KR100270508B1 - Novel cephalosporin diacetylase gene and protein production method using the same - Google Patents

Abstract

New strains that secrete new cephalosporin diacetylase from the soil were purely isolated and identified as Bacillus sp. As a result of identifying the new strain, the strain was named Bacillus DS1152.

New cephalosporin di, involved in the production of diacetyl cephalosporin C, diacetyl 7-ACA and diacetyl 2-methoxymino-2-furyl-acetic cephalosporinic acid from the new strain Bacillus DS1152 (KFCC11026) Acetylase genes were isolated and analyzed for gene sequence and amino acid sequence to reveal new enzymes.

The new enzymes encoded by this novel gene were isolated and purified to examine their biochemical properties, and the genes of the isolated novel enzymes were manipulated by genetic recombination technology to be amplified and expressed in large quantities in E. coli (KFCC11029). At this time, the expressed enzyme had a molecular weight of 24KD, and the structural form was composed of duplexes and had a molecular weight of 48KD.

This novel recombinase was confirmed by immobilization of cells and enzymes having excellent substrate reactivity to various cephalosporin intermediates. By applying this novel technology to the preparation of the cephalosporin-based semisynthetic intermediate, a new technology which is very industrially advantageous over the existing synthetic method has been established.

Description

Novel cephalosporin deacetylase gene and protein production method using the same

The present invention relates to enzymes and genes for promoting deacetylation of cephalosporin C and 7-ACA (7-aminocephalosporanic acid), which are essential precursors for the preparation of cephalosporin-based antibiotics.

Sephalosporin-based compounds such as cephalosporin C and 7-ACA are used as semisynthetic intermediates for the preparation of high value-added cephalosporin antibiotics by eliminating the acetyl group bound to the 3-position. Methods for deacetylation of cephalosporins include chemical and enzymatic methods. Dual enzymatic methods have been reported to improve yields by reaction pH, reaction temperature, reaction byproducts, etc. (USP3,304,236).

This enzyme (Cephalosporin C Deacetylase) has been reported two kinds of enzymes whose biochemical properties are distinguished by the Japanese Shionogi company from microorganisms of Bacillus subtilis origin. Enzymes have recently been reported as mass production by introducing genes into E. coli (EP0454478A1, Applied and Environmental Microbiology.1995.p2224).

The present inventors isolated Bacillus sp. DS1152 having strong cephalosporin C deacetylase activity in soil near Icheon, Gyeonggi-do, Korea, and isolated a novel gene encoding an activating enzyme from this strain. The isolated gene was amplified in a large number of vector systems under E. coli by gene recombination. The results of analyzing the biochemical properties of the amplified enzyme protein are shown in Table 1 below. According to these results, the biochemical properties of the enzyme (Shionogi Co., Ltd.) and enzymes are different from each other, and the substrate affinity and reactivity of cephalosporin seeds are excellent.

Table 1. Biochemical Properties of Enzyme Proteins

Strain Strains of EP0,454,478, A1 Strain KFCC11026 of the Invention Molecular Weight Composition Optimum Temperature Optimum pH Light Km (7-ACA) Km (CPC) Km (MIFACA) 280K Daoctameric 55C8.0-8.55.37.3m24.3mM- 48 KDadimeric 40C8.0-9.04.318.8mM14.6mM17.2mM

By using the enzyme produced in the present invention in the process for producing cephalosporin antibiotics, it is possible to reduce the multi-step reaction process of the existing chemical synthesis process, and to improve the productivity of the cephalosporin antibiotics by enzyme reaction. The purpose is to provide.

1 is a plasmid reconstitution process for introducing a gene into an expression vector used in the present invention.

Bacillus sp. DS1152 (KFCC11026) of the present study is a seed isolated from soil and can be grown in LB (Luria-Bertani) plate medium for 24 hours at 30 ° C. Same as

Table 2. Bacillus sp. DS1152 (KFCC11026) strain characteristics

division Characteristics of the strain shape Rod-shaped Resistant Spore Formation Generate resistant spores motility Mobility Gram dye reaction Gram positive Aerobic presence Aerobic

