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WO1999055881A1 - Cephalosporine desacetylase, gene codant pour celle-ci, et preparation de composes de cephalosporine desacetylee au moyen de celle-ci. - Google Patents

Cephalosporine desacetylase, gene codant pour celle-ci, et preparation de composes de cephalosporine desacetylee au moyen de celle-ci. Download PDF

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Publication number
WO1999055881A1
WO1999055881A1 PCT/KR1998/000487 KR9800487W WO9955881A1 WO 1999055881 A1 WO1999055881 A1 WO 1999055881A1 KR 9800487 W KR9800487 W KR 9800487W WO 9955881 A1 WO9955881 A1 WO 9955881A1
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Prior art keywords
cephalosporin
strain
enzyme
deacetylase
bacillus
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PCT/KR1998/000487
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English (en)
Inventor
Yong Duk Kim
Il Seon Jeong
Sang Hoon Lee
Duk Ho Choi
Kum Soo Han
Bun Sam Lim
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Daesang Corp
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Daesang Corp
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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
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P35/00—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin

Definitions

  • the present invention relates to a new enzyme, a gene coding for it and its use to produce cephalosporin compounds. More particularly, it relates to a new cephalosporin deacetylase, a gene coding for it, and its use to deacetylate cephalosporin C, 7-aminocephalosporanic acid (7-ACA) and 2-memoxyirnino-2- furylacetic cephalosporanic acid (MIFACA) to produce cephalosporin compounds.
  • Cephalosporins such as cephalosporin C and 7-ACA are, after deacetylation in which 3-acetyl group is removed, served as semisynthetic intermediates for producing highly valuable cephalosporin antibiotics.
  • Deacetylation of cephalosporin may be performed by chemical or enzymatic processes.
  • the enzymatic process has advantages of mild reaction conditions of pH and temperature, and high yield (USP 3,304,236).
  • As an enzyme for this purpose two kinds of cephalosporin C deacetylase isolated from Bacillus subtilis have been reported, one of which has been introduced into a E. coli for mass production (EP 0454478A1, Applied and Environmental Microbiology, 1995, p 2224).
  • EP 0454478 A 1 is a giant enzyme of 280KDa consisting of eight monomers of 35KDa and shows a strong reactivity to the substrate such as cephalosporin C and 7-ACA.
  • 7'-substituted-CPC such as 2-memoxyh ⁇ m o-2-furylacetic cephalosporanic acid(MIFACA) which is precursor for cephalosporin antibiotics synthesis.
  • An object of the present invention is to provide a novel strain of Bacillus sp. capable of producing a cephalosporin C deacetylase of high enzyme activity.
  • Another object of the present invention is to provide a gene coding for the novel high activity cephalosporin C deacetylase.
  • Another object of the present invention is to provide an expression vector comprising the gene and a host strain transformed with the expression vector.
  • the present invention provides a method of deacetylating cephalosporin compounds which comprises contacting the compounds with the enzyme or the transformed strain.
  • Figure 1 shows the construction of expression vectors comprising the gene according to the present invention.
  • the present inventors isolated a novel strain of Bacillus sp. showing a potent cephalosporin C deacetylase activity from the soil in Ichon in Kyoungki-province. They also isolated and sequenced a novel gene coding for an active enzyme and cloned into an E. coli using vector systems. They studied biochemical properties of the enzyme coded thereby, which revealed that its properties are distinguished from those of the known deacetylation enzymes and showed a stronger affinity for substrate and a reactivity.
  • the present inventors further transformed a host strain with the recombinant vector comprising the gene.
  • the enzyme and transformed strain may be used to deacetylate cephalosporin compounds for giving useful intermediates for cephalosporin antibiotics synthesis according to the present invention.
  • the enzyme and strain may be immobilized on a proper carrier. The enzymatic process using the enzyme preparation or culture provides advantages that the number of steps can be reduced while the yield can be improved.
  • SEQ. ID. NO. 4 is an artificial sequence used as a Primer No. 1 for PCR.
