Artificial synthesis of cephalosporin c acylase and its mutant applications

Abstract

The application discloses a synthetic cephalosporin C acylase and a mutant gene, an encoding protein and an application thereof. The amino acid sequence of the cephalosporin C acylase is shown as SEQ ID NO. 1. The synthetic cephalosporin C acylase is used for catalyzing synthesis of a cephalosporin C intermediate of cephalosporin drugs, and a mutant is obtained through rational design and protein modification. The cephalosporin C acylase and the mutant can catalyze a cephalosporin analogue to generate a key intermediate mother nucleus of the cephalosporin drugs, and provide a novel acylase for biologically enzymatic preparation of the cephalosporin drugs.

Description

Technical Field

[0001] This invention relates to the application of an artificially synthesized cephalosporin C acylase and its mutant in the synthesis of cephalosporin nuclei, belonging to the field of biotechnology. Background Technology

[0002] 7-Aminocephalosporinic acid (7-ACA) is one of the important intermediates for cephalosporin (β-lactam) drugs. Due to the advantages of cephalosporin antibiotics, such as broad antibacterial spectrum, good efficacy, low toxicity, no cross-resistance with other antibiotics, and low cross-allergic reactions with penicillin, they have become the most important and effective drugs in anti-infective therapy in recent years. Cephalosporin C (CPC) can be produced through bio-fermentation and chemical refining, followed by chemical catalysis or enzymatic hydrolysis to obtain 7-ACA. Antibiotics with high market value abroad, such as cefadroxil, cefadroxil, and cefaclor, currently use 7-ACA as an intermediate in their synthesis. The chemical synthesis of 7-ACA is lengthy, involves many steps, requires harsh reaction conditions, and generates a large amount of waste, and is therefore rarely used now. Currently, enzymatic synthesis of 7-ACA mainly utilizes cephalosporin C acylase, but the synthesis technology still needs improvement, and both yield and quality need to be increased. With the increasing value of cephalosporin C acylase in industrial applications, improving enzyme activity and yield has become a growing concern for researchers. As a key enzyme in the enzymatic production of 7-ACA, in-depth research on the fermentation methods of genetically engineered cephalosporin C acylase strains has significant theoretical and social value.

[0003] Enzymatic production of 7-ACA from CPC mainly includes two-step and one-step enzymatic methods. The two-step method involves converting CPC to glutaryl-7-aminocephalosporinic acid (GL-7-ACA) using D-amino acid oxidase (DAAO), followed by the conversion of GL-7-ACA to 7-ACA using GL-7-ACA acylase. This process suffers from drawbacks such as a long enzymatic hydrolysis route, difficulty in controlling oxidation conditions, numerous byproducts, and lower yields compared to chemical cleavage. Therefore, the one-step enzymatic method, which utilizes CPC acylase to catalyze the hydrolysis of the CPC amide bond, is more commonly used. However, the cephalosporin C acylase strains found in nature that can hydrolyze CPC exhibit low activity. Therefore, there is an urgent need to discover or develop new cephalosporin C acylases with high activity and good stability for industrial application. Summary of the Invention

[0004] The primary objective of this invention is to provide a synthetically produced cephalosporin C acylase and its mutants. The gene sequence of the cephalosporin C acylase is shown in SEQ ID NO: 1.

[0005] The cephalosporin C acylase mutant is a protein obtained by mutating the sequence shown in SEQ ID NO: 1 at positions L161, H296, H309, E320, and A421.

[0006] Proteins obtained by mutating the cephalosporin C acylase mutant with any one, two, three, four, five, or all of the following six mutants:

[0007] (1) Mutate leucine at cephalosporin C acylase 161 to alanine, glutamine, and serine;

[0008] (2) Mutate histidine at position 296 of cephalosporin C acylase to leucine, serine, glutamic acid, or threonine;

[0009] (3) Mutate histidine at position 309 of cephalosporin C acylase to alanine and cysteine;

[0010] (4) Mutate the glutamic acid at position 320 of cephalosporin C acylase to threonine;

[0011] (5) Mutate the alanine at position 421 of cephalosporin C acylase to valine or phenylalanine;

[0012] Preferably, it is a mutant with the following combined mutations: L161A / E320T, L161Q / A421F, L161S / A421V, H296T / A421V, H296S / A421F, H296E / H309A, H296L / H309C, E320T / A421F, L161A / E320T / A421F, L161Q / H296L / H309C / A421F.

