Penicillin G acylase mutant with high synthetic activity and low hydrolytic activity and application of penicillin G acylase mutant

Abstract

The invention discloses a penicillin G acylase mutant with high synthetic activity and low hydrolytic activity and application of the penicillin G acylase mutant, and belongs to the field of bioengineering. The penicillin G acylase gene derived from Escherichia coli is subjected to site-specific mutagenesis, so that the amino acid sequence coded by the penicillin G acylase gene mutates from phenylalanine F to alanine A at the 24th site of an alpha chain, the 146th site of the alpha chain mutates from phenylalanine F to valine V, and the 386th site of a beta chain mutates from serine S to alanine A to obtain the mutant. According to the penicillin G acylase mutant alphaF24A / betaS386A disclosed by the invention, compared with the conversion rate of cefamandole synthesized by catalyzing the reaction of methyl mandelate and 3-(1-methyl-1H-tetrazole-5-yl) thiomethyl-7-aminocephalosporanic acid (7-TMCA) within 4 hours before mutation, the conversion rate of cefamandole synthesized by catalyzing the reaction of methyl mandelate and 3-(1-methyl-1H-tetrazole-5-yl) thiomethyl-7-aminocephalosporanic acid (7-TMCA) is improved by 13.70%, and the yield of a side reaction product mandelic acid is reduced by 82%. The penicillin G acylase mutant disclosed by the invention can be used for catalyzing methyl mandelate and 7-TMCA to synthesize cefamandole while greatly reducing the occurrence of side reactions, and can be applied to preparation of cephalosporin antibiotics.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and specifically relates to a penicillin G acylase mutant with high synthetic activity and low hydrolysis activity, its encoding nucleic acid, a recombinant expression vector containing the nucleic acid sequence, a recombinant expression transformant, and their applications. Background Art

[0002] Penicillin G acylase (PGA, EC 3.5.1.11) is a key enzyme for semi-synthetic β-lactam antibiotics and belongs to the amide bond hydrolase class. It consists of two subunits, an α-chain and a β-chain, and only has enzymatic activity when they are combined in a special form (Mayer J, Pippel J, Guenther G, et al. Crystal structures and protein engineering of three different penicillin G acylases from Gram-positive bacteria with different thermostability [J]. Applied Microbiology and Biotechnology, 2019, 103(18): 7537-7552.). It can not only catalyze the hydrolysis of penicillin and its ring-expanded acid to remove the side chain, but also catalyze the condensation of 6-aminopenicillanic acid (6-APA) or 7-aminocephalosporanic acid (7-ADCA) with the side chain to produce semi-synthetic β-lactam antibiotics, as well as catalyze the hydrolysis and synthesis of penicillin and the first-generation and some second-generation cephalosporin antibiotics. Currently, only one PGA from Escherichia coli or its mutant has been reported to be able to synthesize cefamandole, but due to problems such as its high hydrolysis rate for acyl donors and cefamandole products, it is necessary to add an excessive amount of acyl donors and water-miscible organic solvents to improve production efficiency, but these methods increase the raw material consumption and the cost of downstream purification.

[0003] Cefamandole is one of the important β-lactam antibiotics for inhibiting bacterial infections. Compared with the first-generation cephalosporins, cefamandole is more sensitive to Gram-negative bacteria. It is commonly used clinically to treat pulmonary and urinary tract infections. Currently, the mainstream chemical synthesis method of cefamandole is the acyl chloride method, but multiple steps such as the protection / deprotection of reactive groups and the harsh reaction conditions controlled at low temperature (-40 °C or lower) are still challenges in industrial production. In addition, environmental problems in the chemical synthesis pharmaceutical process are on the rise because some of them involve toxic substances (Bryan M C, Dunn P J, Entwistle D, et al. Key Green Chemistry research areas from a pharmaceutical manufacturers' perspective revisited[J]. Green Chemistry, 2018, 20(22): 5082-5103.). Using penicillin G acylase as a biocatalyst to produce β-lactam antibiotics has good selectivity, high catalytic efficiency and environmental friendliness. However, the hydrolysis rate is relatively high during the enzymatic synthesis of cefamandole. Therefore, it is of great significance to develop an efficient PGA for the enzymatic synthesis of cefamandole.