  • SEQ. ID. NO. 4 is an artificial sequence used as a Primer No. 2 for PCR.
  • Example 1 Isolation of Bacillus sp. strain DS1152
  • a sample obtained from the soil in Ichon in Kyoungki-do, Korea was suspended in 0.9% of NaCl solution. After filtration, the supernatant was plated on nutrient agar and incubated at a temperature of about 30°C for 48 hours. The colonies on the plate were replicated on a solid LB(Luria-Beteani) medium containing 12mg of Fast blue RR salt and 20mg of ⁇ -naphthylacetate and incubated at a temperature of about 30°C for 24 hours. Thus, the strains forming a red-brown zone were selected. The selected strains were liquid cultivated in LB medium.
  • test sample was prepared by adding the supernatant as obtained above to 60mM of sodium phosphate buffer(pH 6.8) containing p- nitrophenylacetate(lmg/ml in methanol). The absorbance at 400nm was measured to select the strains having a high acetyl esterase activity(pNPA method).
  • a cell showing the highest enzyme activity was picked up and characterized. It was identified as Bacillus sp. and named DS 1152.
  • the Bacillus sp. strain DS 1152 was deposited on Korean Federation of Culture Collection in Seoul on April 11, 1998 and given accession number of KFCC 11026. The deposit was converted into one 5 under the Budapest Treaty on December 21 , 1998 and given accession number of KCCM- 10143 .
  • the Bacillus sp. strain DS 1152(KCCM-10143 ) can be grown in a solid LB medium at about 30°C for about 24 hours.
  • the biochemical characteristics of Bacillus sp. strain DS 1152(KCCM- 10143) are summarized in Table 2.
  • Isolation and purification of enzyme LB medium is placed in a 500ml flask and sterilization is carried out by autoclaving at 121°C for 20 minutes.
  • the flask was inoculated with Bacillus sp. strain DS1152 (KCCM -10143) and the resulting culture was grown at 30°C for 9 hours to be used as a seed culture.
  • Cephalosporin deacetylase was purified from the culture broth by following method.
  • the culture broth was centrifuged (5000 rpm X 30 minutes) to collect cells, which was suspended in 50 mM Tris/HCl buffer (pH 8.0) and disintegrated with an ultrasonic homogenizer. The resulting disintegrated cells were subjected to centrifugation (12,000 rpm X 30 rninutes) to remove insoluble fractions. Supernatant was subjected to fractional precipitation using 40 - 80% of saturated ammonium sulfate solution ((NH 4 ) 2 S0 4 ) to give a primarily purified protein. The resulting solution was subjected to ultrafiltration (MWCO 30 kDa) to remove salts and to concentrate.
  • MWCO 30 kDa saturated ammonium sulfate solution
  • resulting protein solution concentrate was loaded onto a column packed with DEAE-sepharose (Pharmacia) equilibrated with 10 mM Tris buffer (pH 8.0) at a flow rate of 2.0 ml/min and at an amount of 40 mg protein per ml of gel. Then, the column was washed with the equilibration buffer and eluted continuous concentration gradient manner using 0 - 300 mM NaCl. Most of cephalosporin deacetylase was eluted at 150 - 200 mM NaCl concentration.
  • Partially purified (95% purity) enzyme was ultraf ⁇ ltered against distilled water 7 to remove salts and freeze-dried.
  • the resulting enzyme was analyzed for its N- terminal 10 amino acid sequence by using Precise Protein Sequencing System (Applied Biosystems) following the standard analysis manual of the manufacturer.
  • the N-terminal 10 amino acid sequence is shown as SEQ. ID. NO. 1 in Sequence Listing.
  • the enzyme activity was measured by following the method described in EP 0,454,478 Al using 0.1M sodium phosphate buffer (pH 7.0). The activity was deterrnined that the amount of the enzyme required for deacetylating 1 mole of 7- AC A for 1 minute at 37°C is defined as one(l) unit. The freeze-dried enzyme was used by dissolving in a buffer.
  • the optimum pH of the enzyme was deterrnined. 7-ACA was used as a substrate, and the amounts of DACA produced and of the substrate consumed were measured by HPLC of the same conditions as above.
  • the enzyme shows the highest activity for pH range of 8.0 - 9.0.
  • Density gradient isoelectrofocusing was carried out with PhastSystemTM (Pharmacia Biotech) by following the Standard User's Manual to determine the isoelectric point(IP) of the enzyme. The experiment was carried out at a pH range from 3.0 to 9.0. Bovine serum albumin having LP of pH 4.7-4.9 is used as a standard. The band containing the enzyme was removed and dissolved into a distilled water to determine pH thereof. Thus, measured pH was 4.3.
