thereafter, using the above produced fluorescently labeled DIG probe, chromosome Southern hybridization was carried out. A single membrane, on which the DNA had been transcribed and immobilized, was immersed in 10 ml of a hybridization buffer (5 x SSC containing 1% by weight of skimmed milk, 0.1% by weight of N-lauroylsarcosine, 0.02% by weight of SDS, and 50% by weight of formamide), and prehybridization was then carried out at 42 0 C for 2 hours. 100 ng of a fluorescently-labeled probe produced as described above was boiled at 95 0 C for 10 minutes and then quenched for heat denaturation. Thereafter, the resultant was added to a prehybridization buffer, and the obtained mixture was then subjected to hybridization at 42 0 C overnight. After completion of the hybridization, the membrane was washed twice with 150 ml of 2 x SSC containing 0.1% by weight of SDS at room temperature. Subsequently, the membrane was washed twice for 5 minutes with 150 ml of 1 x SSC containing 0.1% by weight of SDS, which had been heated to 65 0 C. Subsequently, the membrane was washed with 100 ml of a maleate buffer (0.1 M maleic acid, 0.15 M NaCl; pH had been adjusted to pH 7.5 by addition of NaOH) for 5 minutes. Thereafter, the resultant membrane was subjected to a blocking treatment at room temperature for 30 minutes in ml of a blocking solution (0.1 M maleate buffer containing 0.3% by weight of Tween 0.15 M NaC1, and 1% by weight of skimmed milk; pH Anti-digoxigenin-AP was diluted with 20 ml of a blocking solution, resulting in a concentration of 75 mU/ml, and an antibody reaction was carried out at room temperature for 30 minutes.

Thereafter, the membrane was washed 5 times with 100 ml of a washing buffer (0.1 M maleate buffer containing 0.3% by weight of Tween 20 and 0.15 M NaC1; pH so as to wash out an unbound antibody. The membrane was subjected to an equilibration treatment for 5 minutes in 20 ml of a detection buffer (0.1 M Tris-HC1, 0.1 M NaCl; pH Thereafter, 34 p1 of a 100 mg/ml NBT (nitroso-blue tetrazolium chloride) solution and 35 p of a 50 mg/ml BCIP solution were diluted with 10 ml of a detection buffer, so as to produce a colorimetric substrate solution NBT/BCIP (5-bromo-4-chloro-3-indolyl phosphate). The colorimetric substrate solution was added to the membrane, so that the membrane was completely immersed therein, and it was then incubated for 1 minute to 16 hours, while the light was blocked. During the incubation, the disk was neither moved nor shaken, and coloration was confirmed. As a result, it was revealed that the downstream genes of the nitrile hydratase subunit a portion were contained in an approximately 2.3-kb gene fragment digested with the restriction enzyme HindIII.

Obtainment of clone of interest by colony hybridization Production ofplasmid library used for colony hybridization Subsequently, colony hybridization was carried out using the same fluorescently labeled DIG probe. 10 p.g of the chromosomal DNA of the Geobacillus thermoglucosidasius Q-6 strain was subjected to 1%-by-weight agarose gel electrophoresis. A portion containing a DNA fragment with a size between approximately 2.0 kb and 2.6 kb was cut out of the agarose gel, and the DNA fragment was then extracted and purified by the same method as described above. The obtained DNA fragment was introduced into a HindIII restriction site in the multicloning site of a pUC118 plasmid vector (manufactured by Takara), using a DNA ligation kit (manufactured by Takara). The pUC118 plasmid vector DNA used for ligation was digested with the restriction enzyme HindIII, and then subjected to a phenol/chloroform treatment and purification by ethanol precipitation. Thereafter, the resultant was subjected to a dephosphorylation treatment on the 5'-terminus thereof with alkaline phosphatase (manufactured by Takara), and then subjected again to a phenol/chloroform treatment and ethanol precipitation, followed by agarose gel electrophoresis. Thereafter, resultant DNA was extracted from the agarose gel for repurification, and the obtained product was used as the pUC118 plasmid vector DNA.

A solution containing the pUC118 plasmid vector, to which the chromosomal DNA fragment of the Geobacillus thermoglucosidasius Q-6 strain with a size between approximately 2.0 kb and 2.6 kb was ligated at a HindIII restriction site, was used to transform the Escherichia coli JM109 strain. The obtained transformed cells were dispersed on an LB agar medium by weight of bacto yeast extract, 1% by weight of bacto tryptone, 0.5% by weight of NaCI, and 2.0% by weight of agar; pH containing 50 jpg/ml ampicillin, 1 mM IPTG (isopropyl-P-D-thiogalactopyranoside), and 2% by weight of X-Gal (5-bromo-4-chloro-indolyl-P-D-galactopyranoside). The obtained mixture was cultured at 37 0 C overnight. As a result, a large number of Petri dishes, wherein 50 to 500 white colonies appeared per Petri dish, were obtained.

