Cephalosporin C acylase mutant

Abstract

The invention discloses a cephalosporin C acylase mutant SEQ ID NO: 3. The cephalosporin C acylase mutant has relatively high thermal stability and enzyme activity, can efficiently catalyze a substrate cephalosporin C to synthesize 7-aminocephalosporanic acid, and improves the production economy of the 7-aminocephalosporanic acid.

Description

Technical Field

[0001] The invention belongs to the technical field of biocatalysis, and in particular relates to a cephalosporin C acylase mutant and application thereof in synthesizing 7-aminocephalosporanic acid. Background Art

[0002] Cephalosporin antibiotics are an important class of antibiotics, occupying a large share in the antibiotic market and having a wide range of application value. 7-aminocephalosporanic acid (7-ACA) is one of the important nuclei for producing semi-synthetic cephalosporin antibiotics. It is obtained by removing the molecular side chain from cephalosporin C (Cephalosporin C, CPC) as a raw material. The early 7-ACA production process was mainly prepared by chemical cracking CPC, and the steps included esterification, chlorination, etherification and hydrolysis. However, the use of a large amount of toxic reagents in the reaction process will pollute the environment, and it involves harsh conditions such as low temperature, strong acid and strong alkali (Journal of Medical Chemistry. 1974, 17 (12): 1312-1314), which does not meet the policy requirements of environmental protection and energy conservation, and has been basically replaced by bioenzyme method.

[0003] The bioenzymatic method is divided into two-step enzymatic method, three-step enzymatic method and one-step enzymatic method. The two-step enzymatic method mainly uses D-amino acid oxidase (DAAO) and glutaryl-7-aminocephalosporanic acid (GL-7-ACA) acylase to catalyze CPC to generate GL-7-ACA under the action of DAAO, and then remove the side chain under the action of GL-7-ACA acylase to generate 7-ACA. The two-step enzymatic method has basically replaced the chemical method because of its advantages such as low energy consumption and relatively environmentally friendly process. However, there is a more serious problem in the two-step enzymatic process. The byproduct H 2 O 2 The degradation of CPC substrate results in the generation of more byproducts and the inactivation of biocatalysts, especially DAAO. The three-step enzymatic method is to add catalase to the two-step enzymatic method. The process is to co-immobilize DAAO and catalase and add them to the reaction system at the same time as the GL-7-ACA acylase immobilized enzyme, so that the catalase in situ hydrolyzes the H produced by DAAO. 2 O 2. However, the three-step enzymatic method needs to consider many issues such as multi-enzyme synergistic catalysis, stability of biocatalysts, accumulation and conversion of intermediates, etc., so it is difficult to apply it in actual industry. Since the 1980s, researchers have discovered that strains such as Pseudomonas sp.SE83, Pseudomonas sp.P130, Pseudomonassp.GK16, and Pseudomonas diminuta N176 produce cephalosporin C acylase (CPC acylase) in nature, which can directly catalyze CPC to synthesize 7-aminocephalosporanic acid. However, these enzymes strictly belong to GL-7-ACA acylase, and the activity of CPC acylase is only 2-4% of that of GL-7-ACA acylase, which is difficult to meet the needs of industrial production. Summary of the invention

[0004] The inventor reported a CPC acylase mutant numbered ED3 in the previous Chinese invention patent ZL2016100973700, with the amino acid sequence SEQ ID NO:1 herein, referred to as CPC1 herein, which was originally derived from Pseudomonassp.GK16. It has been put into use by domestic antibiotic manufacturers for several years. However, according to manufacturers, when CPC acylase mutant free enzyme is used to catalyze CPC reaction, if the temperature of the reaction system is out of control and exceeds 30°C, the thermal stability of the enzyme will decrease. Compared with the conventional process temperature of 15°C, the enzyme activity decreases significantly faster under the stirring reaction state, and the increase in the amount of enzyme feed leads to an increase in the production cost of 7-ACA.

[0005] In order to solve this problem, we continue to use genetic engineering technology to structurally modify and screen CPC acylase mutant ED3, i.e. CPC1, to construct CPC acylase mutants with improved thermal stability, and to promote the application and promotion of the current one-step enzymatic production of 7-ACA process. After multiple rounds of mutation and screening by error-prone PCR, a mutant with significantly improved thermal stability was obtained. It was also unexpectedly found that under the conventional process temperature of 15°C, the mutant had higher enzyme activity and even product specificity than the starting enzyme ED3, which was manifested in fewer by-products and more prominent advantages. Therefore, the present invention includes the following technical solutions.

