Cephalosporin c acylase catalyzed acylation reaction and use thereof

Abstract

The application discloses a cephalosporin C acylase and a mutant thereof derived from Roseomonas rosea and application of the mutant in synthesis of a cephalosporin parent nucleus. The mutant of the cephalosporin C acylase disclosed by the application is substituted with one or more than one substitution mutation at L158, L160, G163, R260, Y268, M280, F294, V300, H306, E317, C389, H414 and A418 of the wild-type acylase. The obtained acylase can catalyze the reaction of synthesizing 7-aminocephalosporanic acid from cephalosporin C, and has a good industrial application prospect in synthesis of a cephalosporin intermediate by a biological enzyme method.

Description

Technical Field

[0001] This invention belongs to the field of biocatalysis technology and relates to a method for using a cephalosporin C acylase mutant and its application in the catalytic synthesis of cephalosporin nuclei. Background Technology

[0002] 7-Aminocephalosporanic acid (7-ACA) is a very important intermediate in the synthesis of antibiotics. It can be used to synthesize a variety of cephalosporin antibiotics and has significant medical value.

[0003] Currently, 7-ACA is produced by the cleavage of CPC, the fermentation product of Cephalosporium, mainly through two methods: The traditional process primarily uses a chemical method, involving multiple steps such as carboxyl protection, amide bond breaking, and deprotection of CPC to produce 7-ACA. The chemical cleavage method has been extensively studied and has achieved industrial-scale production. However, the chemical cleavage process is complex, requires large amounts of organic solvents, and causes significant environmental pollution. Driven by the huge market demand for 7-ACA and increasing emphasis on environmental protection, the chemical cleavage method is gradually being replaced by environmentally friendly bio-enzymatic catalysis. The bio-enzymatic production of 7-ACA from CPC has been extensively developed since the late 1960s, mainly including two-step and one-step enzymatic methods. The two-step enzymatic method utilizes D-amino acid oxidase (DAO) and glutaryl-7-aminocephalosporanic acid acylase (GAC) for stepwise catalysis. First, DAO catalyzes the synthesis of glutaryl-7-aminocephalosporanic acid (GL-7-ACA) from CPC, followed by the formation of 7-ACA. Although the process is relatively mature, it still has disadvantages such as a long enzymatic hydrolysis route, difficulty in controlling oxidation conditions, numerous byproducts, and lower yields than chemical cleavage methods. The one-step enzymatic method utilizes CPC acylase to directly catalyze the synthesis of 7-ACA from CPC, eliminating the need for the intermediate GL-7-ACA conversion process. The reaction efficiency is equivalent to that of the chemical method. However, it still has problems such as low specificity of CPC acylase for the substrate CPC, low enzyme activity, difficulty in enzyme extraction, and the need for further improvement in enzyme immobilization. Summary of the Invention

[0004] The technical problem to be solved by the present invention is how to efficiently prepare cephalosporin nucleus 7-ACA using a biocatalytic method.

[0005] To address the above technical problems, the present invention first provides an acylase mutant, wherein the wild-type acylase is derived from Roseomonasrosea and named E745. The cephalosporin C acylase mutant is a protein obtained by substitution mutations at positions L158, L160, G163, R260, Y268, M280, F294, V300, H306, E317, C389, H414, and A418 on the wild-type sequence (SEQ ID NO: 1).

[0006] In the above method, the mutant cephalosporin C acylase can be a protein obtained by mutating E745 with any one, two, three, four, five, six, or all of the following six types of mutations:

[0007] (1) The leucine at position 158 of the amino acid sequence shown in SEQ ID NO: 1 is mutated and replaced with alanine, glycine, glutamine, or serine;

[0008] (2) Mutate the leucine at position 160 of the amino acid sequence shown in SEQ ID NO: 1 to cysteine;

[0009] (3) The glycine at position 163 of the amino acid sequence shown in SEQ ID NO: 1 is mutated to glutamine and threonine;

[0010] (4) Mutate the arginine at position 260 of the amino acid sequence shown in SEQ ID NO: 1 to leucine, methionine or isoleucine;

[0011] (5) Mutate the tyrosine at position 268 of the amino acid sequence shown in SEQ ID NO: 1 to alanine;

[0012] (6) The methionine at position 280 of the amino acid sequence shown in SEQ ID NO: 1 is mutated to arginine, glutamic acid, phenylalanine, or isoleucine;

[0013] (7) Mutate the valine at position 300 of the amino acid sequence shown in SEQ ID NO: 1 to leucine;

[0014] (8) Mutate the histidine at position 306 of the amino acid sequence shown in SEQ ID NO: 1 to alanine, threonine, isoleucine, lysine, or tyrosine.

