Expandase mutants for the synthesis of g-7-adca

Abstract

The application discloses an expandase mutant SEQ ID NO:3 which can efficiently catalyze the expandase reaction of a substrate to generate phenylacetyl-7-amino deacetoxycephalosporanic acid in a reaction system with a penicillin G concentration of up to 10 mM, and can be used for industrial production of G-7-ADCA.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biocatalysis, and particularly relates to a mutant of expandase and application of the mutant in preparation of phenylacetyl-7-amino desacetoxycephalosporanic acid (G-7-ADCA). BACKGROUND

[0002] 7-Aminodesacetoxycephalosporanic acid (7-ADCA) is an important intermediate for semi-synthesis of cephalosporins, and is used in the pharmaceutical industry for synthesis of drugs such as cephalexin, cephradine and cefadroxil.

[0003] The synthesis methods of 7-ADCA include chemical synthesis method and biological synthesis method. The chemical synthesis method is to prepare 7-ADCA from penicillin G (V) as a starting material, i.e. penicillin potassium salt is oxidized, silylated and ring-expanded, and then cleaved to obtain 7-ADCA. However, a large amount of organic reagents are used in the process, it is difficult to meet the environmental protection requirements of today, and the impurity content is high, and the production cost is high. The biological synthesis method (enzyme method or microbial fermentation method) is used for full synthesis of 7-ADCA or replacement of chemical synthesis steps, and has environmental friendliness and cost advantage. However, the main core technology is mastered by foreign companies, and the main synthesis routes reported include the following several kinds:

[0004] Route 1 is to expand the ring of penicillin N as the substrate to synthesize deacetoxycephalosporin C (DAOC), and then to synthesize 7-ADCA through multi-step reactions (Proc Natl Acad Sci USA 1978. 75: 6253-6257.). Patent document CN1357051A discloses that DAOC is converted into glutaryl-7-ADCA by using D-amino acid-oxidase (DAO), and glutaryl-7-ADCA is converted into 7-ADCA by using glutaryltransferase (GLA). Route 2 is to add a side chain to isopenicillin N as the substrate to form a side chain-6-APA, and then to synthesize a side chain-7-ADCA (product) under the action of an expandase, and finally to synthesize 7-ADCA under the action of an acyltransferase. Patent document CN92112278.0 discloses a method for synthesizing 7-ADCA by using adipic acid (or salt (disodium adipate) or ester) as a side chain, and patent document CN94192932.9 discloses a method for synthesizing 7-ADCA by using 3,3'-thiodipropionic acid or salt or ester thereof as a side chain. Route 3 is to synthesize and produce phenylacetyl-7-amino deacetoxycephalosporanic acid (G-7-ADCA) by using a penicillin G strain, and then to synthesize 7-ADCA (see EP-A-0453047, EP-A-0222462). First, a high-yield penicillin G strain of Penicillium chrysogenum is transformed by using an expandase gene, so that penicillin G is expanded in vivo to form G-7-ADCA. Then, G-7-ADCA is recovered from the fermentation broth, and the acyl group is removed. Finally, 7-ADCA is recovered and crystallized.

[0005] Penicillin G has a mature fermentation production process, and is relatively low in price, and is an ideal substrate for preparing 7-ADCA. However, the natural expandase has low activity, which is a rate-limiting step for synthesizing 7-ADCA by using penicillin G as the substrate. Therefore, it is urgent to improve the catalytic activity of the expandase. SUMMARY

[0006] It is known that amino acid mutation is a main means for improving enzyme activity, and changes in key amino acid sites of an enzyme active center and a pocket region sometimes can cause obvious changes in enzyme properties. Based on comparison and screening of expandase data from various microorganisms through bioinformatics analysis, the inventors selected an expandase from Streptomyces clavuligerus (WP_003952493.1) which can catalyze the expansion of penicillin G to form G-7-ADCA as a modification object, tried to mutate the same, obtained some mutants with significantly improved enzyme activity through random mutation by using error-prone PCR and high-throughput screening of the mutant library. Specifically, the present application provides the following technical scheme:

[0007] A mutant of expandase characterized in that it is a mutant of the amino acid sequence SEQ ID NO: 1 of the expandase (WP_003952493.1) derived from Streptomyces clavuligerus, which has been mutated at one or more, preferably two or more, more preferably three or more, more preferably four or more of the following positions: S98, A129, L159, S261, F294, and which has an enzymatic activity higher than the wild-type expandase SEQ ID NO: 1 for the conversion of penicillin G to phenylacetyl-7-aminodeacetoxycephalosporanic acid (G-7-ADCA).

