WO1999055881A1

WO1999055881A1 - Cephalosporin deacetylase, gene coding for it, and preparation method of deacetylated cephalosporin compounds using it

Info

Publication number: WO1999055881A1
Application number: PCT/KR1998/000487
Authority: WO
Inventors: Yong Duk Kim; Il Seon Jeong; Sang Hoon Lee; Duk Ho Choi; Kum Soo Han; Bun Sam Lim
Original assignee: Daesang Corporation
Priority date: 1998-04-24
Filing date: 1998-12-30
Publication date: 1999-11-04
Also published as: KR19990081077A; KR100270508B1

Abstract

An isolated cephalosporin deacetylase derived from a strain of Bacillu sp. which (i) has an optimum pH between 8.0 and 9.0; (ii) has an optimum temperature of about 40 °C; and (iii) has an isoelectric point of about 4.3, and a DNA sequence which codes for the cephalosporin deacetylase are disclosed. An expression vector comprising the DNA sequence and transformed host strain carrying the vector are provided and used to deacetylate cephalosporin compounds to give useful intermediates for cephalosporin antibiotics synthesis.

Description

CEPHALOSPORIN DEACETYLASE, GENE CODING FOR IT,

AND PREPARATION METHOD OF DEACETYLATED

CEPHALOSPORIN COMPOUNDS USING IT

BACKGROUND OF THE INVENTION

1. FIELD OF THE INVENTION

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.

2. DESCRIPTION OF THE PRIOR ARTS

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).

The enzyme disclosed in 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. However, there has not been reported that it show reactivity to 7'-substituted-CPC such as 2-memoxyhτm o-2-furylacetic cephalosporanic acid(MIFACA) which is precursor for cephalosporin antibiotics synthesis.

SUMMARY OF THE INVENTION

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.

According to the present invention, it provides a method of deacetylating cephalosporin compounds which comprises contacting the compounds with the enzyme or the transformed strain.

The objects mentioned above, other features and applications of the present invention would be much more apparent by those of ordinary skills in the art from the following explanation in detail.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the construction of expression vectors comprising the gene according to the present invention.

DETAILED EXPLANATION OF THE 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.

Free Text in Sequence Listing

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.

Examples

The present invention will be described in more detail by way of various Examples, which should not be construed to limit the scope of the present invention.

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 α-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. The cells collected from the medium were suspended in 60mM sodium phosphate buffer(pH 6.8) and disintegrated with an ultrasonic homogenizer. The disintegrated cells were subjected to centrifugation(12,000rpm X 20 rninutes) to obtain a supernatant. For measuring enzyme activity, 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).

Further, the enzyme activity was measured by carrying out enzyme-reaction in lOmM of 7- AC A as a subatrate for 20 rninutes, and then quantitative analysis for deacetyl-7-ACA with HPLC. The activity of the selected strain was summarized in Table 1.

Table 1

pNPA method HPLC assay (μ mole deacetyl-pNPA/ (μ mole deacetyl-ACA/ mg protein/min) mg protein min)

Selected strain 1.033 0.46

Strain of 1.990 0.21

EP0454478A1

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.

Table 2

Characteristics

Shape Rod

Endospore formation Yes

Mobility Yes

Gram-stain Positive

Aerobic or anaerobic Aerobic

Example 2 : Isolation and characterization of enzyme

(1) 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.

Four liters of LB medium is placed in a 7.0 L flask and sterilization is carried out by autoclaving at 121°C for 20 minutes. The flask was inoculated with the seed culture of Bacillus sp. strain DS 1152 (KCCM-10143) obtained above to a final concentration of 1% and the cultivation was carried out at 30°C for 48 hours.

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 ((NH₄)₂S0₄) to give a primarily purified protein. The resulting solution was subjected to ultrafiltration (MWCO 30 kDa) to remove salts and to concentrate. Thus 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. They are pooled and concentrated by subjecting to ultrafiltration (MWCO 30 kDa), and subjected to column chromatography over Sephacryl-S300 (Pharmacia) to obtain a purified desired protein. Its specific activity was measured using 7-ACA as a substrate and the result was 31 unit per mg protein.

(2) Measurement of molecular weight of enzyme

Gel chromatography was carried out on Sephacryl-S300 (Pharmacia) packed in a 1.6 X 100 cm column (Gel volume : 300 ml) to measure the molecular weight of the enzyme. Molecular weight marker proteins of 200 kDa, 150 kDa, 66 kDa, 29 kDa or 19.3 kDa (Sigma) were eluted using a buffer at a flow rate of 2.5 ml/min. The volume of the buffer used to elute the enzyme was compared with that for the marker proteins and the molecular weight of the enzyme was calculated using a standard curve. It was 48 kDa. In addition, the molecular weight of monomer was measured using 12% polyacryl and SDS-PAGE (Lammilis method). It was 24 kDa, indicating that the enzyme was a dimer consisting of two 24 kDa monomers.