[0013] This invention discloses the encoding gene of the cephalosporin C acylase and its mutants, and their applications.

[0014] The present invention correspondingly provides a nucleic acid molecule encoding the above-mentioned mutant cephalosporin C acylase, its expression cassette, a recombinant vector containing the encoding gene or its expression cassette, and a recombinant microorganism containing the gene or the expression cassette or the recombinant vector.

[0015] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0016] The aforementioned nucleic acid expression cassette refers to DNA capable of expressing the mutant cephalosporin C acylase in host cells. This DNA may include not only a promoter to initiate transcription of the gene encoding the mutant cephalosporin C acylase, but also a terminator to terminate transcription of the mutant cephalosporin C acylase. Furthermore, the expression cassette may also include an enhancer sequence.

[0017] The recombinant vector for the nucleic acid molecule may be a bacterial plasmid (such as an expression vector based on the T7 promoter expressed in bacteria, specifically pET-28a), bacteriophage, yeast plasmid (such as YEp series vectors), or retroviral packaging plasmid carrying the gene encoding the mutant cephalosporin C acylase.

[0018] The recombinant vector is a vector obtained by inserting the nucleic acid molecule into an expression vector.

[0019] The recombinant microorganism containing the nucleic acid molecule encoding the mutant cephalosporin C acylase can be a yeast, bacteria, algae, or fungus carrying a gene encoding cephalosporin or the mutant cephalosporin protein, such as Escherichia coli.

[0020] This invention also provides a method for preparing a cephalosporin nucleus, the method comprising: using a cephalosporin analogue as a substrate, and catalyzing a reaction using the cephalosporin C acylase, or a cephalosporin C acylase mutant, or recombinant cells thereof, or the lysis products of the recombinant cells, to obtain a cephalosporin nucleus. Specifically, the reaction catalyzes the formation of compound II from compound I:

[0021]

[0022] Where R represents -OCOCH3 and H.

[0023] Preferably, the present invention employs an immobilized enzyme method, such as resin immobilization of acylase, more specifically using epoxy resin, amino resin, or macroporous adsorption resin as a carrier to immobilize the acylase, thereby obtaining an immobilized enzyme with high conversion activity.

[0024] In the above method, the recombinant cells can be obtained by introducing a recombinant vector capable of expressing the mutant cephalosporin C acylase into biological cells. The biological cells can be microorganisms. The microorganisms can be *Escherichia coli* or other bacteria. In one embodiment of the present invention, the microorganism is *Escherichia coli* BL21(DE3).

[0025] The reaction temperature can be 10-40℃, specifically 20℃; the reaction is carried out in a phosphate buffer solution with a pH of 6.5-9.5, specifically 8.0. The reaction time is generally 0.5-24 hours, specifically 1 hour. The lysis products of the recombinant cells can be obtained by lysing the recombinant cells.

[0026] The artificially synthesized cephalosporin C acylase provided by this invention can be used to catalyze the synthesis of cephalosporin nuclei from cephalosporins. The resulting mutant can catalyze the production of cephalosporin nuclei from cephalosporin analogs, providing a new approach for the preparation of cephalosporin nuclei by biological enzymatic methods.

[0027] The artificially synthesized cephalosporin C acylase disclosed in this invention is the first discovery that can be used to catalyze the synthesis of cephalosporin nuclei. Furthermore, through rational design, this invention modifies proteins and constructs mutants to obtain mutants that can catalyze the production of cephalosporin nuclei from cephalosporin analogs, providing a novel acylase for the bioenzymatic preparation of cephalosporin nuclei. Attached Figure Description

[0028] Figure 1 This is a flowchart of the acylase-catalyzed hydrolysis reaction of cephalosporin C in this invention.

