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WO2005066336A1 - Procede de production en grande quantite de penicilline v acylase - Google Patents

Procede de production en grande quantite de penicilline v acylase Download PDF

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Publication number
WO2005066336A1
WO2005066336A1 PCT/IB2003/006198 IB0306198W WO2005066336A1 WO 2005066336 A1 WO2005066336 A1 WO 2005066336A1 IB 0306198 W IB0306198 W IB 0306198W WO 2005066336 A1 WO2005066336 A1 WO 2005066336A1
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WO
WIPO (PCT)
Prior art keywords
recombinant
penicillin
acylase
site
coding sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/IB2003/006198
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English (en)
Inventor
Hephzibah Sivaraman
Archana Vishnu Pundle
Cheravakkattu Gopalan Suresh
George Guy Dodson
James Antony Brannigan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of York
Council of Scientific and Industrial Research CSIR
Original Assignee
University of York
Council of Scientific and Industrial Research CSIR
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of York, Council of Scientific and Industrial Research CSIR filed Critical University of York
Priority to AU2003298471A priority Critical patent/AU2003298471A1/en
Priority to PCT/IB2003/006198 priority patent/WO2005066336A1/fr
Priority to US10/812,387 priority patent/US20050142652A1/en
Publication of WO2005066336A1 publication Critical patent/WO2005066336A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • 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)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C12N9/84—Penicillin amidase (3.5.1.11)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
    • C12Y305/01011—Penicillin amidase (3.5.1.11), i.e. penicillin-amidohydrolase