(3) Sequencing of N-terminal of the enzyme

Partially purified (95% purity) enzyme was ultrafϊltered 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.

(4) Measurement of enzyme activity

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 quantitative analysis of deacetylated-7- aminocephalosporanic acid (DACA), a byproduct of 7-ACA deacetylation, was performed on HPLC under the following conditions: <HPLC conditions> ^• Mobile phase : 50 mM (NH₄)₂HP0₄ / 55% CH₃CN (pH 3.2)

^• Static phase : Alltech NH₂

^• Column capacity : 4.6 X 250 mm

^• Flow rate : 0.6 miVmin

^• Wavelength : 254 nm ^• Temperature : 45°C

(5) Determination of optimum pH

By using different buffers: 0.1M citrate buffer for pH 3.0 - 5.0; and 0.05M sodium phosphate + 0.05M citrate, followed by adjusting pH with NaOH, for pH 6.0

- 11.0, 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.

(6) Determination of optimum reaction temperature

By using 0.1 M sodium phosphate buffer (pH 7.0), the optimum temperature was deterrnined. Enzyme activity was measured at an interval of 5°C at a range from 20 °C to 50° C using 7-ACA as a substrate. The measurement conditions are the same as those of HPLC in the above (4). The enzyme activity began to increase at 30

°C and showed the highest activity at 40 °C.

(7) Determination of Isoelectric Point of Enzyme

Density gradient isoelectrofocusing was carried out with PhastSystem™ (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.

(8) Substrate Reactivity of Enzyme

The substrate reactivity of enzyme is defined as Michaelis constant (K which 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. By using Lineweaver-Burke formula, substrate reactivity was calculated. 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

Substrate Km(mM) Vmax (mole/min)

7-ACA 18.8 0.59

CPC ^*2 14.6 0.36

MIFACA ^*3 17.2 0.51

* 1 : 7-ACA = 7-aminocephalosporanic acid

* 2 : CPC = cephalosporin C

* 3 : MIFACA = 2-methoxyiπιmo-2-furylacetyl cephalosporanic acid

Further, various properties of the enzyme from the Strain disclosed in EP 0454478 A 1 were also determined, and summarized in Table 4 along with those of the enzyme according to the present invention.

Table 4

Properties \ Source Strain of Bacillus sp. DS 1152 EP 0454478 A 1 (KCCM- 10143 )

Molecular Weight 280 kDa 48 kDa

Structure Octamer Dimer

Optimum Temp. 55°C 40°C

Optimum pH 8.0 - 8.5 8.0 - 9.0

Isoelectric Point 5.3 4.3

Km (for 7-ACA ) 7.3 mM 18.8 mM

Km(for CPC^*2) 24.3 mM 14.6 mM

Km(for MIFACA^*3) - 17.2 mM

* 1 : 7-ACA = 7-aminocephalosporanic acid

* 2 : CPC = cephalosporin C * 3 : MIFACA = 2-methoxyinιmo-2-furylacetyl cephalosporanic acid 10

Example 2 : Separation and Sequencing of gene coding for the enzyme (1) DNA Isolation and Construction of Gene Library

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

25% SDS was added and mixed well at 37°C for 1 hour to give a suspension. A solution (200 L) of proteinase K (20 mg/mL) was added and reacted for 4 hours.

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 μL of 1 mM EDTA in 10 mM Tris buffer (pH 8.0).

Thus isolated DNA was partially digested with Sau3Al and subjected to sucrose density gradient ultracentrifugation at 25000 rpm for 24 hours to fractionize the digestion fragments depending on their sizes. The fragments was precipitated under 70% ethanol and vacuum dried to give DNA fragments of 2 - 6 Kb. The DNA fragments were ligated to pUC18 vector which was treated with

BamHI, and the resulting recombinant pUC18 vector was transformed into the strain of E. coli JM109. The transformed cells were grown in a petri dish containing 50 μg/mL of ampicillin to select grown strains. 1 1

(2) Selection of Acetyl esterase-Positive Strains

To select the strains having Acetyl esterase activity, 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.

(3) Nucleotide Sequencing of Positive Clones

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

Kb site (pSPFl.6). The 1.6 Kb fragment also showed the enzyme activity. The 1.6

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 (Cy5™ AutoRead ™ 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.

(4) Gene Probing

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%.

Example 3 : Gene Expression (1) PCR Amplification of Gene

Two oligonucleotide primers: 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.