[0004] Rational design in protein engineering is based on a certain understanding of the enzymological information of the target protein, such as its sequence, structure, catalytic mechanism, etc. With the help of various bioinformatics software, the effects of mutations at different sites on the stability and catalytic performance of the target protein are predicted. The whole plasmid PCR method is one of the most widely used site-directed mutagenesis methods at present, with the advantages of simple operation, rapidity and high efficiency.

[0005] Currently, the research on penicillin G acylase mainly focuses on aspects such as immobilization, enzymatic properties, and the design and modification of catalytic performance. Site-directed mutagenesis of PGA has been successfully applied to improve the Vs / Vh1 value (the ratio of the initial rate of antibiotic product synthesis to the initial rate of by-product formation) in the biosynthesis of penicillin and amoxicillin. The Vs / Vh1 value of the mutant of AfPGA from Alcaligenes faecalis increased by 4.2 times and the yield of penicillin was 95%, while the wild-type enzyme AfPGA only showed a Vs / Vh1 value of 0.49 and a conversion rate of 23.1% (Li A, Cheng C, Qi W, et al. Combing multiple-site-directed mutagenesis of penicillin G acylase from Achromobacter xylosoxidans PX02 with improved catalytic properties for cefamandole synthesis[J]. Int J Biol Macromol, 2021, 175: 322-329.). In addition, mutants of EcPGA from Escherichia coli showed significant improvements in the yields of cefalexin (99% VS 76% of the wild type), cefaclor (99% VS 65%), and cefprozil (99% VS 60%) (Cecchini D A, Pavesi R, Sanna S, et al. Efficient biocatalyst for large-scale synthesis of cephalosporins, obtained by combining immobilization and site-directed mutagenesis of penicillin acylase[J]. Applied Microbiology and Biotechnology, 2012, 95(6): 1491-1500.).

[0006] Currently, the research on penicillin G acylase focuses on the catalytic synthesis of a few semi-synthetic β-lactam antibiotics, such as ampicillin, amoxicillin, cefalexin, cefadroxil, and cefaclor, that is, penicillin or first-generation cephalosporins. However, since penicillin and first-generation cephalosporin antibiotics have been widely used for more than 70 years, drug resistance has become very obvious. Therefore, it is very important to use penicillin G acylase to synthesize second-generation cephalosporins, such as cefamandole. Summary of the Invention

[0007] The object of the present invention is to provide a penicillin G acylase mutant with high synthetic activity and low hydrolysis activity, its encoding nucleic acid, a recombinant expression vector containing the nucleic acid sequence, a recombinant expression transformant, and their applications, so as to solve the current situation in the prior art where there is a lack of penicillin G acylase with high synthetic activity and low hydrolysis activity.

[0008] In view of the problems of the synthetic and hydrolysis activities of penicillin G acylase, the present invention performs site-directed mutagenesis on it with the aid of bioinformatics software, and further improves the synthetic activity of penicillin G acylase while reducing the hydrolysis activity, laying a foundation for the application of penicillin G acylase in the synthesis of cefamandole.

[0009] In previous research of this application, the gene sequence encoding penicillin G acylase (PGA) was obtained from the Escherichia coli genome in the NCBI database and cloned into Escherichia coli BL21(DE3) for successful expression. At the same time, through molecular modification, its synthetic activity towards cefamandole was improved, and side reactions caused by hydrolysis during the reaction were reduced, laying a foundation for using penicillin G acylase to catalytically synthesize cefamandole.

[0010] Using the idea of rational design and with the assistance of bioinformatics software such as Autodock and Rosetta, the present invention obtained a penicillin G acylase mutant with high synthetic activity and low hydrolysis activity.

[0011] First, the present invention uses molecular simulation software such as Autodock and pymol to perform molecular docking between penicillin G acylase and cefamandole, and then analyzes it in combination with the catalytic mechanism of penicillin G acylase to obtain the sites to be mutated: phenylalanine at position 24 of the α-chain, phenylalanine at position 146 of the α-chain, and serine at position 386 of the β-chain. Then, the enzyme design function of Rosetta is used for calculation, and 576 output results are obtained. The results are screened based on a penalty score less than 30, and a total of 21 mutants with improved theoretical performance are obtained. These 21 mutants are respectively constructed and experimentally verified, and finally 4 mutant enzymes with high synthetic activity and low hydrolysis activity are obtained. The problem of high hydrolysis rate when using penicillin G acylase to synthesize cefamandole is preliminarily solved.