[0008] Preferably, the mutations are selected from S98G, A129S, L159M, S261T and F294I.

[0009] Further, the amino acid sequence of the mutant of expandase is preferably SEQ ID NO: 3, which is a mutant of the amino acid sequence SEQ ID NO: 1 of the wild-type expandase (WP_003952493.1) having the mutations S98G, A129S, L159M, S261T and F294I.

[0010] The second aspect of the present application provides a DNA molecule comprising a gene encoding the mutant of expandase described above.

[0011] In one embodiment, the nucleotide sequence of the gene encoding the mutant of expandase having the amino acid sequence SEQ ID NO: 3 is SEQ ID NO: 4.

[0012] The third aspect of the present application provides a recombinant plasmid comprising the DNA molecule described above. For example, the gene sequence SEQ ID NO: 4 is cloned on the recombinant plasmid.

[0013] The plasmid vector of the recombinant plasmid can be selected from the pMAL series, the pGEX series, the pET series (e.g. pET22b, pET24a, pET28a), the pQE series, the pBAD series, the pCAl series, the pSH series, the pRSFDuet series or other vectors.

[0014] Preferably, the nucleotide sequence of the recombinant plasmid described above can be SEQ ID NO: 5.

[0015] The fourth aspect of the present application provides a microorganism transformed with the recombinant plasmid described above, i.e. a transformant into which the recombinant plasmid described above has been introduced, which is an engineered bacterium expressing the mutant of expandase described above.

[0016] The microbial host of the above transformant is selected from the group consisting of Escherichia coli, Bacillus subtilis, Vibrio natriqum, Corynebacterium glutamicum, Pichia pastoris and Saccharomyces cerevisiae. Preferably, the microbial host is Escherichia coli BL21(DE3).

[0017] The fourth aspect of the present application provides the use of the above expandase mutant or the above microorganism in the production of phenylacetyl-7-amino-deacetoxycephalosporanic acid (G-7-ADCA).

[0018] Specifically, the above expandase mutant or the above microorganism catalyzing the production of phenylacetyl-7-amino-deacetoxycephalosporanic acid (G-7-ADCA) using penicillin G as the reaction substrate.

[0019] Optionally, FeSO4, ascorbic acid and α-ketoglutaric acid can be added to the reaction system. Ascorbic acid (vitamin C) converts the ferrous ion (Fe 2+ ) of ferrous sulfate (FeSO4) to ferric ion (Fe 3 + ) through redox reaction.

[0020] The expandase reaction can be represented by the following reaction formula:

[0021]

[0022] In the reaction system, the concentration of the substrate penicillin G is 5 mM or more, preferably 8 mM or more, preferably 10 mM or more, for example 15 mM or more.

[0023] In one embodiment, the above microorganism expressing the above expandase mutant can be used as the catalyst to catalyze the expandase reaction of the substrate, which is added to the reaction system in the form of microbial cells or cell disruption products. The cell disruption products are, for example, high-pressure disrupted cells, cell ultrasonic disruption products.

[0024] The pH value of the above reaction system is pH 7.0-8.0, for example about pH 7.5.

[0025] The reaction temperature of the above reaction system can be 20-40°C, for example 22-35°C, preferably about 25°C.

[0026] It should be understood that the term "about" or "around" in the expression of numerical characteristics herein means that the indicated number can have an error range or floating range of ±10%, ±9%, ±8%, ±7%, ±6% or ±5%.

[0027] The application screens some expandase mutants with significantly improved enzyme activity, wherein the enzyme activity of the mutant with the amino acid sequence of SEQ ID NO: 3 is 4 times higher than that of the wild-type expandase (WP_003952493.1), and when the mutant catalyzes the expandase reaction of penicillin G to synthesize G-7-ADCA, the concentration of the substrate penicillin G can be more than 10 mM, and the industrial application potential is huge. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 is a structural schematic diagram of the plasmid pET-KH1 for expressing the wild-type enzyme expandase constructed by the application.

