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WO1996023887A1 - Procede pour produire les enzymes thermostables xylanase et beta-glucosidase a partir de bacteries - Google Patents

Procede pour produire les enzymes thermostables xylanase et beta-glucosidase a partir de bacteries Download PDF

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WO1996023887A1
WO1996023887A1 PCT/US1996/000891 US9600891W WO9623887A1 WO 1996023887 A1 WO1996023887 A1 WO 1996023887A1 US 9600891 W US9600891 W US 9600891W WO 9623887 A1 WO9623887 A1 WO 9623887A1
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promoter
thermostable
bacteria
xylanase
gene
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Ethel N. Jackson
Gseping Liu
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EIDP Inc
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EI Du Pont de Nemours and Co
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01021—Beta-glucosidase (3.2.1.21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2445—Beta-glucosidase (3.2.1.21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/248—Xylanases
    • C12N9/2482—Endo-1,4-beta-xylanase (3.2.1.8)

Definitions

  • thermostable enzymes such as xylanase and ⁇ -glucosidase
  • thermostable enzymes are important in the manufacture of petrochemicals and in the paper and pulp industry where they are used for the hydrolysis of cellulose and hemicellulose.
  • thermostable enzymes are produced both intracellularly and extracellularly in small amounts by thermophilic bacteria and fungi.
  • the production of commercial quantities of thermostable enzymes is costly due to the high temperatures needed to culture the microorganisms and the complex purification procedures used for enzyme isolation.
  • Production of thermostable enzymes by easily cultured mesophilic fungi or bacteria would increase the cost effectiveness of production. Additionally, exocellular production of the enzymes would facilitate purification and further contribute to the cost effectiveness of the production process.
  • thermostable enzymes One of the most common thermostable enzymes is xylanase which is useful in the conversion of hemicellulose into fermentable carbohydrates.
  • Xylanase production has been reported for many microorganisms including both fungi and bacteria.
  • Typical xylanase producers include the fungi Trichoderma reesei and Trichoder a harzianum as well as bacteria of the genera Bacillus and Cai ocelluin.
  • Luthi et al. teach the cloning of genes encoding xylan-degrading enzymes from C. saccharolytlcum (Appl . Environ . Microblol . , 56, 1017, (1990) and the expression and purification of these enzymes form recombinant E. coll . (Appl . Environ . Microblol . 56, 1017, (1990). Okadad, in Microbiol . Appl . . Food Blotechnol . [Proc. Congr. Singapore Soc. Microbiol.] 2nd, Meeting Date 1989, 1-12 Nga et al., Eds.
  • thermostable enzyme production As they do not require the harsh conditions needed for enzyme production from native sources. However, the desired enzymes are generally produced intracellularly and must be subjected to expensive and time consuming purification processes. A preferred method of thermostable enzyme production would involve the secretion or release of the desired enzyme into the growth media allowing for a simpler and less expensive purification.
  • thermophilic enzymes have been secreted from mesophilic fungi and bacteria.
  • Morosoli et al. Provide the secretion into the growth media of a xylanase from C. alblduy by Saccharomyces sp. and Hamamoto et al. (Agric. Biol . Chem . 51, 3133, (1987)) have demonstrated that recombinantly produced xylanase from Alkalophilic Bacillus is secreted through the outer member of the E. coll host.
  • thermophilic enzymes Typical examples of the use of Bacillus sp . for the secretion of recombinantly produced thermophilic enzymes are taught by Joergensen et al. (WO 9310248) who disclose the use of genes of thermophilic micro ⁇ organisms, expressed in Bacillus licheniformis and Bacillus subtllis under the control of a variant B . licheiformis .alpha.-amylase promoter. Another example is seen in Jung et al. (Biotechnol . Lett . 15, 115, (1993) who teach the expression of a Clostridium xylanase gene in B. subtllis under the control of a strong B . subtilis promoter. Additionally, Hirata et al. (U.S. 4861718) teach the secretion of a thermo ⁇ stable, B . stearothermophilus ⁇ -galactosidase in
  • B. subtilis under the control of a strong B. stearothermophilus promoter.