[0006] The first aspect of the present invention provides a cephalosporin C acylase (i.e. CPC acylase), which is a polypeptide selected from the following:

[0007] (a) a polypeptide having an amino acid sequence of SEQ ID NO: 3, designated herein as CPC15;

[0008] (b) a conservative variant polypeptide having 90% or more, preferably 92% or more, preferably 95% or more, more preferably 97% or more, more preferably 98% or more, and more preferably 99% or more homology to SEQ ID NO: 3, and having improved enzyme activity and / or thermal stability in catalyzing the reaction of cephalosporin C (CPC) to produce 7-aminocephalosporanic acid (7-ACA) compared to SEQ ID NO: 3.

[0009]

[0010] Among them, the polypeptide with an amino acid sequence of SEQ ID NO: 3 is the above-mentioned CPC acylase mutant ED3, i.e., the (I11V, S50N, K65R, D165A, L166P, L187P, R250W, I488T, N529D, A567E, S604R, S648I) mutant of SEQ ID NO: 1:

[0011] (SEQ ID NO:3).

[0012] The second aspect of the present invention provides a gene encoding the cephalosporin C acylase as described above.

[0013] In a preferred embodiment, the gene encoding cephalosporin C acylase SEQ ID NO:3 is a polynucleotide shown in the nucleotide sequence SEQ ID NO:4, or a polynucleotide having more than 90%, preferably more than 92%, preferably more than 95%, more preferably more than 97%, more preferably more than 98%, and more preferably more than 99% homology with SEQ ID NO:4.

[0014] Another aspect of the present invention provides a DNA molecule comprising an expression cassette / frame encoding a gene as described above, for example, cephalosporin C acylase SEQ ID NO: 3. Preferably, the DNA molecule comprises a promoter located upstream and regulating the expression of the cephalosporin C acylase gene, such as SEQ ID NO: 4, and a terminator located downstream.

[0015] Another aspect of the present invention provides a recombinant plasmid for expressing the coding gene as described above, which comprises the gene as described above or the DNA molecule as described above.

[0016] The plasmid vector of the recombinant plasmid can be selected from the pET series (e.g., pET22b, pET24a, pET28a), pMAL series, pGEX series, pQE series, pBAD series, pCAl series, pSH series, pRSFDuet series or other vectors.

[0017] Another aspect of the present invention provides a microbial engineering bacterium for expressing the cephalosporin C acylase as described above, characterized in that the coding gene as described above or the DNA molecule as described above is integrated into the genome of the microbial engineering bacterium, or the microbial engineering bacterium is a transformant transformed with the recombinant plasmid as described above.

[0018] The host of the microbial engineering bacteria can be selected from Escherichia coli, Bacillus subtilis, Pichia pastoris, and Saccharomyces cerevisiae, preferably Escherichia coli, and more preferably the microorganism is Escherichia coli BL21 (DE3).

[0019] Another aspect of the present invention provides use of the cephalosporin C acylase or the microbial engineering bacteria described above in producing 7-aminocephalosporanic acid.

[0020] As a specific use mode, cephalosporin C (CPC) is used as a reaction substrate, and the cephalosporin C acylase or the microbial engineering bacteria as described above are used to catalyze the reaction to obtain 7-aminocephalosporanic acid.

[0021] In a specific embodiment, conventional process conditions can be used to produce 7-ACA. For example, the concentration of the substrate cephalosporin C (CPC) in the reaction system is 1 to 4 wt%, preferably 1.5 to 3.0 wt%, and more preferably about 2.5 wt%; the reaction temperature is selected to be 10 to 37°C, preferably 12 to 35°C, preferably 13 to 30°C, preferably 14 to 32°C, and more preferably about 15°C.

[0022] It should be understood that when expressing numerical characteristics in this article, the term "around" or "approximately" means that the number represented may have an error range or floating range of ±10%, ±9%, ±8%, ±7%, ±6% or ±5%.

[0023] Furthermore, the pH value of the reaction system is pH 7.0-9.0, preferably pH 7.5-8.5, and more preferably about pH 8.0.

[0024] The cephalosporin C acylase mutant CPC15 obtained by mutation screening through the error-prone PCR method in the present invention has significantly improved thermal stability compared with the starting enzyme ED3 (i.e., CPC1), can meet the requirements of 7-ACA manufacturers for a larger temperature control range of the reaction system, and has higher enzyme activity and product specificity, thereby promoting the one-step enzymatic production of 7-ACA. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a structural map of the recombinant plasmid pET-CPC1 expressing cephalosporin C acylase CPC1.