Example 1 Characterization of Enzymes

(1) Separation and Purification of Enzymes

The 500 ml Erlenmeyer flask to which LB culture was added was sterilized at 121 ° C. for 20 minutes, and then inoculated with Bacillus DS1152 and pre-cultured at 30 ° C. for 9 hours, and used as a seed of the production medium. The culture was performed by sterilizing a 7.0 L fermentation tank containing 4.5 L of LB medium for 20 minutes at 121 ° C., then incubating for 48 hours at 30 ° C. by adding 1% of the above cultured whole culture solution to deacetylase from the culture medium. Purification was carried out as follows. Cells were recovered by centrifugation (5000 rpm x 30 minutes) from the above culture. The recovered cells were suspended using a pH buffer solution of 50 mM Tris / HCl (pH 8.0), and then the cells were crushed using an ultrasonic cell crusher. The pulverized cells were removed again in the insoluble part by centrifugation (12,000 rpm x 30 minutes). After centrifugation, the supernatant was fractionated by using 40-80% (NH ₄ ) ₂ SO ₄ saturated solution. Purification was carried out primarily by precipitation. At this time, since a large amount of salt was present in the solution, the ultrafiltration was performed to remove it and to concentrate the protein. The pore size of the permeable membrane was 30 KDa. This concentrated protein solution was added to DEAE-Sepharose (pharmacia) stabilized in 50 mM Tris / HCl (pH 8.0) at a flow rate of 2.0 ml / min and the amount of 40 mg of protein per ml of gel was added. After complete addition, the column was washed again with the buffer used for stabilization, and the elution of the protein was eluted by flowing 0-300 mM NaCl in a continuous concentration gradient method. At this time, most of the deacetylase was eluted in the NaCl concentration range of 150-200mM, and they were collected in one place and concentrated again by ultrafiltration using a permeable membrane having a pore size of 30KDa. Sephacryl-S300 It was applied to gel chromatography using) and finally separated and purified. In this case, the activity was measured using the substrate as 7-ACA, and 31 units per mg of protein.

(2) determination of molecular weight of enzyme

The overall molecular weight of the enzyme was determined by gel chromatography packed with Sephacryl-S300 in a column of 1.6 × 100 cm (gel volume: 300 ml). The column eluted standard protein of 200, 150, 66, 29, 19.3KDa (Sigma) in buffer at a flow rate of 2.5 ml / min. In the same way, the volume of the buffer solution in which the sample of the enzyme was eluted was compared with the standard sample and the molecular weight was calculated under the standard curve. At this time, the calculated molecular weight of the enzyme was 48KDa, the result is as shown in FIG. In addition, the molecular weight of the monomer was determined by using SDS-PAGE using 12% polyacrylamide and the Rammilis method, and the measured molecular weight of the monomer was 24KDa. From this, it was confirmed that the activating enzyme is an enzyme composed of a monomer having a molecular weight of 24 KDa.

(3) Analysis of amino acid sequence of amino terminal region of enzyme

The partially purified enzyme (purified: 95%) was ultrafiltered with distilled water to remove salts and freeze-dried, and the sample was subjected to the standard analysis manual presented under the Precise Protein sequencing system (Applied Biosystems). The amino acid sequence of the amino terminus 10base was determined accordingly. The determined base sequence of the cephalosporin diacetylase amino terminal was confirmed that the sequence of Ala-Asn-His-Ile-Tyr-Leu-Ala-Gly-Asp-Ser rather than methionine.

(4) Determination of enzyme activity

0.1 M sodium phosphate buffer (pH 7.0) was used as in the example carried out by the Shiono group, and the enzyme activity was defined as 1 unit of deacetylating 1 M of 7-ACA per minute at 37 ° C. The enzyme used in the experiment was vacuum-dried the enzyme prepared by the purification method carried out before, and then dissolved in a certain amount in a buffer solution if necessary. In addition, the quantitative analysis of deacetyl-7-aminocephalosporanic acid (hereinafter referred to as DACA), which is a deacetylation reaction product of 7-ACA, was analyzed by HPLC and the HPLC conditions used were as follows.

Table 3. HPLC Analysis Conditions

Condition condition Moving constant phase column Volume velocity velocity Wave length (℃) 50 mM (NH ₄ ) ₂ HPO ₄ /20% CH ₃ CN pH3.2 Alltech NH ₂ 46 X 250 mm0.5 ml / min 254 nm45

(5) Determination of optimum pH of enzyme

In order to measure the optimum pH of the enzyme, the interval between pH3.0 and 11.0 was investigated, 0.1M citric acid buffer solution was used in the acidic range of pH3.0-5.0 and 0.05M sodium phosphate in the pH6.0-11.0 range. And 0.05M citric acid were added, and then a wide range of buffer solution was adjusted to pH with NaOH. In addition, 7-ACA was used as a substrate, and the produced DACA and the consumed substrate were analyzed by the HPLC conditions presented above. The pH-dependent reactivity of the enzyme showed the highest activity at 8.0-9.0.