  • the substrate reactivity of enzyme is defined as Michaelis constant (K which denotes a reaction speed of the enzyme.
  • K denotes a reaction speed of the enzyme.
  • the reaction is carried out by adding various concentrations of 7-ACA, cephalosporin C (CPC) or 2-methoxyimino-2- furylacetic cephalosporanic acid (MIFACA), as substrates, to 0.1M sodium phosphate buffer (pH 7.0) and allowed the resulting mixture to react at 37°C for 10 minutes, followed by HPLC.
  • K Michaelis constant
  • the results are shown in Table 3, which reveals that the enzyme shows the similar reactivities to all of the three cephalosporin derivatives. Table 3
  • MIFACA 2-methoxyi ⁇ mo-2-furylacetyl cephalosporanic acid
  • the strain of Bacillus sp. DS 1152 (KCCM-10143) was cultivated in LB medium at 30°C for 24 hours and the supernatant was subjected to centrifugation at 5000 rpm for 10 minutes to collect 4 g of cells.
  • the cells suspended in 20 mL of 20 mM Tris buffer (pH 8.0).
  • the resulting suspension was treated with 10 mL of 20% polyethylene glycol 8000 (PEG # 8000), 1 mL of lysozyme solution (100 mg/mL) and allowed to react at 37°C for 4 hours.
  • the resulting reaction mixture was subjected to centrifugation at 3500 rpm for 20 minutes to remove a supernatant, which was mixed with 20 mL of lOmM EDTA in 100 mM Tris buffer (pH 8.0). To the resulting mixture, 1 mL of
  • the resulting mixture was agitated under 15 mL of phenol chloroform/ isoamyl alcohol (25:24: 1) for 12 hours and subjected to centrifugation at 12000 rpm for 30 minutes to obtain a supernatant.
  • the supernatant was treated with 1.5 mL of acetic acid buffer (pH 5.2) and then 25 mL of cold ethanol and allowed to stand at -70°C for 15 minutes. Then, it was centrifuged at 12000 rpm for 10 minutes to remove a supernatant, and the resulting pellet was vacuum dried and dissolved into 500 ⁇ L of 1 mM EDTA in 10 mM Tris buffer (pH 8.0).
  • the transformed cells selected in the above (1) were streaked onto a petri dish to which were added 20 mg of alpha-naphthyl acetate in ethylene glycerol monomethyl ester and 12 mg of Fast blue RR salt in Tris-malate buffer (pH 7.6).
  • the strains forming a red-brown zone were selected by replica method.
  • the recombinant plasmid was isolated from the positive strains selected in the above (2) by using a plasmid isolation kit (Flex-prep kit, Pharmacia). Its restriction map shows that it is a 2.7 Kb DNA fragment having SphI restriction site at about 1.6
  • the 1.6 Kb fragment also showed the enzyme activity.
  • the 1.6 Kb fragment also showed the enzyme activity.
  • Kb fragment was digested with Sau3 Al and the resulting digestion products having
  • 0.4Kb, 0.8 Kb, 1.0 Kb or 1.3 Kb size were sub-cloned into s cloning vector, plasmid pUC18.
  • the nucleotide sequencing of these subclones were carried out with a sequencing kit (Cy5TM AutoRead TM sequencing kit ; Pharmacia) under the standard reaction conditions.
  • An automatic sequencer (ALF express automatic sequencer ;
  • Pharmacia is used to detei ine simultaneously the both of forward and reverse complementary nucleotide sequences of about 500 bases.
  • the nucleotide sequence of the total 1562 bp of the gene was deterrnined by using overlapping sequences and shown as SEQ. ID. NO. 2.
  • a structural gene segment of the full-length gene sequenced in the above (3) was determined based on the enzyme molecular weight (48 KDa for dimer and 24 KDa for subunit) measured in the purification of the enzyme, 10 N-terminal amino acids sequence (SEQ. ID. NO. 1) and open reading frame analysis.
  • the structural gene is between nucleotide No. 581 and nucleotide No. 1235, and its putative amino 12 acid sequence is shown as SEQ. ID. NO. 3.
  • the homology of the structural gene was searched, resulting in 93%.
  • Primer No. 1 (SEQ. ID. NO. 4) which has 21 bp with five Neol sites therein and Primer No. 2 (SEQ. ID. NO. 5) which has 22bp with three Hindlll sites therein were prepared and employed as PCR (Polymerase Chain Reaction) primers for amplifying cephalosporin deacetylase gene.
  • Plasmid pSPF1.6, Primer NO. 1, Primer No. 2 and Taq DNA polymerase were mixed and PCR was carried out for 30 cycles of 55°C 60 sec, 72°C 90 sec and 95°C 60 sec for each cycle to amplify the structural gene (654bp) of cephalosporin deacetylase.
  • the transformed strains of E. coli JM109 containing the recombinant plasmid 13 pDSTA654 (5.24Kb) or pDST654 (4.77 Kb) were cultivated in 100 mL of LB medium at 37°C for 12 hours and 0.5 mM of IPTG was added to induce the expression of the cephalosporin deacetylase gene.