[0012] The object of the present invention can be achieved by the following technical solutions:

[0013] One of the technical solutions of the present invention: Provide a penicillin G acylase mutant with high synthetic activity and low hydrolysis activity, which is a mutant of wild-type penicillin G acylase. The wild-type penicillin G acylase is composed of an α-chain and a β-chain. The amino acid sequence of the α-chain of the wild-type penicillin G acylase is shown in SEQ ID NO.1, and the amino acid sequence of the β-chain of the wild-type penicillin G acylase is shown in SEQ ID NO.2.

[0014] The penicillin G acylase mutant with high synthetic activity and low hydrolysis activity is selected from one of the following:

[0015] (1) Mutate phenylalanine F at position 24 of the α-chain with the amino acid sequence shown in SEQ ID NO.1 to alanine A;

[0016] (2) Mutate phenylalanine F at position 146 of the α-chain with the amino acid sequence shown in SEQ ID NO.1 to valine V;

[0017] (3) Mutate serine S at position 386 of the β-chain with the amino acid sequence shown in SEQ ID NO.2 to alanine A;

[0018] (4) Mutate phenylalanine F at position 24 of the α-chain with the amino acid sequence shown in SEQ ID NO.1 to alanine A, and mutate serine S at position 386 of the β-chain with the amino acid sequence shown in SEQ ID NO.2 to alanine A;

[0019] (5) Mutate phenylalanine F at position 146 of the α-chain with the amino acid sequence shown in SEQ ID NO.1 to valine V, and mutate serine S at position 386 of the β-chain with the amino acid sequence shown in SEQ ID NO.2 to alanine A;

[0020] (6) Mutate phenylalanine F at position 24 of the α-chain with the amino acid sequence shown in SEQ ID NO.1 to alanine A, and mutate phenylalanine F at position 146 of the α-chain with the amino acid sequence shown in SEQ ID NO.1 to valine V;

[0021] (7) Mutate phenylalanine F at position 24 of the α-chain with the amino acid sequence shown in SEQ ID NO.1 to alanine A, mutate phenylalanine F at position 146 of the α-chain with the amino acid sequence shown in SEQ ID NO.1 to valine V, and mutate serine S at position 386 of the β-chain with the amino acid sequence shown in SEQ ID NO.2 to alanine A.

[0022] Preferably, the high synthetic activity and low hydrolysis activity penicillin G acylase mutant is obtained by mutating phenylalanine F at position 24 of the α-chain of the amino acid sequence shown in SEQ ID NO.1 to alanine A, and mutating serine S at position 386 of the β-chain of the amino acid sequence shown in SEQ ID NO.2 to alanine A.

[0023] The high synthetic activity and low hydrolysis activity penicillin G acylase mutant of the present invention is obtained by site-directed mutagenesis of the wild-type penicillin G acylase gene of the parental Escherichia coli (the nucleotide sequence of which is shown in SEQ ID NO.3), and mutations are introduced into phenylalanine F at position 24 of the α-chain, phenylalanine F at position 146 of the α-chain, and serine S at position 386 of the β-chain of penicillin G acylase by whole plasmid PCR technology.

[0024] In the present invention, the naming method of "chain name + original amino acid abbreviation + mutation position + replacement amino acid abbreviation" is used to represent the penicillin G acylase mutant. For example, the mutant αF24A / αF146V / βS386A means that phenylalanine F at position 24 of the α-chain of the amino acid sequence shown in SEQ ID NO.1 is mutated to alanine A, phenylalanine F at position 146 of the α-chain of the amino acid sequence shown in SEQ ID NO.1 is mutated to valine V, and serine S at position 386 of the β-chain of the amino acid sequence shown in SEQ ID NO.2 is mutated to alanine A.

[0025] The high synthetic activity and low hydrolysis activity penicillin G acylase mutant provided by the present invention has higher activity and lower hydrolysis activity than the wild-type penicillin G acylase.