[0026] Figure 2 This is an overlay of HPLC results of the 30-min reaction of synthesizing 7-aminocephalosporanic acid catalyzed by the starting enzyme CPC1 and the mutant CPC15, respectively. DETAILED DESCRIPTION

[0027] Maintaining stability, especially thermal stability, in a homogeneous reaction system under stirring is a prerequisite and basic requirement for industrial production using free enzyme catalysis. The maintenance of the three-dimensional structure, i.e., the higher-order structure, of free enzymes is an important guarantee for maintaining thermal stability. The structural stability of enzymes is related to the interaction within their molecules, among which the sequence of amino acid residues has a decisive influence on the three-dimensional structure of enzymes. Only by maintaining the complete structure of enzyme molecules and the spatial conformation of the active center can the enzymes show stronger stability, especially thermal stability, and thus exert their catalytic function.

[0028] When modifying the starting enzyme CPC1 to improve its thermal stability, we still used the error-prone PCR method to construct a random mutation point library, and used high-throughput screening to find positive mutants with improved thermal stability and even enzyme activity.

[0029] Herein, the terms "(thermal stability, enzyme activity) improvement", "enhancement", "enhancement" or "increase" used above mean an increase of at least 50% or more compared to a reference level, for example, an increase of at least 80% or more, at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, or at least about 5-fold compared to a reference level.

[0030] In this article, the terms "initial enzyme", "starting enzyme" and "starting enzyme" have the same meaning, and all refer to the cephalosporin C acylase with an amino acid sequence of SEQ ID NO: 1, that is, the CPC acylase mutant ED3 disclosed in invention patent ZL2016100973700.

[0031] (SEQ ID NO: 1).

[0032] Correspondingly, the terms "cephalosporin C acylase mutant", "mutant cephalosporin C acylase", "CPC mutant" and "mutant enzyme" have the same meaning, and all refer to mutants of cephalosporin C acylase ED3 with improved thermal stability and / or enzyme activity, such as CPC15 (SEQ ID NO: 3) and CPC4-CPC14, etc. For the sake of convenience, in the present invention, wild-type cephalosporin C acylase and its mutants may be collectively referred to as "cephalosporin C acylase" or CPC, as long as they are not confused with the starting enzyme SEQ ID NO: 1.

[0033] Those skilled in the art can expect that it is reasonable that conservative variant polypeptides of cephalosporin C acylase CPC15 having a high homology (identity) with the amino acid sequence SEQ ID NO: 3, for example, more than 90%, have the same or similar functions.

[0034] As used herein, the term "conservative variant polypeptide" refers to a polypeptide that substantially retains the same biological function or activity of the polypeptide. The variation refers in particular to a small amount of amino acid mutations, such as (1) a polypeptide having the function of cephalosporin C acylase CPC15 formed by substitution, deletion or addition of one or more (e.g., 1-20, preferably 1-10; more preferably 1-5; more preferably 1-3) amino acid residues of the cephalosporin C acylase CPC15 polypeptide as shown in the amino acid sequence SEQ ID NO: 3; (2) a polypeptide having more than 90%, more preferably more than 95%, more preferably more than 98%, more preferably more than 99% identity with the cephalosporin C acylase CPC15 polypeptide as shown in the amino acid sequence SEQ ID NO: 3, and having the function of cephalosporin C acylase CPC15; or (3) a polypeptide formed by adding a tag sequence to the N or C terminus of the cephalosporin C acylase CPC15 polypeptide as shown in the amino acid sequence SEQ ID NO: 3, or adding a signal peptide sequence to its N terminus. According to the teachings of this document, these fragments, derivatives and analogs belong to the scope known to those skilled in the art. The term "mutation" includes, but is not limited to, replacement, deletion, insertion, chemical modification of amino acid residues, preferably a forward mutation, i.e., a mutation that improves function. The substitution may be a non-conservative substitution, a conservative substitution, or a combination of a non-conservative substitution and a conservative substitution. "Conservative" amino acid substitutions or mutations refer to the interchangeability of residues with similar side chains, and therefore generally include substitutions of amino acids in the same or similar amino acid definition classes in polypeptides. However, as used herein, if a conservative mutation may instead be aliphatic to aliphatic, non-polar to non-polar, polar to polar, acidic to acidic, alkaline to alkaline, aromatic to aromatic, or a restricted residue to a restricted residue substitution, then a conservative mutation does not include a hydrophilic to hydrophilic, hydrophobic to hydrophobic, hydroxy-containing to hydroxy-containing, or a small residue to a small residue substitution. It is well known in the art that common cases of conservative substitutions include: mutual substitutions between aromatic amino acids F, W, and Y; mutual substitutions between hydrophobic amino acids L, I, and V; mutual substitutions between polar amino acids Q and N; mutual substitutions between basic amino acids K, R, and H; mutual substitutions between acidic amino acids D and E; and mutual substitutions between hydroxyl amino acids S and T. In addition, A, V, L, or I can be conservatively mutated to another aliphatic residue or another non-polar residue. Exemplary conservative substitutions can be performed, for example, according to the following table.