[0029] Figure 2 is an HPLC detection spectrum of the expandase mutant SEQ ID NO: 3 in the process of catalyzing the expandase reaction of penicillin G. DETAILED DESCRIPTION

[0030] The expandase (WP_003952493.1) from Streptomyces clavuligerus is a 2-oxoglutarate and iron ion-dependent oxygenase, which can catalyze the expandase of penicillin G to form G-7-ADCA, and the inventors refer to it as “expandase” in this paper for its catalytic function. Those skilled in the art can easily understand that as a member of the oxygenase family, its function is not limited to the expandase of penicillin G. However, the enzyme still needs to be greatly improved in enzyme activity before it can be applied to the industrial production of G-7-ADCA, and therefore it must be modified or modified, especially the amino acid sequence.

[0031] One important way to improve the enzyme activity of wild-type enzymes is to mutate the amino acid sequence, change the properties or microenvironment of the enzyme activity center and / or the pocket channel. Therefore, according to the computer-simulated 3D model of the protein sequence, the inventors selected the amino acid sites around the active pocket for site-directed saturation mutation through rational analysis and semi-rational design, and obtained some mutants with significantly improved enzyme activity by random mutation through error-prone PCR and high-throughput screening of mutant libraries, so as to be used for efficient catalysis of the expandase reaction of penicillin G to synthesize G-7-ADCA.

[0032] In the present context, the terms "wild-type", "wild-type (expandase) enzyme" or "original (expandase) enzyme" mean the same and refer to the expandase enzyme WP_003952493.1 having the amino acid sequence of SEQ ID NO: 1. Correspondingly, the terms "mutant", "mutant enzyme", "expandase mutant" mean the same and refer to enzymes which have been modified in their amino acid sequence with respect to the wild-type expandase enzyme, while still maintaining the same catalytic reaction characteristics, in particular to enzymes having an improved enzyme activity, such as the mutant having the amino acid sequence of SEQ ID NO: 3. For the sake of convenience, the wild-type expandase enzyme and its mutants can be collectively referred to as "expandase enzymes" in the present context.

[0033] In the present context, the term "(enzyme activity) improved" means an improvement of at least 50% or 100% compared to a reference level, such as the enzyme activity of the wild-type enzyme, for example at least 1-fold, at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 10-fold, or at least 20-fold compared to the reference level.

[0034] It is to be understood that the term "mutation" as used herein includes, but is not limited to, substitution, deletion, insertion, chemical modification of an amino acid residue, preferably a mutation which is a positive mutation, i.e. a mutation which improves the enzyme activity. The substitution can be a non-conservative substitution, a conservative substitution or a combination of non-conservative and conservative substitutions. A "conservative" amino acid substitution or mutation refers to the interchangeability of residues having similar side chains and thus generally includes the substitution of an amino acid in a polypeptide with another amino acid of the same or similar class. However, as used herein, a conservative mutation does not include a substitution of hydrophilic to hydrophilic, hydrophobic to hydrophobic, hydroxyl-containing to hydroxyl-containing, or small residue to small residue if the conservative mutation can alternatively be an aliphatic to aliphatic, non-polar to non-polar, polar to polar, acidic to acidic, basic to basic, aromatic to aromatic, or a restriction residue to restriction residue substitution. It is well known in the art that common instances of conservative substitutions include: interchanges between aromatic amino acids F, W, Y; interchanges between hydrophobic amino acids L, I, V; interchanges between polar amino acids Q, N; interchanges between basic amino acids K, R, H; interchanges between acidic amino acids D, E; interchanges between hydroxyl amino acids S, 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 made, for example, according to the following table, wherein amino acids belonging to the same partition in the second column can be substituted for one another, and in preferred cases amino acids in the same row in the third column can be substituted for one another:

[0035]

[0036] A "non-conservative substitution" is a substitution or mutation of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. A non-conservative substitution can use amino acids from the groups listed above, but between groups rather than within. In one embodiment, a non-conservative mutation affects (a) the structure of the peptide backbone in the region of the substitution (e.g., a proline in place of glycine), (b) the charge or hydrophobicity, or (c) the bulk of the side chain.