[0026] Using methyl mandelate and 7-TMCA as substrates, cephalothin is catalytically synthesized using the penicillin G acylase mutant. Compared with the wild-type penicillin G acylase (the 4h yield of cephalothin is 70.46%, and the concentration of the side reaction product mandelic acid is 91.78 mM), the 4h yields of cephalothin of the mutants αF24A, βS386A, αF146V / βS386A, αF24A / βS386A, αF24A / αF146V / βS386A are 71.87%, 71.16%, 59.94%, 80.11%, 11.36% respectively, among which αF24A / βS386A shows the best synthetic activity, which is 1.14 times that of the wild-type; the concentrations of the side reaction product mandelic acid are 33.29 mM, 89.54 mM, 20.04 mM, 16.37 mM, 13.84 mM respectively, among which αF24A / βS386A shows the lowest hydrolysis activity towards methyl mandelate, which is 17.84% of the wild-type.

[0027] It can be seen that compared with its wild type, the penicillin G acylase mutant provided by the present invention has improved synthetic activity and decreased hydrolysis activity.

[0028] The second technical solution of the present invention provides an isolated nucleic acid, which is a nucleic acid encoding the penicillin G acylase mutant with high synthetic activity and low hydrolysis activity as described in the first technical solution.

[0029] The method for preparing the nucleic acid of the penicillin G acylase mutant with high synthetic activity and low hydrolysis activity described in the present invention adopts the conventional preparation methods in the art. Preferred preparation methods include: obtaining the coding DNA of the penicillin G acylase mutant with high thermal stability, high synthetic activity and low hydrolysis activity through gene cloning technology, or obtaining the coding DNA of the penicillin G acylase mutant with high synthetic activity and low hydrolysis activity through the method of artificial total sequence synthesis. The method for obtaining the nucleic acid molecule encoding the penicillin G acylase mutant with high synthetic activity and low hydrolysis activity by gene cloning technology described in the present invention is to obtain the coding DNA encoding the penicillin G acylase mutant with high synthetic activity and low hydrolysis activity through polymerase chain reaction (PCR).

[0030] The PCR system and PCR reaction program involved therein can be obtained according to conventional biotechnology means.

[0031] The third technical solution of the present invention: provides a recombinant expression vector containing the nucleic acid of the penicillin G acylase mutant described in the present invention, for example, it can be a recombinant expression plasmid.

[0032] The recombinant expression plasmid can be constructed by conventional methods in the art, and the nucleic acid sequence encoding the penicillin G acylase mutant gene of the present invention is ligated to the commercially available empty plasmid. The plasmid can adopt various common plasmids in the art, including but not limited to expression vectors such as pET expression vector, pRSF expression vector, pUC expression vector or pBR expression vector, etc.

[0033] For different expression hosts, the preferred plasmid vectors may be different. The penicillin G acylase mutant gene can be operably cloned downstream of the expression regulatory sequence in the selected vector to achieve constitutive or inducible expression of the penicillin G acylase mutant. For Escherichia coli host, pET24a plasmid is preferably used as the vector.

[0034] The fourth technical solution of the present invention: provides a recombinant expression transformant containing the penicillin G acylase mutant gene or its recombinant expression vector described in the present invention.

[0035] The recombinant expression transformant described in the present invention can be prepared by transforming the recombinant expression vector described in the present invention into a host cell.

[0036] The host cell is various conventional host cells in the art, including but not limited to any one of Escherichia coli, yeast, Bacillus, Lactobacillus or filamentous fungi. The host cell of the present invention is preferably Escherichia coli. Transforming the recombinant expression plasmid of the present invention into a host cell can obtain the target recombinant expression transformant.

[0037] Preferably, the recombinant plasmid containing the mutated gene is transformed into the host Escherichia coli BL21(DE3) for expression to obtain a recombinant expression transformant containing the penicillin G acylase mutant plasmid of the present invention.

[0038] Technical solution five of the present invention: Provide a recombinant penicillin G acylase mutant catalyst, and the recombinant penicillin G acylase mutant catalyst is any one of the following forms:

[0039] (1) Culturing the recombinant expression transformant of the present invention and separating the transformed cells containing the penicillin G acylase mutant;

[0040] (2) Lyophilized cells obtained by lyophilizing the transformed cells as described in (1);

[0041] (3) Crude enzyme solution prepared by disrupting the transformed cells containing the penicillin G acylase mutant;

[0042] (4) Pure enzyme solution obtained by purifying the cell lysate containing the penicillin G acylase mutant.

[0043] Technical solution six of the present invention: Provide a method for obtaining the recombinant penicillin G acylase mutant, and use the transformant separated containing the penicillin G acylase mutant to express the penicillin G acylase mutant.