Using the above prepared fluorescently-labeled DIG probe, colony hybridization was carried out on a plasmid library of these chromosomal DNA, and clones containing the downstream genes of the nitrile hydratase subunit a of interest were screened.

Obtainment of clone of interest by colony hybridization First, approximately 1,000 clones of the appeared white colonies were streaked on a fresh LB agar medium, using a sterilized pick. This time, such clones were also streaked on an LB agar medium for hybridization of the membrane and on an LB agar medium for conservation. The obtained mixtures were then cultured at 30 0 C overnight.

Subsequently, a nylon membrane Hybond-N+ manufactured by Amersham was gently placed on a Petri dish wherein colonies appeared. One minute later, the membrane was slowly removed from the edge using tweezers. The removed membrane was immersed in a denaturation solution (0.5 M NaOH aqueous solution containing M NaC1) for 7 minutes, with a surface to which the bacterial cell mass was attached upward. Thereafter, the membrane was immersed in a neutralization solution (0.5 M Tris-HCl aqueous solution containing 1.5 M NaCl and 1 mM EDTA-2Na; pH 7.2) for 3 minutes, and it was then further immersed in a fresh neutralization solution for 3 minutes.

Subsequently, the membrane was washed once with a 2 x SSC solution (1L of 1 x SSC contains 8.76 g of NaCl and 4.41 g of sodium citrate), and it was then subjected to air-drying on a dry filter. Moreover, 120 mJ/cm 2 UV was applied to the membrane, so that DNA was immobilized on the membrane.

Detection with DIG antibody and isolation of clone of interest The thus treated membrane on which the DNA had been immobilized was immersed in 10 ml of a hybridization buffer (5 x SSC containing 1% by weight of skimmed milk, 0.1% by weight of N-lauroylsarcosine, 0.02% by weight of SDS, and by weight of formamide), and prehybridization was then carried out at 42 0 C for 2 hours. 100 ng of a fluorescently-labeled probe produced as described above was boiled at 95°C for 10 minutes and then quenched for heat denaturation. Thereafter, the resultant was added to a prehybridization buffer, and the obtained mixture was then subjected to hybridization at 42 0 C overnight. After completion of the hybridization, the membrane was washed twice with 150 ml of 2 x SSC containing 0.1% by weight of SDS at room temperature. Subsequently, the membrane was washed twice for 5 minutes with 150 ml of 1 x SSC containing 0.1% by weight of SDS, which had been heated to 0 C. Subsequently, the membrane was washed with 100 ml ofa maleate buffer (0.1 M maleic acid, 0.15 M NaCl; pH had been adjusted to pH 7.5 by addition of NaOH) for minutes. Thereafter, the resultant membrane was subjected to a blocking treatment at room temperature for 30 minutes in 50 ml of a blocking solution (0.1 M maleate buffer containing 0.3% by weight of Tween 20, 0.15 M NaC1, and 1% by weight of skimmed milk; pH Anti-digoxigenin-AP was diluted with 20 ml of a blocking solution, resulting in a concentration of 75 mU/ml, and an antibody reaction was carried out at room temperature for 30 minutes. Thereafter, the membrane was washed 5 times with 100 ml of a washing buffer (0.1 M maleate buffer containing 0.3% by weight of Tween and 0.15 M NaCl; pH so as to wash out an unbound antibody. The residue was subjected to an equilibration treatment for 5 minutes in 20 ml of a detection buffer (0.1 M Tris-HC1, 0.1 M NaCl; pH Thereafter, 34 l1 of a 100 mg/ml NBT solution and jtl of a 50 mg/ml BCIP solution were diluted with 10 ml of a detection buffer, so as to produce a colorimetric substrate solution NBT/BCIP. The colorimetric substrate solution was added to the membrane, so that the membrane was completely immersed therein, and it was then incubated for 1 minute to 16 hours, while the light was blocked.

During the incubation, the disk was neither moved nor shaken, and coloration was confirmed. As a result, 4 positive signals were found from 1,000 clones on the above membrane, and positive clones overlapping the positions were confirmed on the original Petri dish.

Analysis of positive clones containing downstream genes of nitrile hydratase subunit a portion of Geobacillus thermoglucosidasius Q-6 strain The confirmed positive clones were transferred from the Petri dish, and they were then inoculated into an LB liquid medium containing ampicillin. The mixture was subjected to a shaking culture at 37 0 C at 250 rpm overnight. A bacterial cell mass was recovered by centrifugation, and plasmid DNA was extracted by a common method.