[0029] Figure 2 These are the HPLC detection results of 7-ACA and CPC in this invention.

[0030] Figure 3 In this invention, CPC acylase E742 is used to construct relevant residue sites in mutants. Detailed Implementation

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0032] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0033] Example 1: Acylase Sequence Mining and Enzyme Activity Characterization

[0034] 1.1 Acylase Sequence Mining and Artificial Synthesis

[0035] Potential sequences were mined from the National Center for Biotechnology Information (NCBI) database using BLAST tools through sequence alignment. Based on different species origins and sequence similarity (50%-85%), 125 sequences were selected, and a common amino acid sequence (E742, SEQ ID NO.1) was generated from these. The nucleotide sequence of the E742 amino acid sequence was synthesized using whole-genome synthesis, and the synthesized nucleotides were ligated into the pET-28 vector to obtain an expression vector containing the acylase gene.

[0036] 1.2. Induction, expression, and purification of cephalosporin C acylase

[0037] The expression vector described above was transformed into *Escherichia coli* BL21(DE3). The bacterial culture was evenly spread on LB agar plates containing kanamycin (50 μg / mL) and incubated at 37°C for 14 h. Single colonies grew after incubation, representing transformants containing the cephalosporin C acylase nucleic acid sequence. These transformants were then transferred to test tubes containing 5 mL of LB medium (50 μg / mL kanamycin) and incubated overnight at 37°C with shaking at 220 rpm to obtain the seed culture. This seed culture was then inoculated at a 1% inoculation rate into 100 mL of TB medium (50 μg / mL kanamycin) and incubated at 37°C with shaking at 220 rpm for approximately 3 h. When the OD of the bacterial culture reached a certain level... 600 When the pH reaches 0.6-0.8, add IPTG (0.1 mM) and incubate at 25℃ and 220 rpm for 16 h. Collect the bacterial cells by centrifugation for 10 min (4℃, 4000 rpm). Wash the bacteria with phosphate buffer (50 mM, pH 8.0), centrifuge, and then resuspend in phosphate buffer (10 mL / g bacteria) to obtain a bacterial suspension. After sonication for 15 min, centrifuge for 60 min (4℃, 12000 rpm) and collect the supernatant to obtain the crude enzyme solution.

[0038] 1.3 Purification of Acylase Protein

[0039] Using Ni + Column purification: After pre-equilibration with buffer A (100mM potassium phosphate, 500mM NaCl, 30mM imidazole, pH 8.0), the wild-type crude enzyme solution collected in step 1.2 was filtered and added to Ni. + The column was eluted with buffer B (100mM potassium phosphate, 500mM sodium chloride, 250mM imidazole, pH 8.0) and the target protein was collected. The protein was then concentrated by ultrafiltration, desalted and purified, and stored at 4°C.

[0040] The concentration of the purified target protein was determined using the Bradford method. The target protein was analyzed using SDS-PAGE gel electrophoresis.

[0041] 1.4 Cephalosporin C acylase catalyzes the formation of 7-ACA from CPC.

[0042] The activity of the acylase was tested using the mutant PM2 reported in the invention patent CN103937764B of Amex Technology Co., Ltd. as a control.

[0043] The activity of the pure enzyme obtained in step 1.3 was determined by analyzing the CPC conversion product using HPLC. The selected chromatographic column was an Agilent ZORBAX SB-C18 (4.6*150mm 4um). The reaction system consisted of 0.1 mg of pure enzyme and CPC substrate (40mM). The reaction was carried out at 37℃ and 1000 rpm for 30 min. 100 μL of the sample was taken, diluted 10 times with methanol, and the enzyme activity was detected by HPLC.

[0044] The HPLC mobile phase was prepared as follows: 96% phosphate buffer (pH 7.0, 50 mM), 4% methanol (v / v), flow rate 0.8 mL / min, detection wavelength 254 nm, and column oven temperature 25 °C.

[0045] Enzyme activity is defined as the amount of enzyme required to catalyze the production of 1 micromolar (μmol) 7-ACA per minute from CPC, which is defined as 1 U.