[0037] A "deletion" refers to a modification of a polypeptide by removing one or more amino acids from a reference polypeptide. A deletion can include removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids comprising the reference enzyme, while retaining enzyme activity and / or retaining the improved properties of the engineered expandase. Deletions can be to the interior and / or the ends of the polypeptide. In embodiments, deletions can comprise contiguous segments or can be non-contiguous.

[0038] An "insertion" refers to a modification of a polypeptide by adding one or more amino acids to a reference polypeptide. In some embodiments, the improved engineered expandases include one or more amino acids inserted into a naturally occurring expandase and one or more amino acids inserted into other improved expandase polypeptides. The insertion can be to the interior of the polypeptide, or the carboxy- or amino-terminus. As used herein, insertions include fusion proteins as known in the art. The insertion can be a contiguous segment of amino acids or separated by one or more amino acids in the naturally occurring polypeptide.

[0039] By random mutation through multiple rounds of error-prone PCR, some mutants were screened, and the effective mutation sites include: S98, A129, L159, S261 and F294. Among them, the mutant with the following site mutations has the best enzyme activity: S98G, A129S, L159M, S261T and F294I, and the amino acid sequence of the mutant is SEQ ID NO: 3.

[0040] The amino acid of the expandase mutant of the present application is 311, and the sequence is clear, so that the coding gene, the expression cassette and plasmid containing the gene, and the transformant containing the plasmid can be easily obtained by those skilled in the art.

[0041] In order to optimally express the expandase or its mutant in Escherichia coli which is most commonly used in genetic engineering, the expression genes of these enzymes can be codon-optimized.

[0042] Codon optimization is a technique that can be used to maximize protein expression in an organism by increasing the efficiency of translation of the gene of interest. Different organisms typically show a particular bias for one of the codons that encodes the same amino acid due to mutational bias and natural selection. For example, in fast-growing microorganisms such as E. coli, the optimized codons reflect the composition of their respective genomic tRNA pool. Thus, in fast-growing microorganisms, low-frequency codons for an amino acid can be replaced by codons for the same amino acid but of high frequency. Therefore, the expression of optimized DNA sequences is improved in fast-growing microorganisms.

[0043] For example, to express expandase in E. coli, the codon-optimized gene encoding the amino acid sequence of SEQ ID NO: 3 can be SEQ ID NO: 4.

[0044] These genes, expression cassettes, plasmids, transformants can be obtained by genetic engineering methods well known to those skilled in the art.

[0045] The transformant host can be any microorganism suitable for expressing expandase, including bacteria and fungi. Preferably, the microorganism is B. subtilis, P. pastoris, S. cerevisiae, Vibrio natriq or E. coli, preferably E. coli, more preferably E. coli BL21 (DE3).

[0046] When used as a biocatalyst, the expandase of the present application can be added to the reaction system in the form of an enzyme or in the form of a bacterial cell. The enzyme form includes free enzyme, immobilized enzyme, including purified enzyme, crude enzyme, fermentation broth, carrier immobilized enzyme, etc.; the bacterial cell form includes viable bacterial cells, dead bacterial cells, immobilized bacterial cells, etc.

[0047] When microorganisms such as B. subtilis, P. pastoris, S. cerevisiae or E. coli are no longer used for fermentation proliferation, but for enzyme catalysis, they are a natural immobilized enzyme by themselves, and do not need to be broken down or even extracted and purified, and can be directly used as an enzyme preparation for catalysis. Since the reaction substrates and products are small molecules that can easily pass through the biological barrier of the bacterial cell membrane, the bacterial cells do not need to be broken down, which is economically advantageous.

[0048] The present application will be further described in conjunction with the following specific examples. It should be understood that the following examples are used to illustrate the present application but not to limit the scope of the present application.

[0049] Embodiments

[0050] In this text, the addition amount, content and concentration of various substances are mentioned, wherein the percentage content refers to the mass percentage content unless otherwise specified.

[0051] Materials and methods

[0052] The whole gene synthesis, primer synthesis and sequencing in the examples were completed by Suzhou Genewiz Biotechnology Co., Ltd.