[0035] Initial residue Representative replacement Preferred substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0036] The amino acid sequence of the cephalosporin C acylase mutant CPC15 of the present invention is clear, so those skilled in the art can easily obtain its encoding gene, expression cassette (DNA molecule) and plasmid containing these genes, and transformant containing the plasmid.

[0037] These genes, expression cassettes, plasmids, and transformants can be obtained by genetic engineering construction methods well known to those skilled in the art.

[0038] In order to optimally express cephalosporin C acylase SEQ ID NO: 1 and its mutant SEQ ID NO: 3 in a microbial host such as the Escherichia coli host most commonly used in genetic engineering, the present invention performs codon optimization on the expression gene thereof.

[0039] Codon optimization is a kind of technology that can be used for maximizing protein expression in organism by increasing the translation efficiency of gene of interest.Different organisms usually show the special preference for one of some codons of the same amino acid being encoded due to mutation tendency and natural selection.For example, in fast-growing microorganisms such as Escherichia coli, the optimization codon reflects the composition of its genome tRNA library separately.Therefore, in fast-growing microorganisms, the low frequency codon of amino acid can be used for the codon displacement of the same amino acid but high frequency.Therefore, the expression of optimized DNA sequence is improved in fast-growing microorganisms.

[0040] After codon optimization, the gene encoding the starting enzyme ED3 may be SEQ ID NO:2, and the gene encoding the cephalosporin C acylase mutant SEQ ID NO:3 may be SEQ ID NO:4.

[0041] As used herein, the "DNA molecule", "expression cassette" and "gene expression cassette" refer to a gene expression system comprising all necessary elements required to express the target cephalosporin C acylase mutant SEQ ID NO: 3, which generally includes the following elements: a promoter, a gene sequence encoding cephalosporin C acylase such as CPC15, and a terminator; in addition, it may optionally include a signal peptide coding sequence, etc.; these elements are operably linked.

[0042] As used herein, the term "operably linked" or "operably linked" refers to the functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences. For example, a promoter region is placed at a specific position relative to the target gene nucleic acid sequence SEQ ID NO: 4, so that transcription of the nucleic acid sequence is guided by the promoter region, and thus the promoter region is "operably linked" to the nucleic acid sequence.

[0043] Preferably, the recombinant plasmid (or nucleic acid construct) comprises one or more copies, two or more copies, preferably four or more, six or more, or eight or more copies of the gene encoding the cephalosporin C acylase mutant SEQ ID NO: 3.

[0044] When used as a biocatalyst for synthesizing 7-aminocephalosporanic acid, the cephalosporin C acylase of the present invention can be in the form of an enzyme or a bacterial cell. The enzyme forms include free enzymes, immobilized enzymes, including purified enzymes, crude enzymes, fermentation broths, enzymes immobilized on carriers, etc.; the bacterial cell forms include living bacterial cells and dead bacterial cells.

[0045] The present invention is further described in detail below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0046] Example

[0047] The examples involve the addition amounts, contents and concentrations of various substances, wherein the percentages described therein, unless otherwise specified, are all by mass percentages.

[0048] In the examples herein, if no specific description is given for laboratory temperature or operating temperature, the temperature generally refers to room temperature (15-30° C.).

[0049] Materials and methods

[0050] The whole gene synthesis, primer synthesis and sequencing in the examples were all completed by Suzhou Genewise Biotechnology Co., Ltd.

[0051] The molecular biology experiments in the embodiments include plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, culture medium preparation, etc., and are mainly carried out with reference to Molecular Cloning Experiment Guide (3rd edition), edited by J. Sambrook and DW Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002). If necessary, the specific experimental conditions can be determined by simple experiments.

[0052] PCR amplification experiments were performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. If necessary, adjustments could be made through simple experiments.

[0053] LB medium: 10g / L tryptone, 5g / L yeast extract, 10g / L sodium chloride, pH 7.2. (LB solid medium plus 20g / L agar powder.)

[0054] TB medium: 24g / L yeast extract, 12g / L tryptone, 16.43g / L K 2 HPO 4 .3H 2O, 2.31g / LKH 2 PO 4 , 5g / L glycerol, pH 7.0-7.5.