[0046] Table 1. Acylase activity assay

[0047]

[0048] Example 2: Construction of site-directed mutants of acylase

[0049] 2.1 Single-point mutation construction

[0050] Using acylase E742 as a template, SWISS-MODEL was used for modeling, followed by docking and substrate insertion modeling. Key action sites were analyzed, and the following single-point mutants were constructed: L161A, L161S, L161Q, H296T, H296S, H296E, H296L, H309A, H309C, E320T, A421V, and A421F.

[0051] Primers were designed to target mutation sites for site-directed mutagenesis, and the corresponding primers are shown in Table 2.

[0052] Table 2 Primer sequence list

[0053]

[0054] The specific construction method is as follows:

[0055] 1) PCR

[0056] Using 2 μL of primers L161Q-F, L161A-F, and L161S-F from Table 2 as upstream primers and L161-R from Table 2 as the downstream primer, amplify mutants L161Q, L161A, and L161S; using 2 μL of primers H296T-F, H296S-F, H296E-F, and H296L-F from Table 2 as upstream primers and H296-R from Table 2 as the downstream primer, amplify mutants H296T, H296S, H296E, and H296L; Use 2 μL of primers H309A-F and H309C-F from Table 2 as upstream primers and H309-R from Table 2 as downstream primers to amplify mutants H309A and H309C; use 2 μL of primer E320T-F from Table 2 as upstream primers and E320T-R from Table 2 as downstream primers to amplify mutant E320T; use 2 μL of primers A421V-F and A421F-F from Table 2 as upstream primers and A421V-R from Table 2 as downstream primers to amplify mutant A421V.

[0057] PCR program: 28 cycles of incubation at 94℃ for 2 min, (98℃ for 15 s, 55℃ for 30 s, 72℃ for 5 min), followed by incubation at 72℃ for 10 min.

[0058] 2) Construction of engineered mutant strains

[0059] The obtained PCR product was subjected to the following operations: 1 μL of Dpn I was added to the PCR product to digest the plasmid template, and the mixture was incubated at 37°C for 3 hours. 2 μL of the enzyme-digested PCR product was electroporated into Escherichia coli BL21(DE3), and then the bacterial culture was evenly spread on kanamycin-resistant (50 μg / mL) LB agar plates. After incubation at 37°C for 14 hours, a single colony grew, which was the mutant strain of acylase E742.

[0060] 2.2 Construction of acylase combinatorial mutants

[0061] Based on the mutants constructed in 2.1, and considering the interactions between the site and the substrate, as well as between the sites, the following combined mutants were constructed: L161A / E320T, L161Q / A421F, L161S / A421V, H296T / A421V, H296S / A421F, H296E / H309A, H296L / H309C, E320T / A421F, L161A / E320T / A421F, and L161Q / H296L / H309C / A421F. The primers used for mutant construction are shown in Table 2.

[0062] The specific construction method is as follows:

[0063] 1) PCR

[0064] Using plasmids L161S and H296T as templates, 2 μL of A421V-F and A421-R from Table 2 were used as upstream and downstream primers to amplify the mutants L161S / A421V and H296T / A421V, respectively. Using plasmids L161Q, H296S, and E320T as templates, 2 μL of A421F-F and A421-R from Table 2 were used as upstream and downstream primers to amplify the mutants L161Q / A421F, H296S / A421F, and E320T / A421F, respectively. Using plasmid L161A as a template, 2 μL of primers E320T-F and E320T-R from Table 2 were used as upstream and downstream primers to amplify the mutant L161A / E320T. Using plasmid H296E as a template, 2 μL of A421V-F and A421-R were used as upstream and downstream primers to amplify the mutants. Using 2 μL of primers H309A-F and H309-R from Table 2 as upstream and downstream primers, the mutant H296E / H309A was amplified; using plasmid H296L as a template, 2 μL of primers H309C-F and H309-R from Table 2 as upstream and downstream primers, the mutant H296L / H309C was amplified; using plasmid L161A as a template, 2 μL of primers E320T-F and A421F-R from Table 2 as upstream and downstream primers, the mutant L161A / E320T / A421F was amplified; using plasmid L161Q / A421F as a template, 2 μL of primers H296L-F and H309-R from Table 2 as upstream and downstream primers, the mutant L161Q / H296L / H309C / A421F was amplified.