[0015] (9) Mutate the glutamic acid at position 317 of the amino acid sequence shown in SEQ ID NO: 1 to threonine;

[0016] (10) The cysteine ​​at position 389 of the amino acid sequence shown in SEQ ID NO: 1 is mutated to isoleucine and serine;

[0017] (11) Mutate the histidine at position 414 of the amino acid sequence shown in SEQ ID NO: 1 to phenylalanine and valine;

[0018] (12) Mutate the alanine at position 418 of the amino acid sequence shown in SEQ ID NO: 1 to valine and glutamine.

[0019] (13) Preferably, it is a mutant with the following combination of mutations: L159G / V300L, L160C / V300L, L158S / A418V, L158Q / A418V, G163T / A418V, C389S / A418V, V300L / C389I, L160C / C389S, C389S / A418Q, L160C / C389I / A418V, L158S / C389I / A418Q.

[0020] The present invention correspondingly provides a nucleic acid molecule encoding the above-mentioned cephalosporin C acylase mutant, 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] The recombinant vector is a vector obtained by inserting the nucleic acid molecule into an expression vector.

[0025] 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.

[0026] The present invention also provides another method for preparing cephalosporin nuclei, the method comprising: using a cephalosporin analogue as a substrate, and catalyzing a reaction using the cephalosporin C acylase mutant, or a recombinant cell of the cephalosporin C acylase mutant, or the lysis product of the recombinant cell, to obtain a cephalosporin nucleus.

[0027] Specifically, a method for preparing compound II is characterized by comprising a catalytic reaction using compound I as a substrate and the cephalosporin C acylase mutant as described in any one of claims 1 to 2 as a catalyst, to obtain compound II:

[0028]

[0029] Where R represents acetoxy, hydroxyl, or hydrogen.

[0030] Preferably, the acylase of the present invention is an immobilized enzyme, for example, 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.

[0031] In the above method, the recombinant cells can be obtained by introducing a recombinant vector that can express the cephalosporin C acylase mutant into biological cells.

[0032] The biological cells may be microorganisms. The microorganisms may be *Escherichia coli* or other bacteria. In one embodiment of the present invention, the microorganism is *Escherichia coli* BL21(DE3).

[0033] The reaction temperature can be 10–40°C, specifically 20°C; the reaction is carried out in a phosphate buffer solution with a pH of 6.5–9.5, specifically pH 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.

[0034] This invention constructs a mutant library through semi-rational design, obtaining mutants with significantly improved effects. The resulting mutants can catalyze the production of cephalosporin nuclei from cephalosporin derivatives, showing promising industrial application prospects in the biocatalytic preparation of cephalosporin nuclei. Attached Figure Description

[0035] Figure 1 This is a flowchart of the acylase-catalyzed hydrolysis reaction of cephalosporin C in this invention.

[0036] Figure 2 This is a standard curve of 7-ACA and CPC content in this invention.

[0037] Figure 3 These are the HPLC detection results of 7-ACA and CPC in this invention.

[0038] Figure 4 The CPC acylase E744 in this invention is used for the construction of substrate saturated mutant libraries and related residue sites. Detailed Implementation

[0039] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0040] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0041] Example 1. Acylase Sequence Mining and Enzyme Activity Characterization

[0042] 1.1 Acylase Sequence Mining

[0043] An NCBI search revealed an acylase derived from Roseomonasrosea, whose amino acid sequence is shown in SEQ ID NO.1 (named E745).

[0044] 1.2 Induced expression of cephalosporin C acylase

[0045] The nucleic acid sequence containing the amino acid sequence of SEQ ID NO.1 was transformed into Escherichia coli BL21(DE3) bacterial culture and evenly spread on kanamycin-resistant (concentration of 50 μg / mL) LB plates. After incubation at 37℃ for 14 h, single colonies grew, which were the transformants containing the cephalosporin C acylase nucleic acid sequence.