(6) determination of optimum reaction temperature of enzyme

0.1 M sodium phosphate buffer (pH 7.0) was used for the optimal temperature analysis of the enzyme, and the temperature range used for the test was performed at intervals of 5 ° C. in the range of 20-45 ° C. In addition, the substrate used in the experiment was carried out by the method described above using the substrate 7-ACA and the analysis was carried out through the HPLC conditions presented above. Enzyme activity was increased from 30 ℃, the highest activity at 40 ℃.

(7) Determination of isoelectric point of enzyme

The isoelectric point of the enzyme was analyzed by iso-electrofocusing, and the pH range was pH 3.0-9.0. At this time, bovine serum albumin with isoelectric point pH4.7-4.9 was used as the standard material, and the pH was measured by dissolving the electrolytic material dissolved in the gel in distilled water by cutting off the gel on the enzyme. At this time, the enzyme showed a pI value of 4.3.

(8) substrate reactivity of the enzyme

The substrate reactivity of the enzyme defined the reaction rate of the enzyme as the Michaelis constant. Conditions for the reaction of the enzyme were 0.1M sodium phosphate buffer solution (pH 7.0), 7-ACA, cephalosporin C (hereinafter CPC), 2-methoxymino-2-furylacephacelosporinic acid; 2-Methoxyimino-2-furylacetic cephalosporanic acid; Subsequently, the substrate of MIFACA) was added at different concentrations, followed by reaction at 37 ° C. for 10 minutes, and analyzed by HPLC. Substrate reactivity of the analyzed enzyme was calculated by Lineweaver-Burke equation. The calculated reactivity to the substrate is shown in the following table, and showed particularly even reactivity to the Sepharoserin-based intermediate.

Table 4. Substrate Reactivity of Enzymes

Type of substrate Km (mM) Vmax (mole / min) 7-ACA 18.8 0.59 CPC 14.6 0.36 MIFACA 17.2 0.51

Example 2 Gene Isolation and Sequence Analysis

(1) DNA isolation and gene library preparation

Bacillus DS1152 was incubated for 24 hours at 30 ° C. in LB culture medium, and then 4 g of cells were recovered after centrifugation at 5000 rpm x 10 min, which was suspended in 20 mL of 20 mM Tris buffer (pH 8.0).

This was treated with 10 ml of 20% PEG # 8000 solution and 1 mL of lysozyme solution (100 mg / ml) and reacted at 37 ° C. for 4 hours. The supernatant was removed by centrifugation at 3500 rpm x 20 min, and then mixed under 20 mL of 100 mM Tris buffer solution and 10 mM EDTA solution (pH8.0). Then, 1 ml of 25% SDS was added and mixed for 1 hour at 37 ° C, followed by protease K solution. 200 μL (20 mg / ml) was added and reacted for 4 hours.

The reaction solution was stirred for 12 hours under 15 ml phenol / chloroform / isoamyl alcohol (25: 24: 1), followed by centrifugation at 12000 rpm × 30 min to obtain a supernatant. The solution was treated with 1.5 ml of acetic acid buffer (pH5.2) and 35 ml of cold ethanol, and left to stand at -70 ° C for 15 minutes. The supernatant was removed by centrifugation at 12000 rpm x 10 min, followed by vacuum drying to dissolve in 500 μL of 10 mM Tris buffer, 1 mM EDTA solution (pH 8.0).

Isolated DNA was partially digested with restriction enzyme Sau3AI and fractionated according to the size of the sections by 25000 rpm × 24hr sucrose density gradient ultracentrifugation. Fractionated sections were precipitated and vacuum dried under 70% ethanol to obtain 2-6 Kb DNA fragments.

The obtained DNA fragment was ligated to the pUC18 vector digested with the restriction enzyme BamHI, which was transformed into the E. Coli JM109 strain, and screened for growth bacteria under plate medium containing antibiotics (Ampicillin 50ug / ml). It was.