[0044] Preferably, using plasmid pET24a - PGA as a template, designing primers, obtaining a recombinant plasmid encoding the mutant by PCR, inoculating the Escherichia coli seed solution containing the recombinant plasmid into a fermentation medium, and fermenting at 18°C and 120 rpm for 24 h; the composition of the fermentation medium is: yeast extract 36 g / L, glucose 5 g / L, K2HPO4 12.5 g / L, KH2PO4 2.3 g / L, CaCl2 0.078 g / L, pH 8.0.

[0045] Technical solution seven of the present invention: Provide the application of the recombinant penicillin G acylase mutant or the recombinant penicillin G acylase mutant catalyst in the synthesis of cefamandole.

[0046] In an embodiment of the present invention, methyl mandelate and 7 - TMCA are selected as substrates.

[0047] In one embodiment of the present invention, the reaction conditions are as follows: the reaction temperature is 5 - 35°C, preferably 20 - 30°C, more preferably 25°C, and the pH is 5.5 - 9.0, preferably 7.5.

[0048] In the application of the penicillin G acylase mutant provided by the present invention, it is applied to catalyze the synthesis of cefamandole from methyl mandelate and 7 - TMCA. When the concentration of methyl mandelate is 150 mM, the concentration of 7 - TMCA is 50 mM (the solvent is PBS solution), the reaction temperature is 25°C, the pH is 7.5, and the cell amount is 5 g / L. After reacting for 4 h, the yield of cefamandole can reach 80.11%, and the yield of the side - reaction product mandelic acid is 16.37 mM. While under the same conditions, the yield of cefamandole of the wild - type is 70.46%, which is increased by 14%, and the yield of the side - reaction product mandelic acid is 91.78 mM, which is decreased by 82.16%.

[0049] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0050] By using the method of rational design and combining molecular simulation software such as Autodock and Rosetta, site - directed mutagenesis of penicillin G acylase was carried out, and a penicillin G acylase mutant αF24A / βS386A with high synthetic activity and low hydrolysis activity was obtained. When catalyzing the reaction of methyl mandelate and 7 - TMCA to synthesize cefamandole, the yield can reach 80.11%, and at the same time, the side - reaction is reduced by 82.16%. This lays a foundation for the industrial use of this enzyme to synthesize cefamandole. Description of the Drawings

[0051] Figure 1 It is the cefamandole yield when different penicillin G acylase mutants constructed in the present invention synthesize cefamandole;

[0052] Figure 2 It is the yield of the side - product mandelic acid when different penicillin G acylase mutants constructed in the present invention synthesize cefamandole;

[0053] Figure 3 It is the influence of temperature on the cefamandole yield when the penicillin G acylase mutant αF24A / βS386A constructed in the present invention and the wild - type penicillin G acylase PGA synthesize cefamandole;

[0054] Figure 4 It is the influence of temperature on the yield of the side - product mandelic acid when the penicillin G acylase mutant αF24A / βS386A constructed in the present invention and the wild - type penicillin G acylase PGA synthesize cefamandole;

[0055] Figure 5The effect of pH on the yield of cefamandole when the penicillin G acylase mutant αF24A / βS386A constructed in the present invention and the wild-type penicillin G acylase PGA are used to synthesize cefamandole;

[0056] Figure 6 The effect of pH on the yield of by-product mandelic acid when the penicillin G acylase mutant αF24A / βS386A constructed in the present invention and the wild-type penicillin G acylase PGA are used to synthesize cefamandole. Detailed implementation manners

[0057] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0058] The medium formulations involved in the examples are as follows:

[0059] LB liquid medium: Tryptone 10 g / L, yeast extract powder 5 g / L, NaCl 10 g / L, pH 7.0.

[0060] LB solid medium: Add 15 g / L agar to the basic formulation of LB liquid medium.

[0061] Fermentation medium: Yeast extract 36 g / L, glucose 5 g / L, K2HPO4 12.5 g / L, KH2PO4 2.3 g / L, CaCl2 0.078 g / L, pH 8.0.