[0046] The transformants were transferred to test tubes containing 5 mL of LB medium (kanamycin concentration 50 μg / mL) and cultured overnight at 37°C with shaking at 220 rpm to obtain the seed culture. Then, 1% of the seed culture was inoculated into 100 mL of TB medium (kanamycin concentration 50 μg / mL) and cultured at 37°C with shaking at 220 rpm for 3 hours. The culture was then incubated until the OD500 of the transformed culture was reached. 600 When the pH reaches 0.6-0.8, add IPTG to a final concentration of 0.1 mM and continue incubation at 25°C and 220 rpm with shaking for 16 h; collect the bacterial cells by centrifugation at 4°C and 4000 rpm for 10 min. Wash the cells with phosphate buffer (50 mM, pH 8.0), centrifuge, and then resuspend in phosphate buffer (10 mL / g of bacteria) to obtain a bacterial suspension. After sonication for 15 min, centrifuge for 60 min (4°C, 12000 rpm) and collect the supernatant to obtain the crude enzyme solution.

[0047] 1.3 Purification of Acylase Protein

[0048] 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.

[0049] The concentration of the purified target protein was determined using the Bradford method. The target protein was analyzed using SDS-PAGE gel electrophoresis.

[0050] 1.4. Cephalosporin C acylase catalyzes the formation of 7-ACA from CPC.

[0051] Reaction system (1 mL): 40 mM CPC, 0.1 mg acylase, phosphate buffer added to 1 mL. Reaction conditions: 37℃, 1000 rpm, 30 min. Take 100 μL of sample, add 9 times the volume of methanol to terminate the reaction and dilute the system. Detect enzyme activity by HPLC. Analyze the CPC-converted 7-ACA product by HPLC.

[0052] 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℃.

[0053] 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.

[0054] Table 1. Acylase activity assay

[0055]

[0056] Example 2. Construction and high-throughput screening of acylase mutant library

[0057] 2.1 Construction of the mutation library

[0058] Using acylase E745 as a template, modeling was performed using SWISS-MODEL. After modeling, the protein was docked with the substrate molecule CPC, and its binding sites were analyzed. According to the docking results, sites L158, L160, G163, H414, A418, and M280 in the E745 amino acid sequence play a decisive role in binding to the substrate CPC, as these are amino acids near the pocket and affect the CPC binding measurement. Sites R260, Y268, F294, V300, H306, E317, and C389 play an important role in stabilizing the substrate binding pocket. Therefore, a single-point saturation mutant library of the above-mentioned amino acid residue sites was constructed.

[0059] Using the selected primers, a single-point saturation mutation was performed, and the corresponding primers were designed as shown in Table 2.

[0060] Table 2. Primer sequence listing

[0061]

[0062]

[0063]

[0064] The specific construction method is as follows:

[0065] 1. First round of PCR

[0066] Using plasmid E745 as a template, primers L158-F1, L1598-F2, L158-F3, and L158-F4 from Table 2 were mixed in a 12:6:1:1 ratio. 2 μL of this mixture was used as the upstream primer, and L158-G163-R from Table 2 was used as the downstream primer to amplify gene fragment L1. The amplification methods for sites L160 and G163 were the same as for gene fragment L1. For sites R260, Y268, M280, F294, V300, H306, and E317, R260-E317-R was used as the downstream primer; for sites C389, H414, and A418, C389-A418-R was used as the downstream primer for gene fragment amplification.

[0067] The first round of PCR program was as follows: 28 cycles of incubation at 94℃ for 2 min, followed by incubation at 98℃ for 15 s, 55℃ for 30 s, and 72℃ for 30 s, and then incubation at 72℃ for 5 min.

[0068] 2. Second round of PCR

[0069] Using 2 μL of the first-round PCR products L1, L2, L3, and L4 as primers and plasmid E745 as a template, a second-round PCR was performed to construct a single-site saturated mutant library containing L158, L160, G16, R260, Y268, M280, F294, V300, H306, E317, C389, H414, and A418. The second-round PCR program was: 28 cycles of 94℃ for 2 min, (98℃ for 15 s, 60℃ for 30 s, and 72℃ for 5 min), followed by a final incubation at 72℃ for 10 min.