[0065] PCR program: 28 cycles of incubation at 94℃ for 2 min, (98℃ for 15 s, 55℃ for 30 s, 72℃ for 5 min), followed by incubation at 72℃ for 10 min.

[0066] 2) Construction of engineered mutant strains

[0067] The obtained PCR product was subjected to the following operations: 1 μL of Dpn I was added to the PCR product to digest the plasmid template, and the mixture was treated at 37°C for 3 h. 2 μL of the enzyme-digested PCR product was then electroporated into *E. coli* BL21(DE3). The electroporated *E. coli* BL21(DE3) bacterial culture was evenly spread on kanamycin-resistant (50 μg / mL) LB agar plates and cultured at 37°C for 14 h. After the growth of a single colony, this was identified as the acylase E742 gene mutant strain.

[0068] Example 3. Synthesis of 7-ACA catalyzed by cephalosporin C acylase mutant

[0069] 3.1 Cell Culture

[0070] Single clones of the mutants constructed in Example 2 were picked and transferred into test tubes containing 5 mL of LB medium (kanamycin concentration of 50 μg / mL), and cultured overnight at 37°C and 220 rpm with shaking to obtain the seed culture.

[0071] Inoculate 1% of the culture medium into 100 mL of TB medium (kanamycin concentration 50 μg / mL), and incubate at 37°C with shaking at 220 rpm for about 3 hours. When the OD of the bacterial culture reaches a certain value... 600 When the value reaches 0.6-0.8, add IPTG (0.1mM) and continue to incubate at 25℃ and 220rpm for 16h with shaking.

[0072] Collect bacterial cells by centrifugation at 4℃, 4000 rpm for 10 min. Wash the bacterial cells with 50 mM pH 8.0 phosphate buffer, centrifuge, weigh the wet bacterial cells, and then resuspend them in phosphate buffer (10 mL / g bacteria) to obtain a bacterial suspension.

[0073] 3.2 Whole-cell response

[0074] The whole-cell conversion rate was determined by HPLC using an Agilent ZORBAX SB-C18 column (4.6*150mm 4um). The reaction was carried out in a 1 mL system: 800 μL of the whole cells obtained in step 3.1 was used for the reaction, and the substrate CPC (40 mM) was added. The reaction was carried out at 37℃ and 1000 rpm for 30 min. 100 μL of the sample was then diluted tenfold with methanol, and the conversion rate was determined by HPLC.

[0075] The HPLC mobile phase was prepared as follows: 96% phosphate buffer (pH 7.0, 50 mM), 4% methanol (v / v), flow rate 0.8 mL / min, detection wavelength 254 nm, and column temperature oven constant at 25 °C.

[0076] Table 3. Transformation rate of mutant cells to CPC

[0077]

[0078]

[0079] 3.3 Crude enzyme solution catalyzes the synthesis of 7-ACA from CPC

[0080] The crude enzyme solution was prepared according to the method in Example 1, and the conversion rate of the crude enzyme solution that yielded the mutant was verified.

[0081] Reaction system (1 mL): substrate CPC (40 mM), 0.1 mL crude acylase solution, and phosphate buffer added to 1 mL. Reaction conditions: 37 °C, 1000 rpm, 30 min. Take 100 μL of sample, dilute it tenfold with methanol, and determine the conversion rate by HPLC.

[0082] Detection conditions: Agilent ZORBAX SB-C18 column (4.6*150mm 4um); mobile phase: 96% phosphate buffer (pH 7.0, 50mM) with 4% methanol (v / v); flow rate: 0.8 mL / min; detection wavelength: 254 nm; column temperature: constant at 25℃.