Definitions

  • the present invention relates to a recombinant plasmid of figure 1, wherein (1) is pET - 26b(+) cloning /expression region with SEQ ID No. 1 cloned between BamH I site 198 and Nde I site 288, (2) is lac I coding sequence, (3) is pBR322 origin, (4) is Kan coding sequence, and (5) is fl origin. Also, it relates to a recombinant E.Coli strain PTA 2456. Further, it relates to a process for the production of large amount of Penicillin V acylase using recombinant E. Coli strain PTA 2456. BACKGROUND AND PRIOR ART REFERENCES OF THE PRESENT INVENTION
  • Penicillin V acylase is an enzyme used for removing the phenoxyacetic acid group of benzyloxy penicillin (pen V) by hydrolysis to yield 6-aminopenicillanic acid (6-APA) which is used as a precursor in the commercial production of semi-synthetic penicillins.
  • 6-APA 6-aminopenicillanic acid
  • the strategy is similar to the production of 6-APA from benzyl penicillin (pen G) by employing penicillin G acylase (PGA).
  • Penicillin acylase activity was discovered in many microorganisms including both bacteria and fungi as reported by Claridge et al., in "Bacterial Penicillin Amidase.” Nature 187, 237-238 (1960), Rolinson et al., “Formation of 6-Aminopenicillanic Acid from Penicillin by Enzymatic Hydrolysis.” Nature 187, 236-237 (1960), and Sakaguchi et al., "A Preliminary Report on a New Enzyme Penicillin Amidase,” J. Agr. Chem. Soc. Japan, 23, 411 (1950).
  • the inventors have been carrying out detailed studies on a Bacillus sphaericus penicillin acylase with penicillin V specificity. Gene for this enzyme has been cloned in E. coli (Olsson et al., "Molecular Cloning of Bacillus sphaericus Penicillin V Amidase Gene and Its Expression in Escherichia coli and Bacillus subtitis.” Appl. Environ. Microbiol.
  • the main object of the present invention is to develop a recombinant plasmid of figure 1, wherein (1) is p ⁇ T -26b(+) cloning /expression region with S ⁇ Q ID No. 1 cloned between BamH I site 198 and Nde I site 288, (2) is lac I coding sequence, (3) is pBR322 origin, (4) is Kan coding sequence, and (5) is fl origin.
  • Yet another main object of the present invention is to develop a recombinant E. Coli strain PTA 2456.
  • Summary of the present invention The present invention relates to a recombinant plasmid of figure 1, wherein (1) is p ⁇ T - 26b(+) cloning /expression region with S ⁇ Q ID No.
  • the present invention relates to a recombinant plasmid of figure 1, wherein (1) is pET -26b(+) cloning /expression region with SEQ ID No. 1 cloned between BamH I site 198 and Nde I site 288, (2) is lac I coding sequence, (3) is pBR322 origin, (4) is Kan coding sequence, and (5) is fl origin; a recombinant E.Coli strain PTA 2456; and lastly, a process for the production of large amount of Penicillin V acylase using recombinant E. Coli strain PTA 2456.
  • a recombinant plasmid of figure 1 wherein (1) is pET -26b(+) cloning /expression region with SEQ ID No. 1 cloned between BamH I site 198 and Nde I site 288, (2) is lac I coding sequence, (3) is pBR322 origin, (4) is Kan coding sequence, and (5) is fl origin.
  • SEQ ID No. 1 is the sequence of Bacillus subtilis gene of figure 2, encoding conjugated bile acid hydrolase.
  • a recombinant E. Coli strain PTA 2456 In still another embodiment of the present invention, wherein the recombinant stain produces an amount of Penicillin V acylase about 57 to 65 times more than in the ordinary conditions.
  • the strain comprises recombinant plasmid of figure 1, whereby (1) is pET -26b(+) cloning /expression region with SEQ ID No. 1 cloned between BamH I site 198 and Nde I site 288, (2) is lac I coding sequence, (3) is pBR322 origin, (4) is Kan coding sequence, and (5) is fl origin.
  • a process for the production of large amount of Penicillin V acylase using recombinant E.Coli strain PTA 2456 comprising steps of: • preparing a recombinant plasmid of figure 1, wherein (1) is pET -26b(+) cloning /expression region with SEQ ID No. 1 cloned between BamH I site 198 and Nde I site 288, (2) is lac I coding sequence, (3) is pBR322 origin, (4) is Kan coding sequence, and (5) is fl origin, • transforming the competent cells of E.
  • the fermentation medium comprises bacto-tryptone of concentration ranging between 8-10 g/1, bacto-yeast extract of concentration ranging between 5-8 g/1, sodium chloride of concentration ranging between 3-5, and an antibiotic of concentration ranging between 30-50 ⁇ g/ml.
  • the E. coli strain is BL-21 DE3.
  • the present invention relates to a Bacillus subtilis gene cloned in Escherichia coli and a process for the production of penicillin V acylase using the said clone. More particularly it relates to a method for increasing the production of penicillin V acylase by cloning in E. coli, a gene from B. subtilis whose product is known as conjugated bile acid hydrolase, but which also showed penicillin V acylase activity.
  • Fig. 1 shows recombinant vector produced in accordance with the present invention wherein (1) is pET -26b(+)cloning /expression region and nucleotide sequence described in Fig2 is cloned between BamH I (198) and Nde 1(288), (2) is lac I coding sequence, (3) is pBR322 origin, (4) is Kan coding sequence & (5) is fl origin Fig. 2 shows complete nucleotide of Bacillus subtilis gene encoding conjugated bile acid hydrolase inserted between sites of restriction enzymes Bam H I and Nde I of cloning/expression region (1), in Fig. 1.