  • the grown cells were collected by centrifugation at 5000 rpm for 10 minutes and suspended in 10 mL of 1 mM EDTA in 10 mM Tris-HCl buffer (pH 7.0). The resulting suspension was treated with an ultrasonicator for 10 seconds four times and centrifuged at 13000 rpm for 10 minutes to give a supernatant, which will be served as a material for gene expression evaluation.
  • the expression levels of the cephalosporin deacetylase gene (24 KDa) in the transformed E. coli JM109 containing pUC18 (Control), pDST654 or pDSTA654, or the strain Bacillus sp. DS1152 (KCCM- 10143) were evaluated by electrophoresis on SDS-polyacrylamide gel. Although, the number of copy of the gene amplified in the host slightly varies depending on the kind of the plasmid, both the plasmids show a high expression level.
  • the transformed strain E. coli JM109 (pDST654) (KCCM- 10140) was inoculated to 200 mL of seed culture medium containing glucose 0.2%, yeast extract 3% and NaCl 0.25% (pH 7.0) and cultivated at 28°C, 200 rpm for 12 hours.
  • seed culture was cultivated in 20 L of the same culture medium in a 50L fermentor at 28°C, 400 rpm and 1 v/v/m for 12 hours.
  • 10L of the same medium was additionally added to final concentrations of yeast extract 5.6%, NaCl 0.4% and glucose 3.4%.
  • 0.5 mM of IPTG was added to initiate the gene expression, followed by additional 4 hour fermentation.
  • the culture broth was centrifuged at 4000 rpm for 30 rninutes to harvest about 2 Kg of cells.
  • the cells (100 g, wet weight) were resuspended in 200 mL of solution of 0.9% NaCl in 0.2% benkol and stirred for 1 hour. The resulting suspension was centrifuged at 4000 rpm for 20 minutes to collect the cells, which were resuspended in 100 mL of 0.9% NaCl. A solution (800 mL) of 3% K-carrageenan, which was melted at 80°C, was cooled to 45°C and mixed with the cell suspension obtained above. The resulting mixture was immobilized as a thread shape in 0.3M KC1 solution. To improve the strength of the gel containing the cells, the gel was stored in 2L of solution of 85 mM of glutaraldehyde in 85 mM of hexamethylene diamine.
  • the cells (1 Kg) were suspended into a solution of 1 mM EDTA in 10 mM Tris-HCl buffer (pH 7.0) to a concentration of 20%, and disintegrated with a hydraulic cell disintegrator (Microfluidizer : Microfiuidics) at 1,000 psi. 20 mM MgCl 2 was added and treated with 1 U/mL of deoxyribonuclease I at room temperature for 1 hour to hydrolyze nucleic acids. The reaction solution was centrifuged at 4000 rpm for 30 rninutes to remove cell debris and cells. The resulting supernatant (4 L) was concentrated over microfilter and used as a material for enzyme immobilization.
  • a hydraulic cell disintegrator Microfluidizer : Microfiuidics
  • the enzyme immobilization was carried out by following the standard procedure of Eupergit-C (Roehm, Germany). That is, 500 mL of enzyme (titer 105 U/mL, total 25,000 units) was mixed with 100 g of Eupergit-C in 10M Tris-HCl buffer (pH 7.5) and allowed to stand at room temperature for 3 days. The resulting reaction mixture was filtered using 0. IM Tris-HCl (pH 7.5), washed and stored in a refrigerator.
  • reaction substrate (7-ACA) Fifty grams (50g) of reaction substrate (7-ACA) was dissolved in 1L of 100 mM sodium carbonate buffer (pH 7.5) and stirred with the enzyme (lOOg, 30 U/g) in a reactor.
  • 2N NH 4 OH was added to adjust the pH to about 7.8.
  • the reaction temperature was maintained at about 30°C and the reaction was stopped at the termination point of 2N NH 4 OH addition.
  • the reaction yield was 92 mole% by HPLC.
  • the same deacetylation except of using the immobilized cells (lOOg, 6.5 U/g bead) instead of immobilized enzyme showed the decreased reactivity due to the permeability of the substrate through the bead onto which the cells were immobilized. It took about 360 minutes to complete the deacetylation of 5% substrate and the reaction yield was 85 mole% by HPLC, which is slightly lower than that of the immobilized enzyme reaction.
  • the present invention allows a production of novel cephalosporin deacetylase by gene combination technology.
  • the enzyme is used to prepare deacetylated cephalosporin C, deacetylated 7-ACA and deacetylated MIFACA, all of them are intermediates for cephalosporin antibiotics synthesis.
  • the preparation process is more simple and gives a higher yield than the conventional chemical synthesis by inhibiting byproduct formation and intermediate decomposition.