[0055] Fermentation medium for 5L fermenter: 24g / L yeast extract, 12g / L tryptone, 16.43g / L K 2 HPO 4 .3H 2 O, 2.31g / L KH 2 PO 4 , 5g / L glycerol, 0.5g / L defoaming agent, pH7.0-7.5, the fermentation liquid volume in each tank is 2L.

[0056] Feed medium for 5 L fermenter: 60% glycerol.

[0057] Kanamycin (Kan, 50 μg / mL) was used as required according to the antibiotic gene carried by the plasmid.

[0058] It should be noted that, for the convenience of description, in the embodiments, the strain number, plasmid number, enzyme number, and enzyme encoding gene number may share one number, which is easy for those skilled in the art to understand, that is, the same number can refer to different biological forms in different environments. For example, CPC1 can represent the expression strain (EcCPC1) of the starting enzyme CPC1, and can also represent the plasmid pET-CPC1 number, the starting enzyme SEQ ID NO:1 number, and the starting enzyme encoding gene SEQ ID NO:2 number.

[0059] Example 1: Construction of initial CPC acylase expressing recombinant E. coli

[0060] Based on the CPC acylase mutant ED3 reported in the invention patent ZL2016100973700, according to its amino acid sequence SEQ ID NO:1, codon optimization was performed according to the codon preference of Escherichia coli to obtain its coding gene sequence SEQ ID NO:2. Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to perform full gene synthesis and cloned into the NdeI and XhoI sites of plasmid pET24a to obtain the recombinant plasmid pET-CPC1. The plasmid schematic diagram is shown in the figure Figure 1 shown.

[0061] The recombinant plasmid pET-CPC1 was electroporated into the competent host Escherichia coli BL21 (DE3), and the positive clones were screened to construct the recombinant Escherichia coli EcCPC1 expressing the initial CPC acylase CPC1.

[0062] Example 2: Construction and screening of CPC acylase random mutation point library by error-prone PCR

[0063] 2.1 Construction of random mutation point library by error-prone PCR

[0064] The random mutant library was constructed using error-prone PCR technology with plasmid pET-CPC1 as template.

[0065] The following primer pair CPC-5 / CPC-3 was designed:

[0066] Forward primer CPC-5: 5'-ATGGAACCGACGAGCACCC-3',

[0067] Reverse primer CPC-3: 5′-CGGTTTAAAGTTAAACGGGG-3′.

[0068] Using plasmid pET-CPC1 as a template, PCR amplification was performed to obtain a CPC acylase mutant DNA sequence of about 2.1 kbp.

[0069] 50 μL error-prone PCR reaction system includes: 10 ng plasmid (pET-CPC1) template, 50 pmol primer pair CPC-5 and CPC-3, 1× Taq buffer, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP, 1 mM dTTP, 7 mM MgCl 2 , (0mM, 0.05mM, 0.1mM, 0.15mM, 0.2mM)MnCl 2 , 2.5 units of Taq enzyme (Takara).

[0070] The PCR reaction conditions were: 95°C for 5 min; 94°C for 30 s, 55°C for 30 s, 72°C for 2 min / kbp, 30 cycles; 72°C for 10 min.

[0071] The PCR product was electrophoresed and recovered by gel (Axygen DNA gel recovery kit AP-GX-50). Using plasmid pET-CPC1 as template and the recovered product of about 2.1 kbp (random mutation fragment) as large primer, MegaPrimer PCR was performed with KOD-plus DNA polymerase: 94℃5min; 98℃10s, 60℃30s, 68℃2min / kb, 25 cycles; 68℃10min. The plasmid template was digested with DpnI restriction endonuclease (Thermo Company) and electroporated into E. coli BL21 (DE3), and more than 10 4 A random mutation library of 10 clones.

[0072] 2.2 High-throughput screening of mutant libraries

[0073] Transformants from the mutant library were selected and inoculated into a 96-well plate (700 μL LB medium containing 100 μg / mL kanamycin and 0.1 mM IPTG). After incubation at 37°C and 400 rpm for 6 hours, the temperature was lowered to 25°C and incubated for 16 hours. Centrifuge at 5000 rpm for 10 minutes, discard the supernatant, freeze at -70°C for 1 hour, and thaw at room temperature for 30 minutes. Add 200 μL of 0.1 M potassium phosphate buffer (pH 8.0) containing 1 mg / mL lysozyme, resuspend the bacteria, incubate at 37°C for 1 hour, heat-treat at 45°C for 0.5 hour, then cool, centrifuge at 4°C and 5000 rpm for 20 minutes, and take 20 μL of supernatant for CPC activity determination.