[0070] 3. Construction of engineered mutant strains

[0071] The obtained second-round PCR product was processed as follows: 1 μL of Dpn I was added to the second-round PCR product to digest the plasmid template, and the mixture was treated at 37°C for 3 hours. 2 μL of the enzyme-digested second-round 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 hours. After the growth of a single colony, this was identified as the engineered strain of the acylase E745 gene mutant.

[0072] 2.2 High-throughput screening and activity assay of acylase mutant libraries

[0073] Single colonies from the mutant library were inoculated into 96-well plates and cultured in 300 μL of LB medium (containing 50 μg / mL kanamycin) at 37°C for 8–12 h. A negative control group (empty vector of pET-28a) was also included. Then, 200 μL of seed culture was inoculated into 800 μL of TB medium (containing 0.1 mM IPTG and 50 μg / mL kanamycin). Expression was performed at 25°C for 16 h. Cells were collected by centrifugation, frozen at -80°C for 1 h, and thawed at room temperature for 30 min. 200 μL of potassium phosphate buffer (0.1 M pH 8.0, containing 1 mg / mL lysozyme) was added to resuspend the cells, and incubated at 37°C for 1 h. 200 μL of substrate CPC (20 mM) was added to the cell lysis buffer, and the reaction was carried out at 37°C for 4–6 h. Transfer 40 μL of the reaction solution to a new 96-well plate, add 200 μL of stop reaction solution (20% glacial acetic acid: 0.05M NaOH = 2:1), and centrifuge for 10 min (4℃, 5000 rpm). Take 200 μL of the supernatant, add 40 μL of colorimetric reagent (0.5% p-DAB dissolved in methanol), react at room temperature for 10 min, and then measure the absorbance at 415 nm.

[0074] Table 3. Relative viability test of random mutant strains

[0075]

[0076] Example 3. Construction of acylase combinatorial mutants

[0077] Based on the high-activity mutants screened in Example 2, and the mutants with significant regional influence, the following mutants were constructed: L159G / V300L, L160C / V300L, L158S / A418V, L158Q / A418V, G163T / A418V, C389S / A418V, V300L / C389I, L160C / C389S, C389S / A418Q, L160C / C389I / A418V, and L158S / C389I / A418Q. Primers for constructing the mutants are shown in Table 4.

[0078] Table 4. Primers for site-directed mutagenesis

[0079]

[0080] The specific construction method is as follows:

[0081] 1) PCR

[0082] Using plasmids L158G and L160C as templates, and V300L-F and V300L-R from Table 4 as upstream and downstream primers, respectively, the mutants L159G / V300L and L160C / V300L were amplified. Using plasmids L158S, L158Q, G163T, and C389S as templates, and A418V-F and A418V-R from Table 4 as upstream and downstream primers, the mutants L158S / A418V, L158Q / A418V, G163T / A418V, and C389S / A418V were amplified. Using plasmid L160C as a template, and C389S-F and C389S-R from Table 4 as upstream and downstream primers, the mutant L160C / C389S was amplified. Using plasmid V300L as a template, C389I-F and C389I-R from Table 4 were used as upstream and downstream primers to amplify the mutant V300L / C389I; using plasmid C389S as a template, A418Q-F and A418Q-R from Table 4 were used as upstream and downstream primers to amplify the mutant C389S / A418Q; using plasmid L160C as a template, C389I-F and A418V-R from Table 4 were used as upstream and downstream primers to amplify the mutant L160C / C389I / A418V; using plasmid L158S as a template, C389I-F and A418Q-R from Table 4 were used as upstream and downstream primers to amplify the mutant L158S / C389I / A418Q.

[0083] 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.

[0084] 2) Construction of engineered mutant strains

[0085] 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 mutant strain of the acylase SEQ ID NO.1.

[0086] Example 4. Whole-cell catalytic synthesis of 7-ACA by cephalosporin C acylase

[0087] 4.1 Cell Culture

[0088] Single clones of the mutants constructed in Example 3 were transferred to test tubes containing 5 mL of LB medium (kanamycin concentration of 50 μg / mL) and cultured overnight at 37°C with shaking at 220 rpm to obtain the seed culture. 1% of this seed culture was then inoculated into 100 mL of TB medium (kanamycin concentration of 50 μg / mL) and cultured at 37°C with shaking at 220 rpm for 3 hours. The culture was then incubated until the OD500 of the bacterial culture was... 600 When the pH reaches 0.6-0.8, add IPTG (0.1 mM) and continue incubation at 25°C and 220 rpm with shaking for 16 h; collect the bacterial cells by centrifugation at 4°C and 4000 rpm for 10 min. Wash the bacterial cells with phosphate buffer (50 mM pH 8.0), centrifuge, weigh the wet bacterial cells, and then resuspend them in phosphate buffer (10 mL / g bacteria) to obtain a bacterial suspension.