  • the present invention also provides a process for the preparation of Penicillin V acylase using the said recombinant PTA 2456, which comprises of growing the said PTA 2456 in a conventional fermentor medium for a period of 4 to 18 hrs. at a temperature ranging between 30 to 40 °C, separating the biomass and recovering the product by conventional methods used for separation of wild type enzyme.
  • the conventional fermentation medium consists of following composition (g/1): bacto-tryptone, 8-10; bacto-yeast extract, 5-8; sodium chloride, 3-5 and an antibiotic30-50 ⁇ g/ml.
  • the product is separated by conventional methods such as sonication of microbial cell mass, ammonium sulphate fractionation of the sonicate followed by hydrophobic interaction chromatography.
  • a method is described wherein DNA recombinant technology has been exploited to achieve an improved production of penicillin V acylase (PVA).
  • PVA penicillin V acylase
  • the yields obtained using the new strain are substantially higher than those reported so far.
  • the method of invention comprises of isolating a selected chromosomal fragment from B. subtilis NCIMB 11621 containing the gene encoding the reported enzyme identified as conjugated bile acid hydrolase (CBH), incorporating this DNA fragment into a multi copy vector and subsequently transforming the competent cells of E. coli (BL-21 DE3) using the modified plasmid.
  • CBH conjugated bile acid hydrolase
  • a method of cloning a B. subtilis hydrolase enzyme into E. Coli A selected lkb chromosomal DNA fragment from B. subtilis NCIMB 11621, which represented the gene encoding the enzyme originally known as conjugated bile acid hydrolase and having PVA activity, as observed by the inventors of the present invention, has been inserted into the vector plasmid and then the modified plasmid was introduced into E. coli.
  • a new strain of E. coli namely (PTA 2456) having enhanced penicillin V acylase productivity which includes within it a cloning vector p ⁇ T-26b containing an insert of a lkb chromosomal DNA fragment of B. subtilis NCIMB 11621.
  • the multi copy plasmid construct used for the transformation of E. coli cells has been designed from the vector pET-26b by inserting a fragment of about lkb DNA drawn from B. subtilis NCIMB 11621 in the cloning region between the sites of restriction enzymes BamH I and Nde I.
  • the chromosomal DNA fragment, 1-10 kb length, of B. subtilis that include the appropriate gene coding the enzyme responsible for conjugated bile acid hydrolase activity could be inserted into the pET 26b vector, following the procedure described by Sambrook et al., Molecular Cloning, A Laboratory Manual, Vol.l, pl82 (1988) to form an appropriate plasmid.
  • the plasmid thus formed may be then cloned in E. coli using the procedure described therein.
  • Bacillus sphaericus NCIM 2478, ATCC 14577 Bacillus subtilis : NCIMB 11621 , BGSC 1 A436
  • Escherichia coli ATCC for deposit accession number: PTA 2456
  • Example 1 A plasmid containing a chromosomal DNA fragment from B. subtilis NCLMB 11621 was prepared as follows: The vector pET-26b(+) 5360 bp used for the cloning of conjugated bile acid hydrolase gene in E. coli. The vector has two restriction enzyme sites, one for Nde I and another for BamH I, and it also confers resistance towards kanamycin. These characteristics were made use for inserting the conjugated bile acid hydrolase gene and testing the expression of the vector. The reference conjugated bile acid hydrolase gene was amplified through PCR using chromosomal DNA of B.
  • subtilis as template along with upstream and downstream primers.
  • the vectors as well as the amplified insert were subjected to the restriction enzyme digestion by Nde I and BamH I, followed by ligation using T4 DNA ligase (Sambrook et al , Molecular Cloning, A Laboratory Manual, Vol.l, pl82 (1988)).
  • the construct thus obtained was used for the transformation of competent cells of E. coli BL- 21(DE3) in order to achieve the expression of the desired protein (Sambrook et al , Molecular Cloning, A Laboratory Manual, Vol.l, pl82 (1988)).
  • the transformed cells were selected by picking up colonies grown on Luria-Bertani agar medium containing kanamycin.
  • the plasmid was isolated from the transformants and digested with Nde I and BamH I.
  • the digestion product was subjected to electtophoresis on 1% Agarose gel which showed bands corresponding to the size of the plasmid and that corresponding to the size (lkb) of the insert.
  • Examples 2 The resultant transformed E. coli PTA 2456 with plasmid containing conjugated bile acid hydrolase gene within was tested for penicillin V acylase production.
  • the 500ml Erlenmyer flasks containing 100ml LB medium were inoculated aseptically using 1 ml each of the overnight grown culture of the said clone.
  • the flasks were incubated at 37 °C and 150 rpm.
  • Isopropyl ⁇ -D Thiogalactopyranoside (IPTG) was added aseptically when the culture reached OD 595 value between 0.2-0.6.
  • the incubation was continued for next 3-4 hrs and the cells were harvested by centrifugation.
  • the penicillin V acylase activity was checked using whole cells and cell-free extracts.
  • the crude extract was incubated with 2% potassium salt of penicillin V in citrate buffer of 0.1 molarity and pH 5.8 at 40°C for 10 minutes.
  • 6-aminopenicillanic acid (6-APA) formed was estimated using PDAB by the method of Bomstein & Evans "Automated calorimetric determination of 6-aminopenicillanic acid in fermentation".