[0074] 2.3 High-throughput enzyme activity assay

[0075] Substrate reaction solution: 0.1 M potassium phosphate buffer (pH 8.0) containing 2 wt% CPC sodium salt,

[0076] Termination solution: 0.05M NaOH, 20% v / v glacial acetic acid,

[0077] Color developer: methanol solution containing 0.5 wt% of PDAB (p-dimethylaminobenzaldehyde).

[0078] Definition of enzyme activity: The amount of enzyme required to catalyze the production of 1 μmol of 7-ACA from CPC per minute is defined as 1 unit (U).

[0079] Add 20 μL of the supernatant from the above step to 20 μL of the substrate reaction solution, react at 37°C for 12 hours, add 200 μL of the termination reaction solution, and then centrifuge at 5000 rpm for 10 minutes. Take 200 μL of the centrifugal supernatant, add 40 μL of the color developer, react at room temperature for 10 minutes, and then detect the absorbance at 415 nm. The larger the absorbance value, the higher the activity of CPC acylase.

[0080] In the random mutation library, a total of about 20,000 mutant clones were screened in two rounds, and the screening results are shown in Table 1. Strains with significantly improved enzyme activity were selected, plasmids were extracted, and Suzhou Jinweizhi Company was commissioned to perform nucleic acid sequencing. The CPC acylase-related fragments were compared with SEQ ID NO: 2 to determine the amino acid sequence changes of CPC acylase. The preferred strain with the highest improvement in enzyme activity was selected as the starting strain for the next round of random mutant library construction. The results of two consecutive rounds of random mutation library screening by error-prone PCR are listed in Table 1.

[0081] Table 1. Results of high-throughput screening of the first and second rounds of random mutation libraries

[0082]

[0083] Note: "+" represents that the percentage of activity is greater than 0% and less than or equal to 25% relative to the respective starting strain; "++" represents that the percentage of activity is greater than 25% and less than or equal to 50% relative to the respective starting strain; "+++" represents that the percentage of activity is greater than 50% and less than or equal to 75% relative to the respective starting strain; "++++" represents that the percentage of activity is greater than 75% and less than or equal to 100% relative to the respective starting strain; "+++++" represents that the percentage of activity is greater than 100% relative to the respective starting strain.

[0084] Example 3: Construction and screening of CPC acylase random mutation point library by the third and fourth rounds of error-prone PCR

[0085] Furthermore, in order to screen mutant enzymes with higher thermal stability and / or higher enzyme activity, two rounds of error-prone PCR mutation screening were performed using EcCPC7 as the starting strain.

[0086] 3.1 Construction of random mutation point library by error-prone PCR

[0087] The random mutation library was constructed using the screened mutant strain EcCPC7 plasmid as a template. The construction method was based on the method for constructing a CPC acylase random mutation point library in Example 2.

[0088] 3.2 High-throughput screening of mutant libraries

[0089] Transformants from the mutant library were selected and inoculated into a 96-well plate (700 μL LB medium containing 100 μg / mL kanamycin and 0.1 mM IPTG). After incubation at 37°C and 400 rpm for 6 hours, the temperature was lowered to 25°C and incubated for 16 hours. Centrifuge at 5000 rpm for 10 minutes, discard the supernatant, freeze at -70°C for 1 hour, and thaw at room temperature for 30 minutes. Add 200 μL of 0.1 M potassium phosphate buffer (pH 8.0) containing 1 mg / mL lysozyme, resuspend the bacteria, incubate at 37°C for 1 hour, heat treat at 45°C for 1 hour, then cool, centrifuge at 4°C and 5000 rpm for 20 minutes, and take 20 μL of supernatant for CPC activity determination.

[0090] 3.3 High-throughput enzyme activity assay

[0091] Referring to the method in Example 2, 20 μL of supernatant was added to 20 μL of substrate reaction solution, reacted at 37°C for 12 h, 200 μL of termination reaction solution was added, and then centrifuged at 5000 rpm for 10 min. 200 μL of centrifugal supernatant was taken, 40 μL of color developer was added, reacted at room temperature for 10 min, and then the absorbance at 415 nm was detected.

[0092] The third and fourth rounds of random mutation libraries were screened by screening a total of about 20,000 mutant clones, and the screening results are shown in Table 2. Strains with significantly improved enzyme activity were selected, plasmids were extracted, and Suzhou Jinweizhi Company was commissioned to perform nucleic acid sequencing. The CPC acylase-related fragments were compared with SEQ ID NO: 2 to determine the amino acid sequence changes of CPC acylase. The preferred strain with the highest improvement in enzyme activity was selected as the starting strain for the next round of random mutant library construction. The results of two consecutive rounds of random mutation library screening by error-prone PCR are listed in Table 2.