The plasmid DNA was digested with the restriction enzyme HindII, and it was then subjected to 1.5%-by-weight agarose gel electrophoresis. When the size of an insert fragment was measured, it was found to be approximately 2.3 kb. It was confirmed by several patterns of PCR and digestion patterns with restriction enzymes that the insert fragment contained the nitrile hydratase subunit a portion of Geobacillus thermoglucosidasius Q-6 strain.

The thus obtained plasmid was named as pUC118-Q6Hin2.3. The entire nucleotide sequence of the insert fragment was determined. Figure 1 shows the restriction maps and gene structures of the nitrile hydratase of the Geobacillus thermoglucosidasius Q-6 strain and a group of downstream genes thereof.

As a result, it was confirmed that an open reading frame (hereinafter referred to as ORF3) consisting of a 339-bp nucleotide sequence exists downstream of the side of the nitrile hydratase subunit a (ORF2) in the same direction in the insert fragment. The space between the translational stop codon of ORF2 and the translational start codon of ORF3 was found to be 12 bp. The space between the translational stop codon of ORF3 and the translational start codon of ORF located further downstream thereof was found to be 145 bp. ORF3 encoded 112 amino acids, and it had low homology with the following proteins in known database. With regard to the identity in terms of amino acids between ORF3 and a sequence with high homology therewith in such known database, it showed identity of 31% with genus Bacillus BR449 strain P12K, 31% with Rhodococcus rhodochrous J1 strain NhhG, 21% with Rhodococcus rhodochrous J1 strain NhlE, and 23% with Pseudonocardia thermophila JCM3095 strain P16.

Example 14: Construction of expression plasmids for nitrile hydratase subunit a and subunit p and downstream gene ORF3 of Geobacillus thermoglucosidasius Q-6 strain pET-26b(+)-pa and pET-28a(+)-pa plasmids were digested with the restriction enzyme HindlII, and then subjected to a dephosphorylation reaction, followed by extraction with phenol chloroform, so as to conduct a dephosphorylation treatment. The digested product was subjected to 1%-by-weight agarose electrophoresis, and a DNA fragment with a size of approximately 6.1 kb was then extracted by a common method.

This DNA fragment comprises a pET vector and a portion up to a HindIII restriction site corresponding to amino acid at position 60 of the nitrile hydratase subunit P and subunit a of the Geobacillus thermoglucosidasius Q-6 strain.

Subsequently, the pUC118-Q6Hin2.3 plasmid was digested with the restriction enzyme HindIII, and it was then subjected to 1%-by-weight agarose electrophoresis.

Approximately 2.3-kb insert DNA was extracted. This insert was ligated to the previously extracted fragment, and the Escherichia coli JM109 strain was transformed therewith. Thereafter, a plasmid containing the insert DNA was selected from the transformant selected in terms of kanamycin resistance. By confirming the direction of the insert by PCR, plasmids wherein the nitrile hydratase subunit a and subunit P of the Geobacillus thermoglucosidasius Q-6 strain, the downstream gene ORF3, and regions further downstream thereof were contained in pET-26b(+) and pET-28a(+) vectors, were obtained. Hereinafter, the thus completed plasmids are referred to as pET-26b(+)-p1l2 and pET-28a(+)-pa12, respectively.

Subsequently, the nitrile hydratase subunit a and subunit P of the Geobacillus thermoglucosidasius Q-6 strain and a portion up to the translation stop codon of the downstream gene ORF3 were introduced into the above plasmids, so as to construct expression plasmids for allowing 3 types of proteins to co-express. The previously constructed plasmid pET-26b(+)-pal2 was digested with the restriction enzymes NdeI and BglII, and the resultant was then subjected to 1.5%-by-weight agarose gel electrophoresis. A 1.7-kb gene fragment containing the subunit a, the subunit P, and the translation stop codon of the downstream gene ORF3, was extracted by a common method. At the same time, the expression vectors pET-26b(+) and pET-28a(+) were digested with NdeI and BamHI, and the resultant was then subjected to l%-by-weight agarose gel electrophoresis. A 5.3-kb gene fragment corresponding to a vector portion was extracted by a common method. Using, as an insert, the previously extracted 1.7-kb gene fragment containing the subunit a, the subunit P, and the translation stop codon of the downstream gene ORF3, the above vectors were subjected to a ligation reaction according to a common method. During this reaction, a terminus digested with the restriction enzyme BglII was adhered to a terminus digested with the restriction enzyme BamHI. The Escherichia coli JM109 strain was transformed with a solution obtained after the ligation reaction, and plasmid DNA was then extracted from the transformant selected in terms of kanamycin resistance. Thereafter, a plasmid, into which the insert had been introduced, was selected. As stated above, expression plasmids, into which the nitrile hydratase subunit a and subunit 1 of the Geobacillus thermoglucosidasius Q-6 strain and the downstream gene ORF3 are introduced as inserts, and which allow 3 types of proteins to co-express, were obtained. Hereinafter, these plasmids of interest are referred to as pET-26b(+)-pal and pET-28a(+)-pal.