[0062] The reaction conditions for synthesizing cefamandole using methyl mandelate and 7-TMCA as substrates:

[0063] The reaction system is 1 mL: Cells 5 g / L. Weigh 0.764 g of 7-TMCA and dissolve it in 20 mL of PBS with pH = 7.5. After adding an equimolar amount of 2 M sodium hydroxide to completely dissolve it, adjust the pH to 7.5 by adding orthophosphoric acid while mixing. Add 1.159 g of methyl mandelate, place it in an oven until it dissolves, take 430 μL, add 570 μL of crude enzyme solution, mix well, pH 7.5, set the temperature at 25 °C, and react for 4 h. After the reaction is completed, take 100 μL of the mixed solution, add 200 μL of methanol containing 3 mM internal standard (p-nitrophenol) and 700 μL of 50 mM disodium hydrogen phosphate with pH = 6.5, and perform high-performance liquid chromatography (HPLC) to analyze the reaction results.

[0064] HPLC analysis method

[0065] Analysis method (HPLC method): The chromatographic column is a reversed-phase column C18 (Diamonsil plus, 4.6 mm * 250 mm * 5 μm); mobile phase: methanol: phosphate (50 mM disodium hydrogen phosphate, pH 6.5) = 2:8; flow rate: 0.8 mL / min; detection wavelength: 224 nm; column temperature: 25 °C; sample injection volume: 10 μL; the retention times of mandelic acid, 7-TMCA, cefamandole, and methyl mandelate are 4.9 min, 8.4 min, 18.7 min, and 21.1 min respectively.

[0066] Calculation method for the yield of cefamandole:

[0067] Yield α1 = (32.623 * A1 / A0 + 0.1014) / B * 100%

[0068] Where A1 represents the peak area of cefamandole, A0 represents the internal standard (p-nitrophenol), B represents the concentration of the substrate 7-TMCA, and the relational expression y = 32.623x + 0.1014 is the standard curve of the internal standard method for cefamandole.

[0069] Calculation method for the concentration of mandelic acid:

[0070] Concentration α2 = 127.95 * A2 / A0 - 4.7024

[0071] Where A1 represents the peak area of mandelic acid, A0 represents the internal standard (p-nitrophenol), and the relational expression y = 127.95x - 4.7024 is the standard curve of the internal standard method for mandelic acid.

[0072] Unless otherwise specified, the specific experiments in the following examples are carried out according to the conventional methods and conditions in the art.

[0073] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific examples. It is necessary to state here that the following specific examples are only used for a clearer description of the present invention and should not be construed as limiting the protection scope of the present invention. Any non-essential improvements made to the present invention are included in the protection scope of the present invention.

[0074] Example 1

[0075] Construction of penicillin G acylase mutants based on homologous recombination PCR method

[0076] Using the recombinant plasmid pET24a-PGA (which can be synthesized by conventional biological methods) stored in the laboratory as a template, plasmids containing mutant genes are amplified in vitro by PCR. PCR amplification uses the PrimeSTAR Max high-fidelity polymerase from Takara Biotechnology (Beijing) Co., Ltd. (Takara China). The PCR reaction system is as follows: (primer concentration is 10 μmol / L):

[0077] The primers for site-directed mutagenesis are (the underlined parts are the mutation sites):

[0078] αF24A primer-F (shown in SEQ ID NO.4):

[0079] 5’-GCGCATACCAGCC CGC CTGCGGACCA-3’

[0080] αF24A primer-R (shown in SEQ ID NO.5):

[0081] 5’-TGGTCCGCAG GCG GGCTGGTATGCGC-3’

[0082] βS386A primer-F (shown in SEQ ID NO.6):

[0083] 5’-TTATATTCAG TGC ACCAGTTGGGCC-3’

[0084] βS386A primer-R (shown in SEQ ID NO.7): <s

[0085] 5’-GGCCCAACTGGT GCA CTGAATATAA-3’

[0086] 68℃primer-F (shown in SEQ ID NO.8):

[0087] 5’-TTGAGTGAGCTGATACCGCTCGCCG-3’

[0088] 68℃primer-R (shown in SEQ ID NO.9):

[0089] 5’-CGGCGAGCGGTATCAGCTCACTCAA-3’

[0090] For PCR amplification, PrimeSTAR Max high-fidelity polymerase from Takara Biotechnology (Beijing) Co., Ltd. (Takara China) was used. The PCR reaction system is as follows: (primer concentration is 10 μmol / L):

[0091]

[0092] The PCR amplification program is: pre-denaturation at 95℃ for 10 min; denaturation at 98℃ for 10 s, annealing at 56℃ for 30 s, extension at 72℃ for 45 s, for 30 cycles; extension at 72℃ for 7 min; storage at 4℃.