(2) Construction of Expression Vectors The amplified gene was digested with Neol and Hindlll, and ligated into pKK223-3 (Amp^R+, Pharmacia) digested with Smal and Hindlll, or pTrc99A (Amp^R+, Pharmacia) digested with Neol and Hindlll to give pDSTA654 (5.24 Kb) or pDST654(4.77 Kb), respectively. The recombinant plasmids were transformed into E. coli JM109 and the transformed strain containing pDST654 was deposited with Korean Federation of Culture Collections on April 24, 1998 and given accession number of KFCC-11029. This deposit was converted into one under Budapest Treaty on November 11, 1998 and assigned an accession number of KCCM- 10140.

The flow of constructing the expression vectors pDSTA654 (5.24 Kb) and pDST654(4.77 Kb) is shown in Figure 1.

(3) Gene Expression

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. 7- AC A, as a substrate, was dissolved in 10 mM Tris-HCl buffer (pH 8.0) to a concentration of 5 mg/mL and 7- AC A was added to 50 mM Tris-HCl buffer (pH 7.0) to a final concentration of 2.5 mg/mL. The resulting mixture was allowed to react at 30°C for 30 minutes and the amount of deacetylated 7-ACA produced was analyzed by HPLC. The amount of protein from cell homogenate was measured by Bradford Assay by comparing with standard protein level, and the specific enzyme production titer expressed as μmole of deacetylated 7-ACA produced by one(l) mg of protein for 1 minute was shown in Table 5. 14

Table 5

Specific Enzyme

Host Cell Plasmid Inducer Production Titer

(μmole deacetylated 7-

ACA/mg protein/min) pUC18 - 0.05

E. coli JM109 pDST654 0.5 mM IPTG 4.72 pDSTA654 0.5 mM IPTG 3.20

Bacillus sp. - - 0.46

DS 1152

Example 4 : Fermentation and Immobilization (1) Fermentation

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. Thus obtained 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. At the point of sugar depletion, 10L of the same medium was additionally added to final concentrations of yeast extract 5.6%, NaCl 0.4% and glucose 3.4%. When the fermentation reached to log phase, 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.

(2) Cell Immobilization

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.

(3) Enzyme Immobilization

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 MgCl₂ 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.

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.

Example 5 : Deacetylation by Immobilized Enzyme or Cell

The reaction scheme of deacetylation by cephalosporin deacetylase according to the present invention is as follows: R - Rl : H- 7-Aminocephalosporanic acid O

OAc

R2 : H2N-CH-(CH2)₃- C- Cephalosporin C

COOH CO2H

Cephalosporin R3

11 Deacetylase ^~v O

C- C 2-Methoxyimino-2-furyl- " acetic cephalosporanic acid

O NOCH3 (MIFACA)

R-NH

CH3COOH

// ^N\ ^\^ OH O Υ

COOH

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.

To avoid the decrease of pH of the reaction mixture due to acetic acid generated during deacetylation, 2N NH₄OH 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 NH₄OH addition.

It took about 90 minutes to complete the deacetylation of 5% substrate and 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. As described above, 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.

Although preferred embodiments of the present invention have been described in detail herein above, it should be clearly understood that many variations and/or modifications of the basic inventive concepts herein taught which may appear to those skilled in the art will still fall within the spirit and scope of the present invention as defined in the appended claims.

Claims

1. An isolated cephalosporin deacetylase derived from a strain of Bacillu sp. which: (I) has an optimum pH between 8.0 and 9.0;

(ii) has an optimum temperature of about 40°C; and (iii) has an isoelectric point of about 4.3.

2. A cephalosporin deacetylase according to Claim 1 which is a dimer having a molecular weight of about 48Kda.

3. A cephalosporin deacetylase according to Claim 1, wherein the strain is Bacillus sp. DS 1152 (KCCM- 10143 ).

4. A cephalosporin deacetylase according to claim 1 which has an N-terminal sequence : SEQ. ID. NO. 1.

5. A DNA sequence which codes for a cephalosporin deacetylase according to claim 1 and comprises a fragment between nucleotide No. 581 and nucleotide No. 1235 in SEQ. ID. NO. 1.

6. A DNA sequence according to claim 5 which is shown as SEQ. ID. NO. 2.

7. An expression vector comprising a DNA sequence according to claim 5.

8. A host strain transformed with an expression vector according to claim 7.

9. A transformed strain according to claim 8, wherein host strain is E. coli JM109. 19

10. A transformed strain according to claim 9 which is a strain of E. coli JM109 (pDST654) (KCCM- 10140).

11. An isolated strain of Bacillus sp. which has the same biochemical characteristics as those of Bacillus sp. DS1152 (KCCM-10143).

12. A method of deacetylating cephalosporin compounds, comprising contacting the compounds with an enzyme according to claim 1 or a transformed strain according to claim 8.

13. A method according to claim 12, wherein said enzyme or strain is immobilized on a carrier.