  • thermostable enzymes are useful, however all suffer from the need to use a signal peptide for membrane translocation and correct post-secretional processing.
  • the selection of an appropriate signal peptide for the protein to be secreted is unpredictable and further complicates the method of production.
  • a preferred method would allow for the secretion of the desired protein in the absence of the signal protein.
  • Proteins that can be secreted without the signal peptide while maintaining biological activity are known, but are rare. There are several examples of proteins containing an C-terminal gene extension that appears to function in membrane translocation (Koronakis et al., EMBO, 8, 595, (1989)). Even more rare are proteins which lack any distinguishable export signal (Rubartelli et al., EMBO, 9, 1503, (1990)), and yet are translocated. Genes encoding two proteins, a xylanase and a ⁇ -glucosidase of the thermophile C. saccharolytlcum, have been cloned and sequenced and analysis of the predicted amino acid sequence indicate that both of these proteins lack a conventional signal peptide. (Perlman et al., J. Mol . Biol . 167, 391, (1983))
  • thermostable enzymes may be produced exocellularly in the absence of the generally required signal peptide.
  • recombinant mesophilic Bacillus subtilis was used to achieve exocellular enzyme production of both xylanase and ⁇ -glucosidase.
  • thermostable enzyme that has been secreted from a recombinant host in the absence of an appropriate signal sequence.
  • the present invention provides a method for the exocellular production of thermostable proteins from bacteria.
  • the method for the exocellular production of a thermostable protein from bacteria preferably Bacillus sp . , comprises the steps of:
  • thermostable protein is expressed.
  • the invention also concerns a method for making a transformation vector for transforming bacteria, so as to exocellularly produce a thermostable protein, comprising the steps of: (i) creating a DNA fragment comprising
  • Figure 1 illustrates the construction of plasmid pBE119 containing the xylanase gene (xynA) C. saccharo- lyticu- ⁇ under the control of the Bacillus apr promoter (aprp) .
  • Figure 2 illustrates the construction of plasmid pBE145 containing the xylanase gene of C. saccharo ⁇ lytlcum under the control of the Bacillus npr promoter (nprp) .
  • Figure 3 illustrates the construction of plasmid pBE164 containing the ⁇ -glucosidase ( ⁇ -glu) gene under the control of the bacillus apr promoter.
  • Figure 4 illustrates the construction of plasmid pBE158 containing the xynA gene downstream of the bacillus apr promoter fused to the apr signal sequence
  • Figure 5 is an SDS-PAGE gel, stained with commassie blue comparing the accumulation of xylanase in the supernatant of pBE119 (apr-xynA) transformed Bacillus grown in three different media.
  • Figure 6 is a Western blot using anti-xylanase antiserum as the primary antibody. This figure demonstrates exocellular production of xylanase in B. subtilis without a signal peptide and reduced production of xylanase associated with the apr S s .
  • Figure 7 is a coomassie-stained PAGE gel of the supernatant fraction and cell associated fraction of cells transformed with pBE164 illustrating exocellular ⁇ -glucosidase production is possible without a signal sequence.
  • ATCC refers to the American Tissue Culture Collection depository located at 12301 Parklawn Drive, Rockville, MD 20852 U.S.A.
  • ATCC No. is the accession number to the following cultures on deposit under terms of the
  • the present invention provides a method for the exocellular production of commercially useful thermostable enzymes in high yields from recombinant bacteria.
  • the present invention also provides vectors for the transformation of host bacteria wherein these vectors are devoid of the signal sequence typically required for translocation of proteins across the cell membrane.
  • thermostable enzyme refers to an enzyme capable of withstanding temperatures in the range of 45°C-115°C without significant loss of biological activity (Bergquist et al., Biotech Genet . Engin . Rev. , 5, 199, (1987)).
  • Typical thermostable enzymes may include but are not limited to xylanases, amylases, transferases, glucosidases, galactosidases, dehydrogenases, polymerases and Upases.