[0093] After verifying the correctness of the 10 μL PCR product by nucleic acid electrophoresis, the reaction solution was recovered using a universal DNA purification and recovery kit. The two fragments of the plasmid were ligated using a ligase: 5 μL of each plasmid fragment and 10 μL of ligase, and incubated in a 50 °C water bath for 15 min. Then, the ligated plasmid was transformed into competent Escherichia coli DH5α cells, spread on an LB resistant solid plate (containing 50 μg / mL kanamycin Kan), cultured at 37 °C for 15 h, and 1 - 3 transformants were picked and transferred to an LB liquid medium (containing 50 μg / mL Kan) for culturing for 12 h. After that, the bacterial solution was taken and the plasmid was extracted using a plasmid extraction kit. The extracted plasmid was transformed into competent Escherichia coli BL21(DE3) cells. Spread on an LB resistant solid plate (containing 50 μg / mL Kan), cultured at 37 °C for 15 h, and 1 - 3 transformants were picked and transferred to an LB liquid medium (containing 50 μg / mL Kan) for culturing for 12 h. After that, the bacterial solution was sent for sequencing.

[0094] The double-site mutant was constructed using the successfully mutated single-site mutant plasmid as a template in the same way, and the triple-site mutant was constructed using the successfully mutated double-site mutant plasmid as a template in the same way.

[0095] Example 2

[0096] Cultivation and expression of the parent and mutant strains

[0097] Plate activation: Dip the glycerol tube bacterial solution with an inoculation loop, streak it in four areas on an LB resistant solid plate (containing 50 μg / mL Kan), and incubate it inverted at 37 °C in a constant temperature incubator for 12 h.

[0098] Seed culture: Scrape a loopful of bacteria from the solid plate and inoculate it into an LB liquid medium (containing 50 μg / mL Kan), with a liquid loading of 50 mL / 250 mL, and incubate it at 37 °C and 200 rpm on a shaker for 12 h.

[0099] Flask fermentation: Inoculate the 12 h cultured seed liquid into the fermentation medium (containing 50 μg / mL Kan) at an inoculation amount of 1%, with a liquid loading of 50 mL / 250 mL. First, incubate it at 37 °C and 200 rpm on a shaker for 4 h, place the medium in an ice-water bath for 5 min. After the temperature drops to 4 °C, add an IPTG inducer with a final concentration of 0.25 mM, lower the temperature to 18 °C and the rotation speed to 120 rpm, and culture for 24 h.

[0100] Example 3

[0101] Extraction of crude enzyme solutions of wild-type and mutant penicillin G acylase

[0102] Control the cell amount to 5 g / L, according to V 发酵液 ×OD 600 ×0.4 = V PBS ×5 g / L, calculate the amount of the fermentation broth taken.

[0103] Take a certain amount of the fermentation broth, centrifuge at 12000 rpm for 15 min, pour off the supernatant, wash with 0.8% physiological saline, centrifuge at 12000 rpm for 15 min, repeat twice, and finally resuspend the cells with 10 ml of 50 mM PBS at pH 7.5. Use an ultrasonic cell disruptor to disrupt the cells. The disruption conditions are: ultrasonic for 3 s, intermittent for 4 s, power 50%, and time 10 min. After disruption, centrifuge at 12000 rpm for 30 min, and the supernatant is the crude enzyme solution.

[0104] Example 4

[0105] Comparison of the synthetic activities of different mutants and wild-type enzymes

[0106] Use the above-extracted crude enzyme solution to catalyze the reaction of methyl mandelate and 7-TMCA to synthesize cefamandole according to the above method. As shown in the appendix Figure 1 As shown, the cefamandole yield of wild-type PGA is 70.46%, and the cefamandole yields of mutants αF24A, βS386A, αF146V / βS386A, αF24A / βS386A, and αF24A / αF146V / βS386A are 71.87%, 71.16%, 59.94%, 80.11%, and 11.36% respectively, which are 1.02, 1.01, 0.85, 1.14, and 0.16 times that of the wild-type respectively. Among them, αF24A / βS386A shows the best synthetic activity. As shown in the appendix Figure 2 As shown, at the end of the reaction, the concentration of the side reaction product mandelic acid of wild-type PGA is 91.78 mM, and the concentrations of the side reaction product mandelic acid of mutants αF24A, βS386A, αF146V / βS386A, αF24A / βS386A, and αF24A / αF146V / βS386A are 33.29 mM, 89.54 mM, 20.04 mM, 16.37 mM, and 13.84 mM respectively, which are 36.27%, 97.56%, 21.83%, 17.84%, and 15.08% of the wild-type respectively. Among them, αF24A / βS386A shows the lowest hydrolysis activity towards methyl mandelate.