[0053] The molecular biology experiments in the examples, including plasmid construction, enzyme digestion, ligation, preparation of competent cells, transformation, medium preparation, etc., were mainly performed according to the Molecular Cloning Laboratory Guide (3rd edition), J. Sambrook, D. W. Russell (USA) edited, Huang Peitang et al. translation, Science Press, Beijing, 2002. If necessary, the specific experimental conditions can be determined by simple tests.

[0054] The PCR amplification experiments were performed according to the reaction conditions or kit instructions provided by the plasmid or DNA template supplier. If necessary, it can be adjusted by simple test.

[0055] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.2. (LB solid medium plus 20 g / L agar powder.)

[0056] TB medium: 24 g / L yeast extract, 12 g / L tryptone, 16.43 g / L K2HPO4.3H2O, 2.31 g / L KH2PO4, 5 g / L glycerol, pH 7.0-7.5. (TB solid medium plus 20 g / L agar powder.)

[0057] Fermentation medium for 5L fermenter: 24 g / L yeast extract, 12 g / L tryptone, 16.43 g / L K2HPO4.3H2O, 2.31 g / L KH2PO4, 5 g / L glycerol, antifoaming agent 0.5 g / L, pH 7.0-7.5, the liquid volume of each tank of fermentation broth is 2L.

[0058] Feeding medium for 5L fermenter: 60% glycerol.

[0059] Kanamycin (Kan, 50 μg / mL) was used according to the requirements of the antibiotic gene carried by the plasmid.

[0060] Penicillin G and G-7-ADCA standard were gifted by Sinopharm Weichida Pharmaceutical Co., Ltd.; iron sulfite, ascorbic acid, α-ketoglutaric acid and other reagents were purchased from Aladdin Chemical Reagent Co., Ltd.

[0061] HPLC detection conditions of substrate penicillin G and product phenylacetyl-7-aminodesacetoxycephalosporanic acid (G-7-ADCA):

[0062] Agilent 1260 high performance liquid chromatograph; chromatographic column: Agilent RX-C18 (250*4.6mm, 5μm); mobile phase: 20mM potassium phosphate buffer (pH 3.5): acetonitrile (90:10); flow rate: 1.0mL / min; detection wavelength: 215nm.

[0063] It should be noted that for the convenience of description, in the examples, the strain number, plasmid number, enzyme / number, enzyme coding gene number can share a number, which is easily understood by those skilled in the art, i.e. the same number can refer to different biological forms in different environments.

[0064] Example 1: Construction and activity detection of recombinant E. coli of wild-type expandase gene

[0065] 1. Construction of vectors and strains

[0066] Based on the amino acid sequence SEQ ID NO: 1 of the expandase (WP_003952493.1) from Streptomyces clavuligerus, the codon optimization was carried out according to the codon preference of E. coli, and the coding gene sequence was SEQ ID NO: 2. The full gene synthesis was entrusted to Suzhou Jinyuizhi Biological Technology Co., Ltd., and was cloned into the NdeI and XhoI sites of plasmid pET24a to obtain plasmid pET-KH1. The plasmid schematic diagram is shown in Figure 1 .

[0067] The recombinant plasmid pET-KH1 was transformed into the expression host E. coli BL21 (DE3) by electroporation to obtain the recombinant E. coli EcKH1 expressing the initial expandase. At the same time, the negative control group (transformed with pET24a empty vector) was constructed to obtain E. coli EcKH0.

[0068] 2. Enzyme expression

[0069] A single colony was picked from the LB plate of the transformant and transferred to LB medium containing 50μg / mL kanamycin and incubated at 37℃, 220rpm overnight. Then, it was transferred to 100mL TB medium containing 50μg / mL kanamycin at a 1% v / v inoculation amount and incubated at 37℃ until the OD 600 When the OD reached about 1.2, 0.2mM IPTG was added to induce protein expression at a final concentration, and the recombinant bacteria expressing wild-type expandase were obtained after induction at 25℃ for 16h.