  • thermophilic microorganism or “thermo ⁇ philic bacteria” or “thermophile” will refer to microorganisms which produce enzymes and are capable of living at elevated temperatures of between 45°C and
  • thermostable enzymes isolated to date are similar in function to the more typical mesophilic enzymes with the exception of being able to function at unusually high temperatures.
  • xylanase will refer to a thermostable enzyme capable of the hydrolysis of cellulose and hemicellulose and typically produced by a variety of fungi and bacteria. Sources of xylanase and xylanase genes may include but are not limited to members of the genera Caldocellum, Bacillus, Trichoderma and Clostrldium.
  • ⁇ -glucosidase will refer to a thermostable enzyme capable of the hydrolysis of glucose and glucosides as well as cellulose-based substrates.
  • exocellular protein or extracellular protein will refer to any protein produced by a microorganism which is secreted, transported or released in either an active or passive fashion through the cellular membrane to an exocellular location such as the growth media.
  • precursor protein will refer to a protein which includes the signal peptide and mature protein.
  • mature protein will refer to the final protein product resulting from cleavage of the signal peptide from the precursor.
  • signal peptide will refer to an amino terminal polypeptide preceding the mature protein.
  • the signal peptide is cleaved from and is therefore not present in the mature protein.
  • Signal peptides direct secreted proteins across cell membranes.
  • Signal peptide may also be referred to as "signal protein”.
  • signal sequence will refer to the DNA fragment encoding the signal peptide.
  • promoter and “promoter region” refer to a sequence of DNA, usually 5' to the protein coding sequence of a structural gene, which promotes proper transcription.
  • suitable promoter will refer to any promoter capable of driving the expression of a gene encoding a thermostable enzyme.
  • a “fragment” or “DNA fragment” will constitute a fraction of the DNA sequence of the particular region.
  • construction refers to a plasmid, virus, autonomously replicating sequence, phage or nucleotide sequence, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of DNA fragments have been joined or recombined into a unique entity which is capable of introducing an operably linked promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell.
  • transformation is the acquisition of new genes in a cell by the incorporation of nucleic acid.
  • operably linked refers to the fusion of two fragments of DNA in a proper orientation and reading frame to lead to the transcription of functional RNA.
  • expression as used herein is intended to mean the transcription and translation to gene product from a gene encoding the gene product.
  • plasmid or "vector” as used herein refers to an extra-chromosomal element which is usually in the form of circular double-stranded DNA molecules.
  • restriction endonuclease refers to an enzyme which catalyzes hydrolytic cleavage within a specific nucleotide sequence in double-stranded DNA.
  • compatible restriction sites refers to different restriction sites that when cleaved yield nucleotide ends that can be ligated without any additional modification.
  • apr alkaline protease gene, promoter and protein respectively
  • npr neutral protease gene, promoter and protein respectively.
  • Thermostable enzymes are currently of great commercial usefulness and methods for their reliable production are in high demand.
  • Thermostable xylanases are of increasing importance since they are superior to their thermolabile counterparts for the enzymatic bleach-boosting of wood pulps, because of the high temperature of the incoming pulp.
  • Thermostable enzymes are also used in the food industry where high temperature hydrolysis of carbohydrates is a key process.
  • Thermostable enzymes suitable for expression and exocellular production in the present invention are known in nature and are produced by a variety of thermophilic microorganisms. Suitable thermostable enzymes may include but are not limited to xylanases, amylases, transferases, ⁇ -glucosidases, galactosidases dehydrogenases and Upases.
  • thermostable enzyme activity in the supernatant and cell fractions of cell cultures may be accomplished by any means well know in the art.
  • a variety of methods are available for the determination and quantitation of xylanase.
  • Khan et al. Enzyme Microb . Technol . , 8(6), 373-7, (1986) present an analysis of assay methods for xylanase and xylosidase activities in bacterial and fungal cultures.
  • Tang, et al. Wood Agric . Residues : Res. Use Feed, Fuels, Chem., Proc. Conf. Feed, Fuels, Chem., Wood Agric.