[0083] Table 4. Conversion rate of crude enzyme to CPC by combined mutants

[0084]

[0085]

[0086] Example 4: Whole-cell catalytic hydrolysis of DAOC by cephalosporin C acylase mutant to synthesize 7-ADCA

[0087] DAOC is an analogue of CPC, and the only structural difference between them is the substituent in the six-membered ring side chain of the parent nucleus. In DAOC, the substituent is hydrogen (-H), while in CPC, it is acetoxy (-OCOCH3).

[0088]

[0089] The conversion efficiency of DAOC was obtained by analyzing the DAOC conversion product using HPLC. The reaction was performed in a 1 mL system. 800 μL of the whole cells obtained in step 3.1 were used for the reaction, and DAOC (1 mg / mL) was added. The reaction was carried out at 37°C and 1000 rpm for 30 min. 100 μL of the sample was then diluted tenfold with acetonitrile before the conversion efficiency was determined by HPLC.

[0090] Liquid chromatography detection conditions: Agilent ZORBAX SB-C18 column (4.6*150mm 4um); mobile phase: 95% ammonium formate, 5% acetonitrile; flow rate: 1 ml / min; detection wavelength: 254 nm; column temperature oven: constant at 25℃.

[0091] Table 5. Conversion rate of cephalosporin mutants to DAOC

[0092]

[0093]

[0094] Example 5. Synthesis of 7-ACA by immobilized acylase-catalyzed hydrolysis of CPC.

[0095] (1) Immobilized acylase mutant (H294S / A421F) on epoxy resin and macroporous adsorption resin

[0096] Vector equilibration: Wash three times repeatedly with immobilization buffer at a ratio of 1:5 (mass / volume ratio) of vector / buffer. After washing, dry the container.

[0097] Adjustment of the ratio of carrier to acylase: Dissolve the enzyme in immobilization buffer with a ratio of 1:4 (mass / volume ratio). This ratio can be further optimized between 1:1 and 1:4.

[0098] The enzyme-containing buffer and carrier were added to the reactor and reacted at 25°C and 800 rpm for 3 hours, after which stirring was stopped and the mixture was allowed to stand for 2 hours.

[0099] Immobilized enzymes were collected by filtration after immobilization, and the protein content in the supernatant was determined using a 280 nm absorption wavelength. The immobilization rate and yield were calculated. The enzymes were washed with distilled water, and the process was repeated three times. Subsequently, the reactivity of the epoxy resin-immobilized enzymes was detected using HPLC.

[0100] (2) Amino resin immobilized enzyme

[0101] Carrier equilibration: Immobilization buffer, wash repeatedly 3 times with a carrier / phosphate buffer solution (50mM, pH8.0) ratio of 1:5 (mass / volume ratio), filter and dry after washing.

[0102] Prepare a 2% glutaraldehyde solution using immobilization buffer. Add the carrier and glutaraldehyde buffer at a ratio of 1:4 (mass / volume ratio), and stir at 22°C and 180 rpm for 1 hour. Then wash the carrier three times with buffer, filter, and store at 4°C.

[0103] (3) Conversion rate test of immobilized enzyme catalytic reaction

[0104] Reaction conditions (total volume 4 ml): Immobilized acylase (400 mg) was added to the reaction flask, followed by substrate CPC (40 mM), and buffer was added to a final volume of 4 ml. The reaction was carried out at 37°C and 1000 rpm for 30 min. A 100 μL sample was taken, diluted tenfold with methanol, and the conversion rate was determined by HPLC.