[0070] 3. Enzyme catalytic reaction system

[0071] The bacteria liquid was poured into a 50 mL centrifuge tube, centrifuged at 4000 rpm for 10 min, the bacteria were collected, resuspended and washed once with 100 mM phosphate buffer with pH value of 7.5, centrifuged at 4000 rpm for 10 min to collect the bacteria, and weighed. The reaction liquid formula: 100 mM phosphate buffer with pH value of 7.5, 100 μg / mL FeSO4, 0.5 mM ascorbic acid, 5 mM α-ketoglutaric acid, and 5 mM penicillin G. The wet bacteria were added to the reaction liquid at 5% w / v, and reacted at 25°C and 220 rpm. After 2 h of reaction, centrifugation was performed at 12000 rpm for 1 min, and the supernatant was taken for HPLC detection.

[0072] 4. Enzyme activity analysis

[0073] The conversion rate of the substrate penicillin G or the generation rate of the product G-7-ADCA was used as an index for judging the enzyme activity. The data results of the generation rate of the product G-7-ADCA are shown in the following table

[0074]

[0075]

[0076] For the substrate concentration of 5 mM, since the generation rate of the product G-7-ADCA is only 17.1%, it means that the conversion rate of the substrate penicillin G is very low, which is estimated to be not higher than 18% or even close to 17.1%, indicating that the enzyme activity of the wild enzyme is low, and mutation should be performed to greatly improve the enzyme activity, so that it can be used in practical application.

[0077] Example 2: First round and second round of random mutation point library establishment and high-throughput screening

[0078] 1. Construction of random mutation point library by error-prone PCR

[0079] The plasmid pET-KH1 was used as a template, and an error-prone PCR technique was used to construct a random mutant library.

[0080] The primer pair KH-5 / KH-3 was designed as follows: forward primer KH-5: 5'-CTTTAAGAAGGAGATATACATATG-3',

[0081] reverse primer KH-3: 5'-TTACGCTTTGCTCGTGCGGC-3'.

[0082] The plasmid pET-KH1 was used as a template, and PCR amplification was performed to obtain a DNA sequence of about 1.0 kb of the expandase mutant.

[0083] 50 μL of the error-prone PCR reaction system comprises: 10 ng of plasmid (pET-KH1) template, 50 pmol of a pair of primers KH-5 and KH-3, 1 × Taq buffer, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP, 1 mM dTTP, 7 mM MgCl2, (0 mM, 0.05 mM, 0.1 mM, 0.15 mM, 0.2 mM) MnCl2, 2.5 units of Taq enzyme (Takara).

[0084] The PCR reaction conditions are: 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.

[0085] The PCR product is electrophoresed and gel recovered (Axygen DNA Gel Recovery Kit AP-GX-50). The plasmid pET-KH1 is used as a template, the recovered product (randomly mutated fragment) of about 1.0 kb is used as a large primer, KOD-plus DNA polymerase is used for MegaPrimer PCR: 94 °C for 5 min; 98 °C for 10 s, 60 °C for 30 s, 68 °C for 2 min / kb, 25 cycles; 68 °C for 10 min. The plasmid template is digested by DpnI restriction endonuclease (Thermo), and E. coli BL21 (DE3) is electroporated to obtain more than 10 4 clones of the random mutation library.

[0086] 2. High-throughput screening of the mutant library

[0087] A single colony of the mutant library strain is picked from an LB plate and inoculated into a 96-well plate (each well contains 110 μL of liquid LB-Kan medium), which is incubated at 37 °C, 400 rpm for 12 h. Then 60 μL of the bacterial solution is taken from each well and inoculated into a 96-well deep well plate (each well contains 240 μL of liquid TB-Kan-0.2 mM IPTG), which is incubated at 25 °C, 400 rpm for 12-16 h. The bacterial cells are collected by centrifugation at 4 °C, 4000 rpm for 10 min, and the supernatant is removed. Then the bacterial cells are washed with pre-cooled normal saline, collected by centrifugation at 4 °C, 4000 rpm for 10 min, and the supernatant is removed. Each well is added with 200 μL of enzyme reaction solution (100 mM phosphate buffer with pH value of 7.5, 100 μg / mL FeSO4, 0.5 mM ascorbic acid, 5 mM α-ketoglutaric acid, 5 mM penicillin G) for reaction. The bacterial cells are resuspended, incubated at 25 °C, 220 rpm for 0.5 h, centrifuged at 12000 rpm for 1 min, and the supernatant is taken for HPLC detection.