Example 15: Nitrile hydratase activity of Geobacillus thermoglucosidasius Q-6 strain, which allows downstream genes to co-express in Escherichia coli Using the expression plasmids pET-26b(+)-pa and pET-28a(+)-pa, the Escherichia coli BL21(DE3)LysE strain of Novagene was transformed. The nitrile hydratase subunit P protein and subunit a protein of the Geobacillus thermoglucosidasius Q-6 strain were allowed to co-express, using a T7 promoter.

Likewise, the Escherichia coli BL21(DE3)LysE strain of Novagene were transformed with pET-26b(+)-pal and pET-28a(+)-pal, and the nitrile hydratase subunit p protein, subunit a protein, and downstream gene ORF3 protein of the Geobacillus thermoglucosidasius Q-6 strain were allowed to co-express, using a T7 promoter.

Transformants obtained by transforming the Escherichia coli BL21(DE3)LysE strain of Novagene with the expression vectors pET-26b(+) and pET-28a(+) were used as controls.

C

overnight, so as to conduct selection in terms of kanamycin resistance. This transformant was inoculated into 2 ml of an LB medium containing 30 [tg/ml kanamycin, and the obtained mixture was then subjected to a shaking culture at 30 0 C at 200 rpm overnight. Thereafter, 2% by weight of the culture solution was inoculated into 10 ml of an LB medium containing 20 gg/ml cobalt chloride hexahydrate (CoC12.6H20) and ug/ml kanamycin, and the obtained mixture was then subjected to a shaking culture at 0 C at 200 rpm for about 3 hours until it resulted in OD600 0.5. Thereafter, 0.1 mM IPTG was added thereto to induce expression from the T7 promoter, and the resultant was then subjected to a shaking culture at 200 rpm for approximately 4 hours.

Thereafter, a bacterial cell mass was recovered.

The thus obtained bacterial cell mass of Escherichia coli, in which nitrile hydratase derived from the Geobacillus thermoglucosidasius Q-6 strain had been allowed to express, was redissolved in a TBS buffer (pH 7.5) containing 20 mM Tris-HCI and mM NaC1. The obtained solution was diluted to result in OD=0.2, so as to produce a reaction solution containing acrylonitrile having a final concentration of 0.2% by weight.

The reaction solution was stirred at 27 0 C, so as to conduct the reaction. Ten minutes later, and thirty minutes later, 100 il of 1 N hydrochloric acid was added to the reaction solution, so as to terminate the reaction. With regard to the unit of enzyme activity, activity necessary for converting 1 pmol acrylonitrile to acrylamide for 1 minute was defined as 1 unit (hereinafter referred to as The hydration activity (U/mg) per wet bacterial cell mass weight was shown in Table 9.

Table 9 Expression plasmid Activity (U/mg) pET-26b(+) vector 0 pET-26b(+)-pa 0.20 pET-26b(+)-pal 2.74 pET-28a(+) vector 0 pET-28a(+)-pa 0.21 pET-28a(+)-pal 4.22 From these result, it was found that when the case of allowing only the nitrile hydratase subunit a and subunit P of the Geobacillus thermoglucosidasius Q-6 strain to express, is compared with the case of allowing these subunits to co-express together with the downstream gene ORF3, the nitrile hydratase activity significantly increased due to the presence of the downstream gene ORF3. Thus, it became clear that the ORF3 has a function to significantly increase the enzyme activity of the nitrile hydratase of the Geobacillus thermoglucosidasius Q-6 strain.

Example 16: Heat stability of nitrile hydratase of Geobacillus thermoglucosidasius Q-6 strain that is allowed to express in Escherichia coli In order to examine the heat stability of activity, a solution (TBS buffer containing 20 mM Tris-HCl and 15 mM NaCl (pH of Escherichia coli having the expression vector pET26b(+)-a, in which the nitrile hydratase of the Geobacillus thermoglucosidasius Q-6 strain had been allowed to express, was subjected to an incubation treatment at 30 0 C, 65 0 C, and 70 0 C for 30 minutes. After completion of the incubation treatment, the resultant was cooled on ice, and it was maintained at 27 0 C, so that the temperature was kept constant. Thereafter, the nitrile hydratase activity was measured at a reaction temperature of 27 0 C. The cells of Escherichia coli, in which the nitrile hydratase of the Geobacillus thermoglucosidasius Q-6 strain had been allowed to express, were suspended at a turbidity of OD 0.2, so as to produce a reaction solution containing acrylonitrile having a final concentration of 0.5% by weight. The reaction solution was stirred at 27 0 C, so as to initiate the reaction. Thirty minutes later, 10% by fluid weight of 1 N hydrochloric acid was added to the reaction solution, so as to terminate the reaction. The activity obtained when an incubation treatment was carried out at 30 0 C was defined as a standard and the obtained activity was indicated relative to such a standard. The results are shown in Table Table Treatment temperature Remaining activity 100.0 102.5 83.9 From these results, it was found that approximately 80% of the enzyme activity of nitrile hydratase can be maintained even at a high temperature of 70°C in Escherichia coli, in which the nitrile hydratase of the Geobacillus thermoglucosidasius Q-6 strain has been allowed to express, and also that it has high heat resistance even in a case where the nitrile hydratase has been allowed to express in Escherichia coli. Thus, it can be said that this nitrile hydratase is an industrially extremely useful enzyme.