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  • Health & Medical Sciences (AREA)
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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
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Abstract

L'invention concerne un plasmide recombiné (voir figure), dans lequel (1) représente une région de clonage/d'expression pET- 26b(+) de SEQ ID No. 1 clonée entre le site BamH I 198 et le site Nde I 288 ; (2) représente une séquence de codage lac I ; (3) représente une origine de pBR322 ; (4) représente une séquence de codage Kan ; et (5) représente une origine de f1. L'invention concerne également une souche de E.Coli recombinée PTA 2456, et un procédé de production en grande quantité de pénicilline V acylase au moyen de la souche de E.Coli recombinée PTA 2456.
PCT/IB2003/006198 2003-12-24 2003-12-24 Procede de production en grande quantite de penicilline v acylase Ceased WO2005066336A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU2003298471A AU2003298471A1 (en) 2003-12-24 2003-12-24 Process for production of large amount of penicillin v acylase
PCT/IB2003/006198 WO2005066336A1 (fr) 2003-12-24 2003-12-24 Procede de production en grande quantite de penicilline v acylase
US10/812,387 US20050142652A1 (en) 2003-12-24 2004-03-30 Process for production of large amount of penicillin V acylase

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/IB2003/006198 WO2005066336A1 (fr) 2003-12-24 2003-12-24 Procede de production en grande quantite de penicilline v acylase

Related Child Applications (1)

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US10/812,387 Continuation US20050142652A1 (en) 2003-12-24 2004-03-30 Process for production of large amount of penicillin V acylase

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WO2005066336A1 true WO2005066336A1 (fr) 2005-07-21

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AU (1) AU2003298471A1 (fr)
WO (1) WO2005066336A1 (fr)

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL [online] EBI; 16 July 1996 (1996-07-16), "Bacillus subtilis genome sequence between the iol and hut operon", XP002283780, retrieved from EMBL Database accession no. D45912 *
NOVAGEN PRODUCT CATALOGUE, December 1998 (1998-12-01), XP002283778 *
OLSSON A ET AL: "SEQUENCING AND HETEROLOGOUS EXPRESSION OF THE GENE ENCODING PENICILLIN V AMIDASE FROM BACILLUS SPHAERICUS", GENE, vol. 45, no. 2, 1986, pages 175 - 181, XP002283959 *
PUNDLE ARCHANA ET AL: "Bacillus sphaericus penicillin V acylase: Purification, substrate specificity, active-site characterization", CURRENT MICROBIOLOGY, vol. 34, no. 3, 1997, pages 144 - 148, XP002283779, ISSN: 0343-8651 *
ROSENBERG A H ET AL: "VECTORS FOR SELECTIVE EXPRESSION OF CLONED DNAS BY T7 RNA POLYMERASE", GENE, ELSEVIER BIOMEDICAL PRESS. AMSTERDAM, NL, vol. 56, no. 1, 1987, pages 125 - 135, XP001068935, ISSN: 0378-1119 *
STUDIER F W ET AL: "USE OF T7 RNA POLYMERASE TO DIRECT EXPRESSION OF CLONED GENES", METHODS IN ENZYMOLOGY, ACADEMIC PRESS INC, SAN DIEGO, CA, US, vol. 185, 1990, pages 60 - 89, XP000647676, ISSN: 0076-6879 *
YOSHIDA K-I ET AL: "Cloning and Sequencing of a 23-kb Region of the Bacillus subtillis Genome between the iol and the hut Genom", DNA RESEARCH, vol. 2, no. 6, 31 December 1995 (1995-12-31), pages 295 - 301, XP009032064 *

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