[0088] Strains with significantly improved enzyme activity (i.e. substrate penicillin G conversion rate or product G-7-ADCA generation rate) were selected, plasmids were extracted for nucleic acid sequencing, the amidoacylase related fragments in the genome were compared with SEQ ID NO: 2 to determine the amino acid mutation sites, and the strain with the highest enzyme activity improvement was used as the starting strain for the next round of random mutant library construction, and the random mutant library construction and high-throughput reaction screening with penicillin G as the substrate were repeated. The high-throughput screening results of the first two rounds of random mutant libraries are shown in Table 1.

[0089] Table 1, high-throughput screening results of the first to second rounds of random mutant libraries

[0090]

[0091] Note: "+" represents that the enzyme activity is greater than 0% and less than or equal to 50% relative to the respective starting strain; "++" represents that the enzyme activity is greater than 50% and less than or equal to 100% relative to the respective starting strain; "+++" represents that the enzyme activity is greater than 100% and less than or equal to 200% relative to the respective starting strain; "++++" represents that the enzyme activity is greater than 200% relative to the respective starting strain.

[0092] The enzyme activity of the mutant KH8 (i.e. A129S, L159M, S261T mutant) obtained in the first two rounds was greatly improved relative to the wild-type enzyme.

[0093] Example 3: Third and fourth rounds of random mutation point library construction and high-throughput screening

[0094] 1. Construction of random mutation point library by error-prone PCR

[0095] The amidoacylase random mutation point library was constructed according to the method of Example 2.

[0096] 2. High-throughput screening of mutant library

[0097] Single colony from LB plate of mutant library strain was picked to 96-well plate (each well contains 110 μL of liquid LB-Kan medium), after incubation at 37°C, 400 rpm for 12 h, 60 μL of bacterial solution from each well was taken to 96-well deep well plate (each well contains 240 μL of liquid TB-Kan-0.2 mM IPTG), after incubation at 25°C, 400 rpm for 12-16 h, the bacteria were collected by centrifugation at 4°C, 4000 rpm for 10 min, and the supernatant was removed. Then, the bacteria were washed with pre-cooled normal saline, and collected by centrifugation at 4°C, 4000 rpm for 10 min, and the supernatant was removed. Each well was added with 200 μL of enzyme reaction solution (100 mM phosphate buffer with pH value of 7.5, 100 μg / mL FeSO4, 0.5 mM ascorbic acid, 10 mM α-ketoglutaric acid, 10 mM penicillin G) for reaction. The bacteria were resuspended, incubated at 25°C, 220 rpm for 0.5 h, centrifuged at 12000 rpm for 1 min, and the supernatant was taken for HPLC detection.

[0098] Strains with significantly improved enzyme activity (i.e. substrate penicillin G conversion rate or product G-7-ADCA generation rate) were selected, plasmids were extracted for nucleic acid sequencing, amino acid mutation sites were determined by base alignment, and the strain with the highest improvement of enzyme activity was used as the starting strain for the next round of random mutant library construction, and the random mutant library construction and high-throughput reaction screening with penicillin G as the substrate were repeated. The screening results are shown in Table 2.

[0099] Table 2, high-throughput screening results of the third to fourth rounds of random mutant library

[0100]

[0101] Note: "+" represents that the enzyme activity is greater than 0% and less than or equal to 50% relative to the respective starting strain; "++" represents that the enzyme activity is greater than 50% and less than or equal to 100% relative to the respective starting strain; "+++" represents that the enzyme activity is greater than 100% and less than or equal to 200% relative to the respective starting strain.

[0102] Through several rounds of mutation, the mutant KH15 (i.e. S98G, A129S, L159M, S261T and F294I mutant) was obtained, and the enzyme activity of the mutant KH15 was significantly improved relative to the wild-type enzyme. The amino acid sequence of the mutant KH15 is SEQ ID NO: 3.