(2) Selection of Acetyl Esterase Positive Strains

For screening strains with acetylesterase activity from cloned genes, 20 mg of alpha-naphtelacetate dissolved in ethylene glycerol monoethyl ester was tris-maleate buffered with 12 mg of fast blue RR salt. After adding the solution (pH7.6) to the plate medium, the cells were separated by red replicas of reddish brown bacteria with respect to alpha-naphtel acetate by the replica method.

(3) Gene sequencing of positive clones

Recombinant plasmids were separated under a plasmid isolation kit (Flex-prep kit, manufactured by Pharmacia) and examined for plasmid restriction maps, and the restriction enzyme Sph I cleavage site was located at about 1.6 Kb (pSPF1.6) in 2.7 Kb DNA fragment And showed the same enzyme activity in this site. DNA fragments of 1.6 Kb were partially cut into restriction enzyme Sau3AI and the fragments were re-cloned into subclones with DNA fragments corresponding to 0.4 Kb, 0.8 Kb, 1.0 Kb, and 1.3 Kb in the pUC18 plasmid, the cloning vector. . These subclones were subjected to the standard reaction conditions presented under the Cy5TM AutoReadTM sequencing kit (Pharmacia), which were prepared using the ALF express automatic sequencer (Pharmacia). The cloned gene sequence was confirmed by reading about 500bp of complementary sequences in the reverse direction. The identified nucleotide sequences were linked to overlapping portions to identify the entire 1562 bp gene sequence, and the results are shown in SEQ ID NO: 1.

(4) gene search

The gene sequences determined were molecular weight (dimer 48KDa: 24KDa subunit), 10 amino-terminal amino acid sequences (Ala-Asn-His-Ile-Tyr-Leu-Ala-Gly-Asp-Ser) and amino acid transcription identified during enzymatic purification. The structural gene region (581-1235) of the active enzyme was determined by analysis of the open reading frame. This gene region was 93% homologous to 173602 in 172923 of the Bacillus subtilis complete genome, whose function was unknown as a result of homology search in the NIH BLASTN search. It was confirmed that the gene having a.

As a result of analyzing the amino acid sequence encoded by the structural gene of this region and the amino acid sequence encoded by the recombinant gene sequence of the present invention, only four of 217 amino acids (site: 127, 128, 176, 217) were identified. The difference is showing. These results showed that the enzymes of the same function showed partial amino acid sequence differences according to the types of bacteria, and the function would be the same. The result of homology comparison between the putative amino acid sequence of the Bacillus subtilis gene region and the amino acid sequence of the recombinant gene of the present invention is shown in SEQ ID NO: 2.

Example 3: Gene Expression

(1) PCR amplification of genes

Based on the structural gene region estimated in Example 2), oligonucleotide primers of 21bp including restriction enzyme Nco I cleavage site at 5 sites and 22bp containing restriction enzyme Hind III cleavage site at 3 sites were synthesized. This was used as a primer for PCR (Polymerase Chain Reaction) for cephalosporin diacetylase gene amplification. The base sequence of the designed primer is as follows.

5-TGA GCC ATG GCG AAT CAC ATT-3

Nco I

2. 5-TCA CAA GCT TCA CCC TTC TTT G-3

Hind III

The primers designed in Example 4) and the tag DNA polymerase were mixed using pSPF1.6 prasmid as a parent, and repeated cycles of 55 ° C. 60 sec, 72 ° C. 90 sec, and 95 ° C. 60 sec were repeated 30 times. The structural gene of cephalosporin diacetylase corresponding to 654bp was mass amplified.

(2) Construction of Expression Vector and Preparation of Recombinant E. Coli

The amplified gene was digested with restriction enzymes Nco I, Hind III, pKK223-3 (Pharmacia: Ampicillin resistance), pTrc99A (Pharmacia: Pharmacia: expression vector), which were cut by the same method (pKK223-3: Sma I, Hind III). Ampicillin resistance) and pET24d (+) (Novagen product: kanamycin resistance) were ligated into E.Coli JM109 strains to construct pDSTA654, pDST654 and pDSET654, respectively, and pDSET654 was again expressed as BL21 (pLys S). ) And the host-vector system was reconstituted as in FIG. 1 to prepare recombinant E. coli KFCC11029.