[0105] The mobile phase was prepared as 96% phosphate buffer (pH 7.0, 50 mM) with 4% methanol (v / v); the flow rate was 1.0 mL / min; the detection wavelength was 254 nm; and the column temperature was kept constant at 25 °C. The conversion rate reached 69%. <110> Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences <120> Artificial Synthesis of Cephalosporin C Acylase and Application of its Mutants <160> 1 <170> PatentIn version 3.5 <210> 1 <211>774 <212> PRT <213>Artificial sequence <400>1 MTMAAKTDREALRAALPPLDGELRLPGLAAPVTVLRDAWGIPHIRAAGAADAFFALGFVHAQDRLFQMELTRRRALGRAAEWLGAAAAEADILARRLGMEAACRRDFAALGAEARAMLEAYAAGVNAFLASGAPLPVEYALLGATPEPWEGWHSIAVMRRLGLLMGSVWFKLWRAAALPVVGAENVAKLRYDDGGRDLLCIPPGAEAARWEADLAALAPAIAALLEAAGGDASDAAGGGSNNWAVGPARSATGRPVLAGDPHRVFEIPNMYAQHHLACDAFDMIGLTVPGVPGFPHFAHNGRVAYCVTHAFMDIHDLYLERFDAGAAHALFRGGWEPVRRRRERIAVRGGAPREFEVVETRHGPVIAGDPASGTALALRSVQFAETDLSFDCLPRMLRAGSVEALFEATRGWGLIDHNLVAADTAGHIGHLVRARVPRRPRENGWLPVPGWTGEHEWQGWIPHEEMPRVIDPPGGLIVTANNRVVADDHPDYLCTDCHPPYRARRIAERLRADPAFRVEDAAAIHADTLSPNALLLRARLAALPAPGEPAAAALRQRLLAWDGRMEAGSVAATAYIALRRALTRILAERSGLAALAGHPWLAVAPGVAPLNQLWWALPALLRADDAALLGGWSWDEALGAALAEAAAAPAARPWGEAHRPRFAHPLSALFPEAAALLDPPALPIGGDGDTVLANGLVASAGPAATYGALARYVFDVGNWENSRWAVFHGASGHPGSPHYADQHAAWAACRMVPMLYGWDAIEAEARARQELRPA 774

Claims

1. A synthetically produced cephalosporin C acylase mutant, characterized in that, Its amino acid sequence has only undergone the following amino acid mutations based on the amino acid sequence shown in SEQ ID NO:1: L161A, L161Q, L161S, L161Q+A421F, L161S+A421V, L161A+E320T, L161A+E320T+A421F, or L161Q+H296L+H309C+A421F.

2. The biological material according to any one of (1) to (4) of the cephalosporin C acylase mutant as claimed in claim 1: (1) A nucleic acid molecule encoding the cephalosporin C acylase mutant of claim 1; (2) An expression cassette containing the nucleic acid molecule described in (1); (3) A recombinant vector containing the nucleic acid molecule described in (1), or a recombinant vector containing the expression cassette described in (2); (4) A recombinant microorganism containing the nucleic acid molecule described in (1), or a recombinant microorganism containing the expression cassette described in (2), or a recombinant microorganism containing the recombinant vector described in (3).

3. A method for preparing compound II, comprising using compound I as a substrate and catalyzing a reaction with the cephalosporin C acylase mutant as described in claim 1 to obtain compound II: I； II; Where R stands for -OCOCH3 and H.

4. The method according to claim 3, characterized in that: The cephalosporin C acylase mutant as described in claim 1 was obtained through recombinant cell expression.

5. The method according to claim 4, characterized in that: This is achieved by introducing a recombinant vector into biological cells that can express the cephalosporin C acylase mutant as described in claim 1.

6. The method according to claim 5, characterized in that: The cephalosporin C acylase mutant as described in claim 1 catalyzes cephalosporin C acylase in the form of whole cells, crude enzyme solution, crude enzyme powder, immobilized enzyme, or pure enzyme; the whole cells are recombinant microorganisms containing nucleic acid molecules encoding the cephalosporin C acylase mutant as described in claim 1; the recombinant microorganism is Escherichia coli.

7. The method according to claim 6, characterized in that: The recombinant microorganism is Escherichia coli BL21(DE3).

8. The method according to claim 6, characterized in that: The catalytic reaction was carried out in phosphate buffer solutions with concentrations of 40 mmol / L to 60 mmol / L and pH values ​​of 6.5 to 9.

5.

9. The method according to claim 6, characterized in that: The temperature of the catalytic reaction is 20–40°C; the time of the catalytic reaction is 0.5–28 h.

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