  • a preferred method, used in the present invention, for analyzing exocellular xylanase activity involves measuring the release of reducing sugars. Briefly, cells are removed from a culture and a sample of the resulting supernatant is added to a substrate solution containing 0.5% xylan in 50mM sodium citrate buffer. The enzyme is assayed by heating the substrate-supernatant mixture at 70°C and the reaction terminated by boiling for 5 minutes.
  • suitable host bacteria for the vectors comprise gram positive bacteria such as Bacillus sp. and particularly Bacillus subtilis .
  • BE3000 was used as a transformation host.
  • BE3000 can be obtained from its parent strain 1A40 which may be obtained from the Bacillus Genetics Stock Center (BGSC) , the Ohio State University, Columbus, Ohio 43210, U.S.A.
  • BGSC Bacillus Genetics Stock Center
  • Genes encoding thermostable enzymes useful in the present invention may be derived from a variety of sources.
  • Preferred sources are thermophilic microorganisms such as bacteria and fungi.
  • Typical thermophiles will include but are not limited to members of the Caldocellum genus (C. saccharolytlcum) , thermophilic sulfate-reducing bacteria
  • thermophilic Bacillus sp . B. Stearothermophilus, and B. lichenlformls
  • Thermococcus sp. thermophilic Clostridium sp .
  • C. thermocellum thermophilic fungi
  • thermophilic Apsergillus sp. A. foetidus
  • Trichoderma sp. Trichoderma reesei, and Trichoderma harzlanum
  • the present invention provides a variety of plasmids or vectors suitable for the cloning of portions the DNA required for the expression and exocellular production of the thermostable enzymes.
  • Suitable vectors will be those which are compatible with the bacterium employed.
  • Suitable vectors can be derived, for example, from a bacteria, a plasmid and/or a virus (such as bacteriophage T7 or a M-13 derived phage) .
  • Vectors suitable for B. subtllis will have compatible regulatory sequences and origins of replication. They will be multicopy and have a selective marker gene, for example, a gene coding for antibiotic resistance. Vectors of the present invention will be either autonomously replicated or capable of integration into the host genome. In embodiments of the invention vectors compatible with the Bacillus sp. are preferred. Suitable expression vectors will contain a DNA fragment comprising a regulatable promoter sequence which controls transcription, a sequence for a ribosome binding site which controls translation and a heterologous DNA fragment encoding a thermostable enzyme. Notably absent in these vectors are the signal sequences typically necessary for signal peptide expression. Generally, the vectors are constructed so that the promoter region is 5' of the heterologous DNA.
  • the vector may also include a region 3' of the heterologous DNA which controls transcriptional termination. It is most preferred when both the promoter and the transcriptional termination regions are derived from genes homologous to the host bacterium employed, however, it is to be understood that such control regions may be derived from sources other than the host bacterium. Further it will be appreciated by one of skill in the art that a termination control region may be unnecessary for expression of the desired protein.
  • Promoters which are useful for driving the expression of heterologous DNA fragments in Bacillus are numerous and familiar to those skilled in the art. Virtually any promoter capable of transcribing the gene encoding the desired thermostable enzyme is suitable for the present invention, where promoters native to Bacillus sp . are preferred.
  • the promoters in the DNA sequences may be either constitutive or inducible. Suitable promoters may include but are not limited to the alkaline protease promoter (aprp) , the neutral protease promoter (nprp) , and the barnase promoter (iarp) .
  • restriction endonuclease cleavage sites to the 3' or 5' ends of DNA for the purposes of vector construction or modification is also easily accomplished by means well known to those skilled in the art and is described by Sambrook et al., supra . Any restriction endonuclease site may be used but the use of a restriction site unique to that vector is desirable. Suitable compatible restriction sites are well known in the art. (See, for example the Restriction Fragment Compatibility Table of the New England Biolabs 1988-1989 Catalog, New England Biolabs Inc., Beverly, MA 01915 (1988).) Preferred for use herein are Xbal , Ndel, Nhel , Sail and Kpnl .