INDUSTRIAL APPLICABILITY The composition of the present invention exhibits high stability in the face of heat, or a high concentration of nitrile or amide compound, and has an effect of efficiently converting a nitrile compound to an amide compound corresponding thereto.

The composition of the present invention can be preferably used in the field of converting a nitrile compound to an amide compound corresponding thereto even in a reaction that is carried out at a high temperature and in a high concentration of nitrile compound or amide compound.

Claims

1-681 of the nucleotide sequence shown in SEQ ID NO: 3 of the sequence listing; or is a purified and/or isolated DNA which encodes for a protein, said protein comprising a subunit a which is encoded by either a DNA containing a sequence portion 695-1312 of the nucleotide sequence shown in SEQ ID NO: 3 of the sequence listing, or DNA hybridizing with said DNA under stringent conditions; and a subunit 3 encoded by either a DNA containing a sequence portion 1-681 of the nucleotide sequence shown in SEQ ID NO: 3 of the sequence listing, or DNA hybridizing with said DNA under stringent conditions, (provided that the aforementioned case is excluded), and where the purified and/or isolated DNA of any of or (D) encodes a protein having the following physicochemical properties: it has nitrile hydratase activity; 00 0 substrate specificity; it exhibits its activity to acrylonitrile, adiponitrile, c acetonitrile, isobutyronitrile, n-valeronitrile, n-butyronitrile, benzonitrile or C 0D hexanenitrile as substrates; molecular weight; said protein comprising at least the following two types of subunits, and each subunit has the following molecular weight measured by reduced SDS-polyacrylamide electrophoresis: 0 subunit a molecular weight 25,000 2,000; and subunit 3 molecular weight 28,000 2,000; (-i heat stability; after the enzyme in an aqueous solution has been heated 0 10 at a temperature of 700C for 30 minutes, the activity that is 35% or more of that (N before heating is maintained; even if 6%-by-weight acrylonitrile solution is used as a substrate, the activity does not decrease, when compared with a case where the substrate concentration is lower than the case of the above substrate; and even in 35%-by-weight acrylamide aqueous solution, it has activity that is based on the existence of acrylonitrile as a substrate.

2. The purified and/or isolated DNA according to claim 1, further comprising DNA having a nucleotide sequence which encodes for the amino acid sequence shown in SEQ ID NO: 4 of the sequence listing, or DNA encoding a protein which comprises a substitution, deletion, addition, or post-translational modification of one or several amino acids with respect to said amino acid sequence, and is associated with the activation of nitrile hydratase.

3. The purified and/or isolated DNA according to claim 1, which further comprises DNA containing a sequence portion 1325-1663 of the nucleotide sequence shown in SEQ ID NO: 3 of the sequence listing, or DNA hybridizing with said DNA under stringent conditions and which DNA encodes for a protein that is associated with the activation of nitrile hydratase.

4. A purified and/or isolated DNA comprising the gene for the subunit a of nitrile hydratase, which gene encodes for the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing. 00 O 5. A purified and/or isolated DNA comprising a gene for the subunit p of nitrile c hydratase, which gene encodes for the amino acid sequence shown in SEQ ID C NO: 2 of the sequence listing. S6. A purified and/or isolated DNA comprising a gene which is associated with the activation of nitrile hydratase, and which gene encodes for the amino acid sequence shown in SEQ ID NO: 4 of the sequence listing. 00 N 7. The purified and/or isolated DNA according to any one of claims 1 to 6, 0 which is derived from the genus Geobacillus. (N

8. The purified and/or isolated DNA according to any one of claims 1 to 6, which is derived from Geobacillus thermoglucosidasius.

9. The purified and/or isolated DNA according to any one of claims 1 to 6, which is derived from the Geobacillus thermoglucosidasius Q-6 strain (FERM BP- 08658). A recombinant vector, into which the purified and/or isolated DNA according to any one of claims 1 to 9 has been incorporated.