[0103] 3, Construction of an engineering strain expressing the mutant KH15

[0104] The coding gene of mutant KH15, SEQ ID NO: 4, was synthesized by Suzhou Jinyuoshi Biotechnology Co., Ltd. according to the method described in Example 1, cloned into the Ndel, Xhol sites of plasmid pET24a, and the plasmid pET-KH15 was obtained, and the nucleotide sequence thereof was SEQ ID NO: 5; the recombinant plasmid pET-KH15 was transformed into the expression host Escherichia coli BL21 (DE3) by electroporation, and the recombinant Escherichia coli engineering bacteria expressing mutant KH15 were obtained, which were still named as EcKH15.

[0105] The following experiments were performed to investigate the catalytic ability of mutant strain EcKH15.

[0106] Example 4: Fermentor culture of expandase-producing strain

[0107] The engineering strain EcKH1 and the mutant strain EcKH15 were respectively subjected to fermentation culture using a 5L bioreactor. Single colonies of the two strains were respectively picked from LB plates into 5mL liquid LB medium containing Kan, and incubated at 37°C, 220rpm overnight. The next day, the inoculation amount was 5% (v / v), and the seed liquid was transferred to a 5L fermenter after the OD600nm of the culture reached 6. The culture was incubated at 37°C, 400-800rpm, and the dissolved oxygen was controlled within 20-30%. When the OD600 of the bacterial cells reached 20, IPTG was added to induce the expression of expandase, and the final concentration of IPTG was 0.2mM. The culture was continued at 25°C for 16-24h. The pH value was controlled at 6.8-7.2 using ammonia water during the whole fermentation process, and the aeration rate was controlled within 2.5-3.5L / min. After the fermentation was completed, the bacterial cells were collected by centrifugation at 4°C, 10000rpm for 10min, resuspended with 100mM phosphate buffer (pH 7.5), and then subjected to high-pressure disruption to obtain the whole-cell reaction solution.

[0108] Example 5: Cell-catalyzed production of G-7-ADCA

[0109] The 100mL reaction system included: 100mM phosphate buffer (pH 7.5), 100μg / mL FeSO4, 0.5mM ascorbic acid, 10mM α-ketoglutaric acid, 10mM penicillin G, and the whole-cell reaction solution corresponding to 5g wet bacterial cells. The reaction was carried out at 25°C, 300rpm for 2h, and then the sample was taken, filtered and subjected to HPLC detection. Figure 2 The HPLC spectrum of the cell-catalyzed preparation of G-7-ADCA by strain EcKH15 is shown.

[0110] The results show that the production rate of G-7-ADCA in the reaction system of the whole cell reaction solution of strain EcKH15 is 64.8% after 2h reaction, while the production rate of G-7-ADCA in the reaction system of the whole cell reaction solution of strain EcKH1 is only 12.3%. It is indicated that the enzyme activity of mutant KH15 is more than 4 times higher than that of wild enzyme (KH1).

[0111] The above merely describes preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. A mutant expandase enzyme, characterized in that, the amino acid sequence of which is shown as SEQ ID NO:

3.

2. A DNA molecule, characterized in that, which comprises a gene encoding the mutant expandase as claimed in claim 1.

3. The DNA molecule of claim 2, wherein, the nucleotide sequence of the gene encoding the mutant expandase as claimed in claim 1 is shown as SEQ ID NO:

4.

4. A recombinant plasmid, characterized by which comprises the DNA molecule as claimed in claim 3.

5. The recombinant plasmid of claim 4, wherein, the nucleotide sequence of which is shown as SEQ ID NO:

5.

6. A microorganism, characterized in that, which is a transformant transformed with the recombinant plasmid as claimed in claim 5.

7. The microorganism of claim 6, wherein, The microbial host is selected from the group consisting of Escherichia coli, Bacillus subtilis, Vibrio natriegens, Corynebacterium glutamicum, Pichia pastoris and Saccharomyces cerevisiae.

8. The microorganism of claim 7, wherein, The microbial host is Escherichia coli BL21 (DE3).

9. Use of the mutant expandase as claimed in claim 1 or the microorganism as claimed in claim 6 in the production of phenylacetyl-7-amino-deacetoxycephalosporanic acid (G-7-ADCA).

10. The use according to claim 9, characterized in that, The mutant expandase as claimed in claim 1 or the microorganism as claimed in claim 6 is used to catalyze the production of phenylacetyl-7-amino-deacetoxycephalosporanic acid (G-7-ADCA) with penicillin G as the reaction substrate.

Images