  • the combined DNA sequences encoding a promoter, ribosome binding site and termination control regions with a restriction site at its 3' end and the DNA sequences encoding heterologous polypeptides or proteins with a compatible restriction site at its 5' end can be operably integrated by conventional techniques (Sambrook et al., supra; Harwood, supra) .
  • Such amplifications may be accomplished by any of several schemes known in this art, including but not limited to the polymerase chain reaction (PCR) U.S. Patent 4,683,202 (1987, Mullis et al.); or the ligase chain reaction (LCR) (Tabor et al. (Proc. Acad. Sci . USA 82, 1074-1078) (1985)).
  • suitable vectors are constructed they are used to transform suitable bacterial hosts.
  • Introduction of desired DNA fragments into B . subtilis may be accomplished by known procedures such as by transformation, electroporation, or by transfection using a recombinant phage virus. (Sambrook et al., supra) .
  • FIG. 1-4 Construction of Bacillus vectors pertinent to the present invention are illustrated in Figures 1-4.
  • the C. saccharolytlcum xylanase gene (xynA) encoding xylanase was isolated from the plasmid pNZl448 using a PCR protocol and engineered to include a Ndel site and an Xbal site.
  • the Bacillus expression vectors were either pBE20 or pBE60 based (Nagarajan et al.. Gene, 114, 121, (1992) ) and contained either the Bacillus aprp or nprp which are bounded by a 5* Kpnl site and a 3' Ndel site.
  • pBE119 consists of the xynA gene downstream of the aprp ( Figure 1)
  • pBE145 contains the xynA gene downstream of the nprp ( Figure 2)
  • pBE158 contains the xynA gene downstream of the aprp-apr as , also containing the signal sequence ( Figure 4) .
  • the ⁇ -glucosidase ( ⁇ -glu) gene was amplified by PCR from an M13 clone and engineered to incorporate a 5' Ndel site and a 3 • Xbal site. The ⁇ -glu gene was inserted 3' of the aprp to form pBE164 ( Figure 3) .
  • Xylanase activity was measured according to the method of Luthi et al., Appl . Environ . Microbiol . , 56, 2677, (1990) which measures the release of reducing sugars.
  • Cells are removed from an aliquot of culture by centrifugation and a sample of the resulting supernatant is added to a substrate solution containing 0.5% xylan in 50mM sodium citrate buffer.
  • the enzyme is assayed by heating the substrate- supernatant mixture at 70°C for 15 minutes. An aliquot of 50mM hydroxy CaC12, 20mM sodium hydroxide was added and the reaction terminated by boiling for 5 minutes.
  • ⁇ -glucosidase was assayed according to a modification of the method described by Love et al. (Biotechnol . 5, 384, (1987)). The method of Love et al. relies on the release of p-nitrophenol from the su b strate p-nitro-phenol- ⁇ -D-glyucopyranoside (PNPG,
  • BE3000 Bacillus host strains were of the species subtilis and included BE3000 ( trpC2, lys3, ⁇ aprE66, ⁇ npr82, xynA sacB : : ermC) .
  • BE3000 may be derived from its parent strain 1A40 obtainable from the Bacillus Genetics Stock Center (BGSC) , The Ohio State University, Columbus, OH 43210 U.S.A.
  • Bacterial growth conditions Bacillus strains were grown in S7 minimal medium containing 50 ug/ml kanamycin, with 25 mM sodium citrate and yeast extract at concentrations of 0.05-1.00%. The components of S7 media are as follows: 50 mM KPO 4 , pH 7.0 10 mM NH 4 SO 4
  • DNA containing the xylanase gene from C. saccharolytlcum was obtained via PCR amplification of the appropriate regions of plasmid pNZ1448 (a kind gift of Peter Bergquist of Cent. Gene Technol., Univ.