11. A purified and/or isolated microorganism selected from at least one of a microorganism transformed with the purified and/or isolated DNA according to any one of claims 1 to 9, or a Geobacillus thermoglucosidasius Q-6 strain (FERM BP-08658) and a mutant thereof, which microorganism expresses nitrile hydratase having the following physicochemical properties: it has nitrile hydratase activity; substrate specificity; it exhibits its activity to acrylonitrile, adiponitrile, acetonitrile, isobutyronitrile, n-valeronitrile, n-butyronitrile, benzonitrile or hexanenitrile as substrates; molecular weight; said protein comprising at least the following two types of subunits, and each subunit has the following molecular weight measured by reduced SDS-polyacrylamide electrophoresis: subunit a molecular weight 25,000 2,000; and 00 O subunit 3 molecular weight 28,000 2,000; c heat stability; after the enzyme in an aqueous solution has been heated 0 at a temperature of 700C for 30 minutes, the activity that is 35% or more of that before heating is maintained; even if 6%-by-weight acrylonitrile solution is used as a substrate, the activity does not decrease, when compared with a case where the substrate 00 concentration is lower than the case of the above substrate; and even in 35%-by-weight acrylamide aqueous solution, it has activity that is based on the existence of acrylonitrile as a substrate.

12. A method for producing a protein or a bacterial cell mass-treated product containing said protein, wherein a microorganism transformed with the purified and/or isolated DNA according to any one of claims 1 to 9, is cultured in a medium.

13. A method for producing a protein or a bacterial cell mass-treated product containing said protein, wherein a microorganism belonging to the genus Geobacillus and being capable of producing a protein having the following physicochemical properties, is cultured in a medium: it has nitrile hydratase activity; substrate specificity; it exhibits its activity to acrylonitrile, adiponitrile, acetonitrile, isobutyronitrile, n-valeronitrile, n-butyronitrile, benzonitrile or hexanenitrile as substrates; molecular weight; it is a protein comprising at least the following two types of subunits, and each subunit has the following molecular weight measured by reduced SDS-polyacrylamide electrophoresis: subunit a( molecular weight 25,000 2,000; and subunit p molecular weight 28,000 2,000; heat stability; after the enzyme in an aqueous solution has been heated at a temperature of 700C for 30 minutes, the activity that is 35% or more of that before heating is maintained; 00 O even if 6%-by-weight acrylonitrile solution is used as a substrate, the c activity does not decrease, when compared with a case where the substrate C aU concentration is lower than the case of the above substrate; and even in 35%-by-weight acrylamide aqueous solution, it has activity that is based on the existence of acrylonitrile as a substrate.

14. A protein or a bacterial cell mass-treated product containing said protein, oo Irn which is obtained from a microorganism cultured by the production method c according to either claim 12 or 13. l 15. A purified and/or isolated protein which has the following physicochemical properties: it has nitrile hydratase activity; substrate specificity; it exhibits its activity to acrylonitrile, adiponitrile, acetonitrile, isobutyronitrile, n-valeronitrile, n-butyronitrile, benzonitrile or hexanenitrile as substrates; molecular weight; it is a protein comprising at least the following two types of subunits, and each subunit has the following molecular weight measured by reduced SDS-polyacrylamide electrophoresis: subunit a molecular weight 25,000 2,000; and subunit 0 molecular weight 28,000 2,000; heat stability; after the enzyme in an aqueous solution has been heated at a temperature of 700C for 30 minutes, the activity that is 35% or more of that before heating is maintained; even if 6%-by-weight acrylonitrile solution is used as a substrate, the activity does not decrease, when compared with a case where the substrate concentration is lower than the case of the above substrate; and even in 35%-by-weight acrylamide aqueous solution, it has activity that is based on the existence of acrylonitrile as a substrate.

16. A purified and/or isolated protein of either or wherein: 00 is a protein which comprises a subunit a having the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing and a subunit 0 having the amino acid sequence shown in SEQ ID NO: 2 of the sequence listing; and is a protein which comprises a modified or unmodified subunit a and a modified or unmodified subunit P, wherein said modified subunit comprises a substitution, deletion, addition, or post-translational modification of one or several 00 amino acids, with respect to either one of or both of the subunit a having the In amino acid sequence shown in SEQ ID NO: 1 of the sequence listing and the subunit p having the amino acid sequence shown in SEQ ID NO: 2; and which protein has the following physicochemical properties: it has nitrile hydratase activity; substrate specificity; it exhibits its activity to acrylonitrile, adiponitrile, acetonitrile, isobutyronitrile, n-valeronitrile, n-butyronitrile, benzonitrile or hexanenitrile as substrates; molecular weight; said protein comprising at least the following two types of subunits, and each subunit has the following molecular weight measured by reduced SDS-polyacrylamide electrophoresis: subunit a molecular weight 25,000 2,000; and subunit p molecular weight 28,000 2,000; heat stability; after the enzyme in an aqueous solution has been heated at a temperature of 70 0 C for 30 minutes, the activity that is 35% or more of that before heating is maintained; even if 6%-by-weight acrylonitrile solution is used as a substrate, the activity does not decrease, when compared with a case where the substrate concentration is lower than the case of the above substrate; and even in 35%-by-weight acrylamide aqueous solution, it has activity that is based on the existence of acrylonitrile as a substrate.