[0093] Table 2. Results of high-throughput screening of random mutation libraries from round 3 to round 4

[0094]

[0095]

[0096] Note: "+" represents that the percentage of activity is greater than 0% and less than or equal to 25% relative to the respective starting strain; "++" represents that the percentage of activity is greater than 25% and less than or equal to 50% relative to the respective starting strain; "+++" represents that the percentage of activity is greater than 50% and less than or equal to 75% relative to the respective starting strain; "++++" represents that the percentage of activity is greater than 75% and less than or equal to 100% relative to the respective starting strain; "+++++" represents that the percentage of activity is greater than 100% relative to the respective starting strain.

[0097] The results showed that the mutant strain EcCPC15 significantly improved the thermal stability and enzyme activity of CPC acylase. The nucleotide sequence of the corresponding CPC acylase gene was SEQ ID NO:4, and the encoded amino acid sequence was SEQ ID NO:3. Compared with CPC1, it underwent (I11V, S50N, K65R, D165A, L166P, L187P, R250W, I488T, N529D, A567E, S604R, S648I) mutations.

[0098] Example 4: Strain fermentation, enzyme separation and extraction, pure enzyme specific activity and thermal stability determination

[0099] 4.1 Shake flask fermentation

[0100] Pick a single colony of the mutant strain EcCPC15, inoculate it into 5 mL of liquid LB medium containing 50 μg / mL kanamycin sulfate, and culture it overnight at 37°C and 250 rpm. The next day, transfer it to a shake flask containing 200 mL of liquid TB medium at a volume concentration of 1% v / v, and culture it at 37°C and 250 rpm for 2 to 3 hours. When the OD600 is 0.6 to 0.8, add 0.1 mM IPTG, and culture it overnight at 28°C and 200 rpm. Centrifuge at 4°C and 10,000 rpm for 10 minutes to collect the bacteria.

[0101] 4.2 Enzyme extraction

[0102] The bacteria were resuspended with 50 mL of equilibrium buffer (50 mM potassium phosphate buffer, 200 mM NaCl, pH 8.0), and then ultrasonically disrupted. The disrupted bacteria were centrifuged at 4 ° C and 12000 rpm for 20 min, and the supernatant was collected. The supernatant was added to an affinity chromatography column containing 10 mL of Ni-NAT matrix at a rate of 1 mL / min, and then the column material was washed with an equilibrium buffer containing 30 mM imidazole to elute impurities. Finally, the target protein was washed with an equilibrium buffer containing 500 mM imidazole, and the peak eluate was collected. The eluate was desalted through an ultrafiltration tube with a molecular weight cutoff of 10 kDa to obtain CPC15 pure enzyme.

[0103] The initial enzyme CPC1 pure enzyme was prepared in the same way.

[0104] 4.3 Determination of pure enzyme specific activity

[0105] The solution used in this step is the same as the reagent used in Example 2.

[0106] Add 20 μL of the pure enzyme solution desalted in step 4.2 to 20 μL of the substrate reaction solution, react at 37°C for 5 min, add 200 μL of the termination reaction solution, and then centrifuge at 5000 rpm for 10 min. Take 200 μL of the centrifugal supernatant, add 40 μL of the colorimetric agent, react at room temperature for 10 min, detect the absorbance at 415 nm, and compare with the 7-ACA quantitative standard curve for quantification.

[0107] At the same time, the protein concentration of the pure enzyme was determined using the BCA Protein Assay Kit from Thermo Scientific, thereby obtaining the specific activity of the pure enzyme.

[0108] Table 3. Comparison of enzyme activity of pure enzymes at 37°C

[0109]

[0110] It can be seen from Table 3 that, compared with the starting enzyme CPC1, the enzyme activity of the CPC acylase mutant CPC15 of the present invention is increased by 2.7 times.

[0111] 4.4 Thermal stability determination

[0112] The solution used in this step is the same as the reagent used in Example 2.

[0113] The pure enzyme solution after desalting in step 4.2 was heat-treated at 45°C for 0.5h, 20μL was taken and added to 20μL substrate reaction solution, reacted at 37°C for 5min, 200μL termination reaction solution was added, and then centrifuged at 5000rpm for 10min. 200μL centrifugal supernatant was taken, 40μL color developer was added, reacted at room temperature for 10min, and the absorbance at 415nm was detected and compared with the 7-ACA quantitative standard curve for quantification.