  • PCR amplification was accomplished according to the protocol of the manufacturer (GeneAmp PCR Reagent Kit, Perkin-Elmer Cetus, Norwalk, CT) using the following primers: Primer 1, Upper:
  • Reagent concentrations were: IX Buffer 200uM dATP 200uM dCTP 200uM dGTP
  • the amplified product contains the entire xynA gene encoding the xylanase gene, bounded on the 5' end by a Ndel site and on the 3' end by a Xbal site.
  • plasmid pBE1020 Cold, D., J. Bact., 176, 3013, (1994)
  • the xynA PCR fragment were digested with Ndel and Xbal and the appropriate fragments ligated to yield the plasmid pBE105 ( Figure 1) .
  • Plasmid pBE240 and pBE60 are digested with Kpnl and Xbal and the appropriate fragments ligated to form the high copy number plasmid pBE113 ( Figure 1) .
  • pBE105 and pBE113 were digested with Ndel and Xbal and the appropriate fragments were ligated yielding plasmid pBE119 which contains the xylanase gene downstream of the aprp ( Figure 1) .
  • Plasmid pBEl45 Plasmid pBEl45:
  • Plasmid Construction of pBE145, containing the xynA gene under the control of the Bacillus nprp is illustrated in Figure 2.
  • the plasmids pBE105 (containing the xynA gene) and pBE146 (containing the nprp) were digested with the restriction enzymes Ndel and Kpnl.
  • the full nucleic acid sequence of pBE146 is given in SEQ ID NO.3) .
  • the large fragment of pBE105 and the 250 bp fragment of pBE146 were ligated, yielding plasmid, pBE147 ( Figure 2) .
  • nprp-xynA fusion was then transferred from pBE147 to a high copy plasmid by digestion of pBE147 with Kpnl and Xbal and ligation of the appropriate fragment with pBE113 cut with the same enzymes.
  • the resultant plasmid is pBE145 ( Figure 2). Plasmid pBE164:
  • primer 4 5'-GCC CGC TCT AGA TTA TAT TTA CGA ATT TTC C-3' SEQ ID NO.:5
  • the high copy number plasmid pBE60 contains three Ndel sites ( Figure 3) .
  • pBE60 was first digested with Ndel and the two large fragments of plasmid DNA were isolated and treated with the Klenow fragment of DNA polymer as klenow to create blunt ends. The two large fragments were ligated and resulted in pBE928. The loss of the Ndel sites were verified ( Figure 3) .
  • pBE928 and pBE119 were digested with Kpnl and Xbal.
  • PCR amplification using the above primers resulted in a fragment containing a Nhel restriction site at the 5' end and a Sail site at the 3' end.
  • the fragment was digested with Nhel and Sail and ligated to the large fragment of pBE92 which contains the aprp and apr ss ( Figure 4) .
  • the full sequence of pBE92 is given in SEQ ID NO.:8.
  • the resulting plasmid is pBE158 containing the aprp-apr ss -xynA fusion.
  • EXAMPLE 2 Transfnrma ion of B. subtllis Transformation with pBE119. DBE145 and PBE158 Plasmids pBE119 (aprp-xynA) , pBE145 (nprp-xynA) and pBE158 (aprp-apr ss -xynA) were introduced into the B. subtilis strain BE3000 by standard transformation protocols, and bacteria were selected on kanamycin plates and screened by the coupled xylan method described above.
  • Plasmid pBE164 (aprp- ⁇ -glu) was introduced into the B. su_tilis strain BE3000 by standard transformation protocols, and bacteria were selected on kanamycin plates and screened by the release of p-nitrophenol as described above.
  • FIG. 5 shows an SDS-PAGE gel, stained with commassie blue, comparing the accumulation of protein in the supernatant of pBE119 (aprp-xynA) transformed Bacillus grown in S7 media containing 1% glucose (lane 1) ; S7 media containing 25 mM sodium citrate (lane 2) ; or Am3 rich media [equivalent to Penassay Broth, Difco Laboratories, Detroit, MI (lane 3) ] .
  • Lane 4 contains culture supernatant from a Bacillus host cell transformed with an aprp-aprss-phoA (alkaline protease) gene.