[0089] 4.2 Whole-cell response

[0090] The conversion rate of the whole cells obtained in step 4.1 was determined by analyzing the CPC conversion product using HPLC. The reaction was carried out in a 1 mL system. 800 μL of the whole cells obtained in step 4.1 was used for the reaction, and the substrate CPC (40 mM) 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 methanol, and the conversion rate was determined by HPLC.

[0091] HPLC detection conditions: The selected column was an Agilent ZORBAX SB-C18 (4.6*150mm 4um), the mobile phase ratio was 96% phosphate buffer (pH 7.0, 50mM), 4% methanol (v / v), the flow rate was 0.8mL / min, the detection wavelength was 254nm, and the column temperature was kept constant at 25℃.

[0092] Table 5. Transformation rate of CPC in whole cells of the combined mutant

[0093]

[0094] 4.3 Crude enzyme solution catalyzes the synthesis of 7-ACA from CPC

[0095] 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.

[0096] 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.

[0097] 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℃.

[0098] Table 6. Conversion rate of crude enzyme to CPC by combined mutants

[0099]

[0100] Example 5: Cephalosporin C acylase mutant catalyzes whole-cell synthesis of 7-ADCA from DAOC.

[0101] DAOC is an analogue of CPC, the difference in structure being the substituents on the side chains of the six-membered ring of the parent nucleus. In DAOC, the substituent is hydrogen (-H), while in CPC, it is acetoxy (-OCOCH3).

[0102]

[0103] 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.

[0104] 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℃.

[0105] Table 7. Conversion rate of DAOC by cephalosporin C acylase mutant.

[0106]

[0107] Example 7 Immobilization of cephalosporin C acylase

[0108] (1) Immobilization of acylase mutants with epoxy resin and macroporous adsorption resin (taking L158S / C389I / A418Q as an example)

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] (2) Amino resin immobilized enzyme treatment

[0114] 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.

[0115] 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.

[0116] (3) Immobilized enzyme activity test

[0117] 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.

[0118] 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℃. The conversion rate reached 65%.

Claims

1. A cephalosporin C acylase mutant, characterized in that: It is based on the amino acid sequence shown in SEQ ID NO: 1, with only the following mutations: C389S, C389I, C389S / A418V, L160C / C389S, C389S / A418Q, L160C / C389I / A418V or L158S / C389I / A418Q.

2. The biological material related to the cephalosporin C acylase mutant according to claim 1 is any one of (1) to (4) below: (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); (4) Recombinant microorganisms containing the nucleic acid molecules described in (1).

3. A method for preparing compound II, characterized in that, The reaction involves using compound I as a substrate and the cephalosporin C acylase mutant of claim 1 as a catalyst to catalyze the reaction, yielding compound II: I； II; Where R represents acetoxy, hydroxyl, or hydrogen.

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

5. The method according to claim 4, characterized in that: The recombinant cells are achieved by introducing a recombinant vector into microbial cells that can express the cephalosporin C acylase mutant of claim 1.

6. The method according to claim 5, characterized in that: The cephalosporin C acylase mutant as described in claim 1 catalyzes enzymes in the form of whole cells, crude enzyme solution, crude enzyme powder, immobilized enzyme, or pure enzyme.

7. The method according to claim 5, characterized in that: The microbial cells mentioned are yeast, bacteria, or fungi.

8. The method according to claim 7, characterized in that: The microbial cells mentioned are Escherichia coli.

9. The method according to claim 8, characterized in that: The Escherichia coli mentioned is BL21(DE3).

10. The method according to claim 3, characterized in that: The reaction using the cephalosporin C acylase mutant as a substrate to generate the corresponding product was carried out in a phosphate buffer solution with a concentration of 40 mmol / L to 60 mmol / L and a pH of 6.5 to 9.

5.

11. The method according to claim 10, characterized in that: The temperature of the catalytic reaction is 20–40°C; the time of the catalytic reaction is 0.5–28 h.

Images