[0114] At the same time, the protein concentration of the pure enzyme was determined using the BCA Protein Assay Kit from Thermo Scientific, thereby obtaining the specific activity of the pure enzyme.

[0115] Table 4. Comparison of enzyme activities of the directed evolved pure enzymes at 37°C after incubation at 45°C for 0.5h

[0116]

[0117] Through comparative experiments, it is proved that the thermal stability of the CPC acylase mutant CPC15 of the present invention is higher than that of the initial enzyme CPC1, which is manifested in a smaller decrease in enzyme activity under high temperature treatment / reaction conditions.

[0118] Example 5: Application of mutant enzyme in the production of 7-ACA

[0119] 100 mL reaction system includes: 2.5 g CPC sodium salt, dissolved in water, cooled to 15 ° C, adjusted to pH 8.2, added 500 U of mutant CPC15 pure enzyme or initial enzyme CPC1 prepared in step 4.2 of Example 4, stirred for reaction. During the reaction, the temperature was controlled at 15 ± 0.5 ° C, pH 8.0 ± 0.2, reacted for 30 min, and the reaction sample was detected.

[0120] Accurately measure 100 μL of the sample after 40 minutes of reaction, dilute to 10 mL with water, and perform HPLC analysis. The analysis conditions are as follows:

[0121] Agilent 1260 high performance liquid chromatograph; waters C18 200mm×4.6 column, detection wavelength 262nm, mobile phase 0.02M sodium acetate pH5.5: acetonitrile (=93:7), temperature 25°C.

[0122] Results Figure 2 In the reaction, the CPC sodium salt conversion rate of CPC15 was 98.3%, while that of CPC1 was 96.1%. In addition, CPC15 produced less impurity deacetyl-7-aminocephalosporanic acid (D-7-ACA) during the reaction, accounting for 0.32% of the generated 7-ACA; while the D-7-ACA impurity produced by the CPC1 catalytic reaction accounted for 0.45% of the generated 7-ACA.

[0123] In summary, compared with the initial enzyme CPC1, the CPC acylase mutant CPC15 of the present invention has higher thermal stability and enzyme activity, and the product purity is higher and the impurity content is less. Using the mutant CPC15 as a catalyst for one-step enzymatic production of 7-ACA, the reaction temperature is 15±0.5°C for 30 minutes, so that the conversion rate of CPC exceeds 98%, which improves the economic efficiency of industrial application.

Claims

1. A cephalosporin C acylase, which is a polypeptide selected from the following: (a) a polypeptide having an amino acid sequence of SEQ ID NO: 3; (b) a conservative variant polypeptide having more than 90% homology with SEQ ID NO: 3 and having an enzyme activity and / or thermal stability that catalyzes the reaction of cephalosporin C to produce 7-aminocephalosporanic acid that is improved compared to SEQ ID NO: 3, 2. A gene encoding the cephalosporin C acylase according to claim 1.

3. The gene according to claim 2, characterized in that The gene encoding cephalosporin C acylase of SEQ ID NO:3 is a polynucleotide represented by the nucleotide sequence of SEQ ID NO:4, or a polynucleotide having 90% or more homology with SEQ ID NO:

4.

4. A DNA molecule, characterized in that Comprising the gene as claimed in claim 3.

5. A recombinant plasmid, characterized in that: Used to express the gene as claimed in claim 3, comprising the gene as claimed in claim 3 or the DNA molecule as claimed in claim 4.

6. A microbial engineering bacterium for expressing the cephalosporin C acylase according to claim 1, characterized in that: The gene as claimed in claim 3 or the DNA molecule as claimed in claim 4 is integrated into the genome of the microbial engineering bacteria, or the microbial engineering bacteria is a transformant transformed with the recombinant plasmid as claimed in claim 5.

7. The microbial engineering bacteria according to claim 6, characterized in that The microbial host is Escherichia coli.

8. Use of the cephalosporin C acylase according to claim 1 or the microbial engineering bacteria according to claim 6 in producing 7-aminocephalosporanic acid.

9. The use according to claim 8, characterized in that Cephalosporin C is used as a reaction substrate, and the cephalosporin C acylase as claimed in claim 1 or the microbial engineering bacteria as claimed in claim 6 is used to catalyze the reaction to obtain 7-aminocephalosporanic acid.

10. The use according to claim 8, characterized in that The concentration of substrate cephalosporin C in the reaction system is 1-4 wt %; and the reaction temperature is 10-37 DEG C.

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