  • Figure 5 demonstrates that the apparent molecular weight of the predominant protein band in the culture supernatants of a Bacillus host harboring pBE119, but not the control plasmid, was the same as is expected for the xynA gene product.
  • xylanase is the major extracellular protein despite the fact it was synthesized without a signal peptide.
  • Transformants grown in either the S7+ sodium citrate or Am3 rich media produced more extracellular xylanase than those grown in the S7+glucose media.
  • Anti-xvlanase immuno-hlo Anti-xvlanase immuno-hlo :
  • the proteins contained in each fraction were resolved by SDS-PAGE, transferred to a nitrocellulose filter and xylanase was identified by probing with rabbit anti- xylanase anti-serum as the primary antibody.
  • the vector control [pBE60 (panel A) ] demonstrated a lack of endogenous xylanase antigen in B . subtilis strain BE3000.
  • Cells harboring the arp-apr S s ⁇ xynA gene [pBE158 (panel B) ] produced modest levels of xylanase. Approximately 50% of the total xylanase was found in the culture supernatant (s) .
  • Cells transformed with either pBE145, pBE119, pBE158 or the control plasmid pBE60 were grown in S7 media supplemented with sodium citrate as described in the GENERAL METHODS, the culture supernatants were collected and placed on ice and the xylanase assay described in the GENERAL METHODS was used to determine the xylanase activity contained in each supernatant.
  • NAME SIEGELL, BARBARA C.
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:3:
  • AAAGTTCTAA AAGAGCTTTT AGAAAGAGGT ACTCCAATAG ATGGAATTGG TATACAAGCA 1020
  • AAACCGTCTA TCAGGGCGAT GGCCCACTAC GTGAACCATC ACCCAAATCA AGTTTTTTGG 2100
  • GTTCATCCAT AGTTGCCTGA CTCCCCGTCG TGTAGATAAC TACGATACGG GAGGGCTTAC 4140 CATCTGGCCC CAGTGCTGCA ATGATACCGC GAGACCCACG CTCACCGGCT CCAGATTTAT 4200
  • AAACAAAAAA ACCTGCCCTC TGCCACCTCA GCAAAGGGGG GTTTTGCTCT CGTGCTCGTT 5580
  • CACATTAGAA CTGCGAATCC ATCTTCATGG TGAACCAAAG TGAAACCTAG TTTATCGCAA 6180
  • GACACATCCA CTATATATCC GTGTCGTTCT GTCCACTCCT GAATCCCATT CCAGAAATTC 65 0
  • AGATGGTCAT AACCTGAAGG AAGATCTGAT TGCTTAACTG CTTCAGTTAA GACCGAAGCG 6660
  • TCTGTGTCAT CAAGGTTTAA TTTTTTATGT ATTTCTTTTA ACAAACCACC ATAGGAGATT 7560
  • MOLECULE TYPE DNA (genomic)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: GTTTATGCAT ATGAGTTTCC CAAAAGG 27 (2) INFORMATION FOR SEQ ID NO:5:
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 8:
  • GTCTACTAAA ATATTATTCC ATACTATACA ATTAATACAC AGAATAATCT GTCTATTGGT 1080
  • ATGAGTAAAC TTGGTCTGAC AGTTACCAAT GCTTAATCAG TGAGGCACCT ATCTCAGCGA 5400
  • AAATCTCCAC CTTTAAACCC TTGCCAATTT TTATTTTGTC CGTTTTGTCT AGCTTACCGA 7020
  • AAACCACTCA AAATAAAAAA GATACAAGAG AGGTCTCTCG TATCTTTTAT TCAGCAATCG 7140
  • ACGAACTGGC ACAGATGGTC ATAACCTGAA GGAAGATCTG ATTGCTTAAC TGCTTCAGTT 7980
  • TAACTCGTCT TCCTAAGCAT CCTTCAATCC TTTTAATAAC AATTATAGCA TCTAATCTTC 8700
  • ACAAGTTCAA AACCATCAAA AAAAGACACC TTTTCAGGTG CTTTTTTTAT TTTATAAACT 9960

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  • General Health & Medical Sciences (AREA)
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  • Physics & Mathematics (AREA)
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  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne la préparation d'enzymes thermostables, en particulier de la xylanase et de la β-glucosidase. Ces enzymes sont exprimées et sécrétées par des bactéries transformées par les gènes de structure codant pour ces enzymes. L'expression et la sécrétion des enzymes se font en l'absence du peptide signal normalement nécessaire pour la translocation des protéines sécrétées, à travers les membranes cellulaires.