(3) gene expression

Recombinant cells were incubated for 12 hours at 37 ° C. under 100 ml LB medium to induce expression by converting 0.5 mM IPTG or culture temperature according to the characteristics of the vector. The cells were recovered after centrifugation at 5000 rpm, and suspended in 10 mM Tris buffer (Tris HCl buffer, pH 7.0) and 1 mL EDTA solution. This was treated with an ultrasonic crusher for 10 sec x 4 times, and then centrifuged at 13000 rpm x 10 minutes to prepare the supernatant as a material for gene expression.

This was confirmed by SDS-polyacrylamide gel electrophoresis and the expression level of the protein corresponding to 24KDa using E. Coli JM109 (pUC18) as a comparison group. All were confirmed to be expressed in large quantities.

As a substrate for enzymatic reaction, 7-ACA was dissolved at 5 mg / ml in 10 mM Tris buffer (pH8.0), and the final concentration of 1 mg / ml 7-ACA was added to 50 mM Tris buffer (pH7.0) at 30 ° C. Reaction for 30 minutes was performed to quantify the amount of diacetyl7-ACA under HPLC. The amount of the protein from the cell lysate was determined by comparison with the standard protein amount by the Bradford assay, and the expression enzyme production titers of the recombinant genes are shown in the table.

Table 5. Expression enzyme production titers of recombinant genes

host Plasmid Induction of expression Inactivity (mole deacetyl-7ACA / mg protein / min) E.Coli JM109BL21 (DE3) Bacillus sp.DS1152 pUC18PDST654pDSTA654pDSET654 (-) 0.5mM IPTG0.5mM IPTG0.5mM IPTG 0.054.723.203.860.46

Example 4 Cell Culture and Immobilization

(1) Cultivation of Cells

The culture medium used for cell culture was 0.2% glucose, 3% enzyme extract, 0.25% NaCl, pH7.2 and incubated for 12hr at 37 ° C, and then stirred at 400rpm in a 5L fermenter under the same medium. The cells were cultured under the condition of / M, and the cells were cultured by injecting additional medium so that the final concentration was 5.6% of enzyme extract, 0.4% of NaCl, and 3.4% of glucose. Gene expression was induced by the addition of 500uM IPTG at the time of reaching the maturation stage on the growth curve of the cells, and after 4 hours of incubation, the cells were collected by centrifugation at 4000rpm x 20min in a centrifuge. .

(2) Cell fixation

The collected bacteria were resuspended in 10 mM Tris buffer solution (pH9.0), buffer solution under 1 mM EDTA, stirred at 40 ° C for 1 hour, and then collected by centrifugation. The collected 8 g (wet weight) cells were resuspended at 8 ° C. at 40 ° C., 0.9% NaCl, 0.2% Triton X-100. At the same time, 2.07g κ-carrageenan was dissolved in 45 ml of 0.9% NaCl, 80 ° C, cooled to 45 ° C, and the two solutions were mixed and cooled again at 10 ° C for 30 minutes. At this time, in order to increase the strength of the gel, it was soaked under 0.3 M KCl to obtain a fixed bacteria.

(3) enzyme fixation

The collected cells were suspended at 20% concentration in 10 mM Tris buffer (pH 7.0), 1 mM EDTA solution. The cells were crushed in a hydraulic cell crusher, 20mM MgCl 2 was added, and treated with deoxyribonuclease I at a concentration of 1 U / ml, and stirred at room temperature for 1 hour to hydrolyze the nucleic acid. This solution was microfiltered to remove cell debris and inactive peptides and used as a sample for enzyme fixation.

Enzyme immobilization process was carried out using 100 ml (titer 880 U / ml, total 88,000 units) in 20 ml of 1.0 M (K2HPO4 + KH2PO4) (pH7.5) buffer according to the standard method of Eupositit C (Eupergit-C: German Rhm). The mixture was allowed to stand for 3 days at room temperature by mixing with the seed carrier.

The reaction mixture was filtered and washed with 0.1 M (K2HPO4 + KH2PO4) (pH7.5) buffer, and stored in the refrigerator.

Example 5 Deacetylation by Immobilization

The structure of the product converted by the enzymatic reaction of the invention is as follows.

20 g of the reactive mass (7-ACA) was quantified and dissolved in 1 L of 100 mM sodium carbonate buffer (pH 7.5), followed by stirring with immobilized enzyme of the reactor.