17. A purified and/or isolated protein of either or wherein: is a protein which comprises a subunit a encoded by DNA containing a sequence portion 695-1312 of the nucleotide sequence shown in SEQ ID NO: 3 of the sequence listing, and a subunit p encoded by DNA containing a sequence 00 O portion 1-681 of the nucleotide sequence shown in SEQ ID NO: 3 of the cl sequence listing; and aU is a protein which comprises a subunit a encoded by either DNA Scontaining a sequence portion 695-1312 of the nucleotide sequence shown in SEQ ID NO: 3 of the sequence listing, or DNA hybridizing with said DNA under stringent conditions, and a subunit p encoded by either DNA containing a o0 sequence portion 1-681 of the nucleotide sequence shown in SEQ ID NO: 3 of the sequence listing, or DNA hybridizing with said DNA under stringent conditions S(provided that the aforementioned case is excluded), and which protein has 0 10 the following physicochemical properties: it has nitrile hydratase activity; substrate specificity; it exhibits its activity to acrylonitrile, adiponitrile, acetonitrile, isobutyronitrile, n-valeronitrile, n-butyronitrile, benzonitrile or hexanenitrile as substrates; molecular weight; said protein comprising at least the following two types of subunits, and each subunit has the following molecular weight measured by reduced SDS-polyacrylamide electrophoresis: subunit a molecular weight 25,000 2,000; and subunit 1 molecular weight 28,000 2,000; heat stability; after the enzyme in an aqueous solution has been heated at a temperature of 700C for 30 minutes, the activity that is 35% or more of that before heating is maintained; even if 6%-by-weight acrylonitrile solution is used as a substrate, the activity does not decrease, when compared with a case where the substrate concentration is lower than the case of the above substrate; and even in 35%-by-weight acrylamide aqueous solution, it has activity that is based on the existence of acrylonitrile as a substrate.

18. A purified and/or isolated protein which comprises at least either one of, or both of, a polypeptide containing a subunit a having the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing, or a post-translationally modified product thereof; and a polypeptide containing a subunit p having the amino acid sequence shown in SEQ ID NO: 2 of the sequence listing, or a post- 00 O translationally modified product thereof, wherein the protein has the following c physicochemical properties: aD it has nitrile hydratase activity; substrate specificity; it exhibits its activity to acrylonitrile, adiponitrile, acetonitrile, isobutyronitrile, n-valeronitrile, n-butyronitrile, benzonitrile or hexanenitrile as substrates; 0 molecular weight; said protein comprising at least the following two types of subunits, and each subunit has the following molecular weight measured Sby reduced SDS-polyacrylamide electrophoresis: 10 subunit a molecular weight 25,000 +2,000; and subunit p molecular weight 28,000 2,000; heat stability; after the enzyme in an aqueous solution has been heated at a temperature of 700C for 30 minutes, the activity that is 35% or more of that before heating is maintained; even if 6%-by-weight acrylonitrile solution is used as a substrate, the activity does not decrease, when compared with a case where the substrate concentration is lower than the case of the above substrate; and even in 35%-by-weight acrylamide aqueous solution, it has activity that is based on the existence of acrylonitrile as a substrate.

19. A method for producing an amide compound, wherein a protein having the following physicochemical properties or a bacterial cell mass-treated product containing said protein, is allowed to act on a nitrile compound, so as to obtain an amide compound induced from the above nitrile compound, said physicochemical properties including: it has nitrile hydratase activity; substrate specificity; it exhibits its activity to acrylonitrile, adiponitrile, acetonitrile, isobutyronitrile, n-valeronitrile, n-butyronitrile, benzonitrile or hexanenitrile as substrates; molecular weight; it is a protein comprising at least the following two types of subunits, and each subunit has the following molecular weight measured by reduced SDS-polyacrylamide electrophoresis: subunit a molecular weight 25,000 2,000; and 00 Ssubunit p molecular weight 28,000 2,000; c heat stability; after the enzyme in an aqueous solution has been heated 0 at a temperature of 70 0 C for 30 minutes, the activity that is 35% or more of that before heating is maintained; even if 6%-by-weight acrylonitrile solution is used as a substrate, the o activity does not decrease, when compared with a case where the substrate 00 concentration is lower than the case of the above substrate; and even in 35%-by-weight acrylamide aqueous solution, it has activity that is based on the existence of acrylonitrile as a substrate.