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Abstract

L'invention concerne une céphalosporine désacétylase dérivée d'une souche de Bacillu sp. qui présente (i) un pH optimal se situant entre 8,0 et 9,0.(ii) une température optimale d'environ 40 °C et (iii) un point isoélectrique d'environ 4,3. L'invention concerne également une séquence d'ADN codant pour cette céphalosporine désacétylase ainsi qu'un vecteur d'expression comprenant cette séquence d'ADN et une souche hôte transformée portant ce vecteur, ces derniers étant utilisés pour désacétyler les composés de céphalosporine ce qui permet d'obtenir des intermédiaires utiles pour la synthèse d'antibiotiques céphalosporine.
PCT/KR1998/000487 1998-04-24 1998-12-30 Cephalosporine desacetylase, gene codant pour celle-ci, et preparation de composes de cephalosporine desacetylee au moyen de celle-ci. Ceased WO1999055881A1 (fr)

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KR1019980014790A KR100270508B1 (ko) 1998-04-24 1998-04-24 신규세파로스포린디아세틸라아제유전자및이를이용한단백질제조방법
KR1998/14790 1998-04-24

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000061767A1 (fr) * 1999-04-09 2000-10-19 Antibioticos, S.A.U. Protease extracellulaire d'acremonium chrysogenum a activite cpc-acetylhydrolase et utilisation dans la synthese de derives desacetyles de cephalosporine, et inactivation du gene pour augmenter la production de cephalosporine
WO2009013611A3 (fr) * 2007-07-26 2009-04-02 Orchid Chemicals & Pharm Ltd Estérase modifiée et ses applications

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0454478A1 (fr) * 1990-04-27 1991-10-30 SHIONOGI SEIYAKU KABUSHIKI KAISHA trading under the name of SHIONOGI & CO. LTD. Gène codant pour une acétylhydrolase de la céphalosporine et protéine codée par celui-ci
JPH0453499A (ja) * 1990-06-19 1992-02-21 Shionogi & Co Ltd デアセチル―７―アミノセファロスポラン酸の製造法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0454478A1 (fr) * 1990-04-27 1991-10-30 SHIONOGI SEIYAKU KABUSHIKI KAISHA trading under the name of SHIONOGI & CO. LTD. Gène codant pour une acétylhydrolase de la céphalosporine et protéine codée par celui-ci
JPH0453499A (ja) * 1990-06-19 1992-02-21 Shionogi & Co Ltd デアセチル―７―アミノセファロスポラン酸の製造法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PATENT ABSTRACTS OF JAPAN vol. 16, no. 244 1992 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000061767A1 (fr) * 1999-04-09 2000-10-19 Antibioticos, S.A.U. Protease extracellulaire d'acremonium chrysogenum a activite cpc-acetylhydrolase et utilisation dans la synthese de derives desacetyles de cephalosporine, et inactivation du gene pour augmenter la production de cephalosporine
US6815189B1 (en) 1999-04-09 2004-11-09 Antibiotics, S.A.U. Gene from acremonium chrysogenum encoding a protein with cephalosporin C acetylhydrolase activity and methods of use of such gene
WO2009013611A3 (fr) * 2007-07-26 2009-04-02 Orchid Chemicals & Pharm Ltd Estérase modifiée et ses applications

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KR19990081077A (ko) 1999-11-15

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