At this time, 1N KOH was added to prevent the pH from dropping by the deacetylated acetic acid generated as the reaction proceeded, and the reaction proceeded by adjusting the reaction pH to 7.9-8.2 level. The reaction temperature was maintained at 30 ℃, the reaction was terminated at the end of the addition of KOH.

It took about 40 minutes to deacetylate the reactor (2% concentration). As a result of confirming the reaction product by HPLC analysis, the reaction yield was 92%.

Reactions containing immobilized cells in the same way were limited in reactivity by the permeability of the substrate through the cell membrane, and the reaction yield by analysis of the reactants was 44% lower than that of the immobilized enzyme.

[Sequence list]

SEQ ID NO .: 1

Sequence length: 1562

Type of sequence: nucleic acid

Number of chains: 2 prints

Topology: linear

Type of sequence: Gemonic DNA

origin

Biology: Bacillus Species

Main Name: DS1152 (KFCC11026)

order:

ATCGAGCTCG GTACCGGGGA TCATGACTGG GACGGAACGG TCGGCAGGCC 50

GAAGATTGAA AAGTGGGATG CTGAAAACCG CTGTCTGAAG ACGATTTTTC 100

AGCCGGACGG CGTGCTGTCC AACAACGGCA CAAAAGGAAA TCCTGTTCTT 150

CAGGCCAACC TGTTCGGAGA CTGGCGGGAA GAAGTGATAT GGAGAACGGA 200

AGACAGCAGC GCACTCCGCA TCTATACAAC GACACATCTC ACCCGCCATC 250

GCTTTTACAC GCTTATGCAC GATCCGGTTT ACAGGCTCGG CATCGCCTGG 300

CAGAATACCG CCTACAACCA GCCGCCGCAC ACGGGCTTTT ATCTCGGAAC 350

GGGAATGAAA AAACCACCGA AGCCCGCCCT GTACATAGCG GGCAGCAAAG 400

CGGAGGCGCC GCTATAGGAG GAACTAAGGG ATAAGAAGCA AAGGCTGAAC 450

AGTACGACAA GATGACTTAT CAAAGCCAGG ACGACTAATG TTCAGGCGCT 500

GAAATCACCG GTTTTTTCAA GGGAAGAGCT GTCCATGATT GATGCCATAT 550

GAAACGAAAA AAACGAGGGG GAATGAGAA ATG GCG AAT CAC ATT TAT 597

1 Met Ala Asn His Ile Tyr 6

CTT GCT GGC GAT TCA ACT GTT CAA ACG TAT GGA GAC AGC ACA 639

Leu Ala Gly Asp Ser Thr Val Gln Thr Tyr Gly Asp Ser Thr 20

AAT CAA GGG GGG TGG GGG CAG TTT CTC GGC TCG CAT CTG CCG 681

Asn Gln Gly Gly Trp Gly Gln Phe Leu Gly Ser His Leu Pro 34

GAG CAT ATT CAA GTG ATT AAC AGA GCA ATC GGG GGA AGA AGC 723

Glu His Ile Gln Val Ile Asn Arg Ala Ile Gly Gly Arg Ser 48