PCT/US1996/000891 1995-01-30 1996-01-24 Procede pour produire les enzymes thermostables xylanase et beta-glucosidase a partir de bacteries Ceased WO1996023887A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP96903629A EP0807180A1 (fr) 1995-01-30 1996-01-24 Procede pour produire les enzymes thermostables xylanase et beta-glucosidase a partir de bacteries

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US38052195A 1995-01-30 1995-01-30
US08/380,521 1995-01-30

Publications (1)

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WO1996023887A1 true WO1996023887A1 (fr) 1996-08-08

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WO (1) WO1996023887A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1195437A1 (fr) * 2000-10-04 2002-04-10 Mitsui Chemicals, Inc. Procédé pour la production recombinante des protéines en utilisant un promoteur hybride Plac/Np
US9926584B2 (en) * 2013-06-25 2018-03-27 Novozymes A/S Expression of natively secreted polypeptides without signal peptide
CN115838681A (zh) * 2022-11-02 2023-03-24 中国水产科学研究院黄海水产研究所 北极来源β-葡萄糖苷酶的重组菌及其应用
WO2023117970A1 (fr) * 2021-12-20 2023-06-29 Basf Se Procédé de production améliorée de protéines intracellulaires dans bacillus

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993010248A1 (fr) * 1991-11-14 1993-05-27 Novo Nordisk A/S PROCEDE D'EXPRESSION DES GENES DANS $i(BACILLUS LICHENIFORMIS)

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
WO1993010248A1 (fr) * 1991-11-14 1993-05-27 Novo Nordisk A/S PROCEDE D'EXPRESSION DES GENES DANS $i(BACILLUS LICHENIFORMIS)

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
D. LOVE ET AL: "Molecular cloning of a beta-glucosidase gene from an extremely thermophilic anaerobe in E. coli and B. subtilis", BIOTECHNOLOGY, vol. 5, April 1987 (1987-04-01), pages 384 - 387, XP002006104 *
DONALD K A G ET AL: "Production of a bacterial thermophilic xylanase in Saccharomyces cerevisiae.", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 42 (2-3). 1994. 309-312. ISSN: 0175-7598, XP000573855 *
E. LÜTHI ET AL: "Xylanase from the extremely thermophilic bacterium Caldocellum saccharolyticum", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 56, no. 9, September 1990 (1990-09-01), pages 2677 - 2683, XP000572753 *
JUNG K K ET AL: "EXPRESSION OF A CLOSTRIDIUM- THERMOCELLUM XYLANASE GENE IN BACILLUS-SUBTILIS.", BIOTECHNOL LETT 15 (2). 1993. 115-120. CODEN: BILED3 ISSN: 0141-5492, XP000573872 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1195437A1 (fr) * 2000-10-04 2002-04-10 Mitsui Chemicals, Inc. Procédé pour la production recombinante des protéines en utilisant un promoteur hybride Plac/Np
US9926584B2 (en) * 2013-06-25 2018-03-27 Novozymes A/S Expression of natively secreted polypeptides without signal peptide
WO2023117970A1 (fr) * 2021-12-20 2023-06-29 Basf Se Procédé de production améliorée de protéines intracellulaires dans bacillus
CN115838681A (zh) * 2022-11-02 2023-03-24 中国水产科学研究院黄海水产研究所 北极来源β-葡萄糖苷酶的重组菌及其应用

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