20. A method for producing an amide compound, wherein a protein obtained by culturing in a medium either a microorganism transformed with the purified and/or isolated DNA according to any one of claims 1 to 9, or a microorganism belonging to the genus Geobacillus and being capable of producing a protein, having the following physicochemical properties, or a bacterial cell mass-treated product containing said protein, is used and where said physicochemical properties include: it has nitrile hydratase activity; substrate specificity; it exhibits its activity to acrylonitrile, adiponitrile, acetonitrile, isobutyronitrile, n-valeronitrile, n-butyronitrile, benzonitrile or hexanenitrile as substrates; molecular weight; it is a protein comprising at least the following two types of subunits, and each subunit has the following molecular weight measured by reduced SDS-polyacrylamide electrophoresis: subunit (a molecular weight 25,000 2,000; and subunit p molecular weight 28,000 2,000; heat stability; after the enzyme in an aqueous solution has been heated at a temperature of 70 0 C for 30 minutes, the activity that is 35% or more of that before heating is maintained; even if 6%-by-weight acrylonitrile solution is used as a substrate, the activity does not decrease, when compared with a case where the substrate concentration is lower than the case of the above substrate; and 00 0 even in 35%-by-weight acrylamide aqueous solution, it has activity that C is based on the existence of acrylonitrile as a substrate.

21. A purified and/or isolated DNA which encodes for a protein having the 0 following physicochemical properties: it has nitrile hydratase activity; substrate specificity; it exhibits its activity to acrylonitrile, adiponitrile, 00oo acetonitrile, isobutyronitrile, n-valeronitrile, n-butyronitrile, benzonitrile or c hexanenitrile as substrates; S(c) molecular weight; said protein comprising at least the following two C 10 types of subunits, and each subunit has the following molecular weight measured by reduced SDS-polyacrylamide electrophoresis: subunit a molecular weight 25,000 2,000; and subunit 3 molecular weight 28,000 2,000; heat stability; after the enzyme in an aqueous solution has been heated at a temperature of 70°C for 30 minutes, the activity that is 35% or more of that before heating is maintained; even if 6%-by-weight acrylonitrile solution is used as a substrate, the activity does not decrease, when compared with a case where the substrate concentration is lower than the case of the above substrate; and even in 35%-by-weight acrylamide aqueous solution, it has activity that is based on the existence of acrylonitrile as a substrate; and said DNA is substantially as hereinbefore described with reference to the Examples and SEQ ID Nos.

22. A microorganism transformed with a purified and/or isolated DNA according to claim 21.

23. A method for producing a protein or a bacterial cell mass-treated product containing said protein from a microorganism cultured in a medium, and wherein the protein is substantially as hereinbefore described with reference to the Examples and has the following physicochemical properties: it has nitrile hydratase activity; 00 O substrate specificity; it exhibits its activity to acrylonitrile, adiponitrile, c acetonitrile, isobutyronitrile, n-valeronitrile, n-butyronitrile, benzonitrile or C aD hexanenitrile as substrates; molecular weight; said protein comprising at least the following two types of subunits, and each subunit has the following molecular weight measured by reduced SDS-polyacrylamide electrophoresis: Ssubunit a molecular weight 25,000 2,000; and oo Ssubunit p molecular weight 28,000 2,000; heat stability; after the enzyme in an aqueous solution has been heated 0 10 at a temperature of 700C for 30 minutes, the activity that is 35% or more of that (N before heating is maintained; even if 6%-by-weight acrylonitrile solution is used as a substrate, the activity does not decrease, when compared with a case where the substrate concentration is lower than the case of the above substrate; and even in 35%-by-weight acrylamide aqueous solution, it has activity that is based on the existence of acrylonitrile as a substrate.

24. A purified and/or isolated protein which has the following physicochemical properties: it has nitrile hydratase activity; substrate specificity; it exhibits its activity to acrylonitrile, adiponitrile, acetonitrile, isobutyronitrile, n-valeronitrile, n-butyronitrile, benzonitrile or hexanenitrile as substrates; molecular weight; said protein comprising at least the following two types of subunits, and each subunit has the following molecular weight measured by reduced SDS-polyacrylamide electrophoresis: subunit a molecular weight 25,000 2,000; and subunit p molecular weight 28,000 2,000; heat stability; after the enzyme in an aqueous solution has been heated at a temperature of 700C for 30 minutes, the activity that is 35% or more of that before heating is maintained; 00 0 even if 6%-by-weight acrylonitrile solution is used as a substrate, the C activity does not decrease, when compared with a case where the substrate CD concentration is lower than the case of the above substrate; and even in 35%-by-weight acrylamide aqueous solution, it has activity that is based on the existence of acrylonitrile as a substrate; substantially as hereinbefore described with reference to the Examples and SEQ ID Nos. 00 C 25. A method for producing an amide compound substantially as hereinbefore 0 described with reference to the Examples. ASAHI KASEI KABUSHIKI KAISHA WATERMARK PATENT TRADE MARK ATTORNEYS P26511AU00