TCG AAA ACA TTT GTG GAA GAG GGC AGG CTT CAG GCG ATT CTT 765

Ser Lys Thr Phe Val Glu Glu Gly Arg Leu Gln Ala Ile Leu 62

GAT GTG ATT GAG CCG GAT GAC TGG CTG TTT GTG CAG ATG GGC 807

Asp Val Ile Glu Pro Asp Asp Trp Leu Phe Val Gln Met Gly 76

CAT AAT GAC GCG TCG AAA AAT AAG CCG GAG CGC TAC ACC GAG 849

His Asn Asp Ala Ser Lys Asn Lys Pro Glu Arg Tyr Thr Glu 90

CCC TAT ACC ACC TAT AAA CAA TAT TTA AAG CAG TAT ATC GCA 891

Pro Tyr Thr Thr Tyr Lys Gln Tyr Leu Lys Gln Tyr Ile Ala 104

GGC GCG CGG GAA AAA GGC GCC CAT CCG CTT CTC ATT ACC CCT 933

Gly Ala Arg Glu Lys Gly Ala His Pro Leu Leu Ile Thr Pro 118

GTA GCA CGC TTT CAT TAT GAA AAT GAC ATG TTT TTG AAC GAC 975

Val Ala Arg Phe His Tyr Glu Asn Asp Met Phe Leu Asn Asp 132

TTT CCT GAC TAT TGC ATT GCC ATG AAG CAG ACG GCT GCT GAG 1017

Phe Pro Asp Tyr Cys Ile Ala Met Lys Gln Thr Ala Ala Glu 146

GAG AAT GTC CAG CTC ATT GAT CTG ATG GAG AAA AGT CTT GCT 1059

Glu Asn Val Gln Leu Ile Asp Leu Met Glu Lys Ser Leu Ala 160

TTC TTT ACC GAG AAG GGC GAG GAA AAA GTG TAC ACC TAT TTT 1101

Phe Phe Thr Glu Lys Gly Glu Glu Lys Val Tyr Thr Tyr Phe 174

ATG GTG TCA GAA GGA ATT AAC GAT TAC ACG CAC TTT ACA AAA 1143

Met Val Ser Glu Gly Ile Asn Asp Tyr Thr His Phe Thr Lys 188

AAA GGC GCA AAT GAA ATG GCG AAA CTT GTG GCA AAA GGC ATA 1185

Lys Gly Ala Asn Glu Met Ala Lys Leu Val Ala lys Gly Ile 202

AAG GAA CTC GGC CTG CCA TTG ACA GAA TCG ATC ATC AAA GAA 1227

Lys Glu Leu Gly Leu Pro Leu Thr Glu Ser Ile Ile Lys Glu 216

GGG TGA AAA ATG TGA GGA GAAAACTG TATCACGGCG CTTGTTATTA 1273

Gly *** 217

TCCCGAATTA TGGGATGAAG AGACGATTCA GCAGGACATT GACATCATGC 1323

GTGAAGTTGG CGTGAATGTT GTGCGGATCG GTGAATTTGC CTGGTCTGTC 1373

ATGGAACCGG AAGAAGGAAA AATTGACGTC AGTTTTTTCA AAGAGATCAT 1423

CACCCGTCTG TATGATAACG GCATCGAAAC GATCATGTGC ACGCCGACGC 1473

CTACACCGCC GATTTGGCTG TCGCACGGCC GGCCGGAGCG TATGCATGTA 1523

AACGAAAAAA GAGAGGTCAT GGGGCATGGC TCCCGTCAG 1562

[Sequence list]

SEQ ID NO .: 2

Length of sequence: 217

Type of sequence: amino acid

Number of chains: 1 chain

Topology: linear

Type of sequence: protein

origin

Biology: Bacillus Species

Main Name: DS1152 (KFCC11026)

order:

A Met Ala Asn His Ile Ty Leu Ala Gly Asp Ser Thr Val Gln Thr Tyr 16

B Met Ala Asn His Ile Ty Leu Ala Gly Asp Ser Thr Val Gln Thr Tyr 16

A Gly Asp Ser Thr Asn Gln Gly Gly Trp Gly Gln Phe Leu Gly Ser His 32

B Gly Asp Ser Thr Asn Gln Gly Gly Trp Gly Gln Phe Leu Gly Ser His 32

A Leu Pro Glu His Ile Gln Val Ile Asn Arg Ala Ile Gly Gly Arg Ser 48

B Leu Pro Glu His Ile Gln Val Ile Asn Arg Ala Ile Gly Gly Arg Ser 48

A Ser Lys Thr Phe Val Glu Glu Gly Arg Leu Gln Ala Ile Leu Asp Val 64

B Ser Lys Thr Phe Val Glu Glu Gly Arg Leu Gln Ala Ile Leu Asp Val 64

A Ile Glu Pro Asp Asp Trp Leu Phe Val Gln Met Gly His Asn Asp Ala 80

B Ile Glu Pro Asp Asp Trp Leu Phe Val Gln Met Gly His Asn Asp Ala 80

A Ser Lys Asn Lys Pro Glu Arg Tyr Thr Glu Pro Tyr Thr Thr Tyr Lys 96

B Ser Lys Asn Lys Pro Glu Arg Tyr Thr Glu Pro Tyr Thr Thr Tyr Lys 96

A Gln Tyr Leu Lys Gln Tyr Ile Ala Gly Ala Arg Glu Lys Gly Ala His 112

B Gln Tyr Leu Lys Gln Tyr Ile Ala Gly Ala Arg Glu Lys Gly Ala His 112

A Pro Leu Leu Ile Thr Pro Val Ala Arg Phe His Tyr Glu Asn Asp Met 128

B Pro Leu Leu Ile Thr Pro Val Ala Arg Phe His Tyr Glu Asn Gly Val 128

A Phe Leu Asn Asp Phe Pro Asp Tyr Cys Ile Ala Met Lys Gln Thr Ala 144

B Phe Leu Asn Asp Phe Pro Asp Tyr Cys Ile Ala Met Lys Gln Thr Ala 144

A Ala Glu Glu Asn Val Gln Leu Ile Asp Leu Met Glu Lys Ser Leu Ala 160

B Ala Glu Glu Asn Val Gln Leu Ile Asp Leu Met Glu Lys Ser Leu Ala 160

A Phe Phe Thr Glu Lys Gly Glu Glu Lys Val Tyr Thr Tyr Phe Met Val 176

B Phe Phe Thr Glu Lys Gly Glu Glu Lys Val Tyr Thr Tyr Phe Met Ile 176

A Ser Glu Gly Ile Asn Asp Tyr Thr His Phe Thr Lys Lys Gly Ala Asn 192

B Ser Glu Gly Ile Asn Asp Tyr Thr His Phe Thr Lys Lys Gly Ala Asn 192

A Glu Met Ala Lys Leu Val Ala lys Gly Ile Lys Glu Leu Gly Leu Pro 208

B Glu Met Ala Lys Leu Val Ala lys Gly Ile Lys Glu Leu Gly Leu Pro 208

A Leu Thr Glu Ser Ile Ile Lys Glu Gly *** 217

B Leu Thr Glu Ser Ile Ile Lys Glu Arg *** 217

In the above table, the upper amino acid sequence (A) is the amino acid sequence of the present invention, and the lower amino acid sequence (B) is the amino acid sequence estimated from the gene of the Bacillus subtilis genomic region searched by the gene bank.

The present invention mass-produced new enzymes from microorganisms by genetic recombination. The enzyme produced produces diacetyl cephalosporin C and diacetyl7-ACA, which are essential precursors for the preparation of antibiotics of the cephalosporin family, by enzymatic methods.

By using the enzyme of the present invention in the deacetylation reaction of cephalosporin C and 7-ACA, it is possible to remove and shorten some of the existing multi-step chemical synthesis processes, and to prevent the formation of reaction by-products and decomposition of intermediates. It can contribute to the improvement of production yield.

Claims (11)

Cephalosporin deacetylase structural gene represented by the DNA base sequence of SEQ ID NO: 1 Recombinant vector comprising the cephalosporin deacetylase structural gene of claim 1 E. coli KFCC11029 comprising the recombinant vector of claim 2 The protein of SEQ ID NO: 2 (A) produced by the transformant E. coli KFCC11029 of claim 3 Bacillus DS1152 (KFCC11026) with DNA fragment of SEQ ID NO: 1 and cephalosporin deacetylase activity 5. The protein according to SEQ ID NO: 2 (A) produced by the transformant E. coli KFCC11029, characterized in that the amino acid sequence containing methionine at the amino terminus and the mutant protein with amino acid partially substituted. The protein according to SEQ ID NO: 2 (A) produced by the transformant Escherichia coli KFCC11029 according to claim 4, which is an enzyme composed of a duplex or a multimer. The protein according to SEQ ID NO: 2 (A) produced by the transformant Escherichia coli KFCC11029 according to claim 4, which is a protein construct used for the cephalosporin deacetylation reaction. The protein according to SEQ ID NO: 2 (A) produced by the transformant E. coli KFCC11029 according to claim 4, which is an enzyme used for the cephalosporin deacetylation reaction. The protein according to SEQ ID NO: 2 (A) produced by the transformant E. coli KFCC11029, according to claim 8, which is an enzyme immobilized on a carrier and used for the cephalosporin deacetylation reaction. E. coli KFCC11029 was incubated at 37 ° C for 12 hours with glucose 0.2%, enzyme extract 3%, NaCl 0.25%, and pH 7.2 as the medium. After culturing under conditions, injecting additional medium so that the final concentration was 5.6% of enzyme extract, 0.4% of NaCl, and 3.4% of glucose at the time when the sugar was depleted, 500uM when reaching the maturity stage on the growth curve of the cells. Method for producing cephalosporin deacetylase by inducing gene expression by adding IPTG.