[0107] It can be seen that the effect of mutant αF24A / βS386A is the best. Compared with its wild-type, its synthetic activity is significantly improved, and its hydrolysis activity is significantly decreased. In addition, compared with their wild-type, the synthetic activities of mutants αF24A and βS386A are also improved, and their hydrolysis activities are decreased.

[0108] Example 5

[0109] Comparison of the catalytic synthesis of cefamandole from methyl mandelate and 7-TMCA by mutant αF24A / βS386A and wild-type penicillin G acylase at different temperatures

[0110] According to the above method, the temperatures were set at 5°C, 10°C, 15°C, 20°C, 25°C, and 30°C respectively for the reaction. The results are as shown in the appendix Figure 3 As shown, the cefamandole yields of wild-type PGA were 17.30%, 59.96%, 59.55%, 63.39%, 70.46%, and 57.87% respectively. At the end of the reaction, as shown in the appendix Figure 4 As shown, the concentration of the side reaction product mandelic acid of wild-type PGA was 95.54 mM, 73.42 mM, 71.39 mM, 79.04 mM, 91.78 mM, and 75.79 mM respectively. The cefamandole yields of mutant αF24A / βS386A were 40.44%, 52.97%, 55.49%, 66.66%, 80.11%, and 69.85% respectively. At the end of the reaction, the concentration of the side reaction product mandelic acid of mutant αF24A / βS386A was 8.84 mM, 10.84 mM, 13.70 mM, 22.55 mM, 16.37 mM, and 43.72 mM respectively.

[0111] It can be seen that 25°C is a more suitable reaction temperature for the mutant αF24A / βS386A to catalyze the synthesis of cefamandole from methyl mandelate and 7-TMCA.

[0112] Example 6

[0113] Comparison of the catalytic synthesis of cefamandole from methyl mandelate and 7-TMCA by mutant αF24A / βS386A and wild-type penicillin G acylase at different pH values

[0114] According to the above method, the pH values were set at 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0 respectively for the reaction. The results are as shown in the appendix Figure 5 As shown, the cefamandole yields of wild-type PGA were 61.40%, 61.47%, 59.73%, 64.47%, 70.46%, 63.06%, 57.56%, and 41.26% respectively. At the end of the reaction, the results are as shown in the appendix Figure 6As shown, the concentrations of the wild-type PGA side reaction product mandelic acid were 54.54 mM, 53.59 mM, 50.02 mM, 61.63 mM, 91.78 mM, 76.97 mM, 84.21 mM, 82.52 mM, and the cefamandole yields of the mutant αF24A / βS386A were 5.45%, 11.85%, 27.11%, 47.55%, 80.11%, 62.73%, 58.90%, 59.01%, respectively. At the end of the reaction, the concentrations of the side reaction product mandelic acid of the mutant αF24A / βS386A were 0.63 mM, 1.82 mM, 5.08 mM, 5.67 mM, 16.37 mM, 28.90 mM, 29.49 mM, 31.78 mM.

[0115] It can be seen that pH 7.5 is a more suitable reaction pH for the mutant αF24A / βS386A to catalyze the synthesis of cefamandole from methyl mandelate and 7-TMCA.

[0116] The above description of the embodiments is for those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative labor. Therefore, the present invention is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art without departing from the scope of the present invention according to the disclosure of the present invention should be within the protection scope of the present invention.

[0117] The sequence information related to the present invention is summarized as follows:

[0118] SEQ ID NO.1: Amino acid sequence of the α-chain of wild-type penicillin G acylase

[0119] EQSSSEIKIVRDEYGMPHIYANDTWHLFYGYGYVVAQDRLFQMEMARRSTQGTVAEVLGKDFVKFDKDIRRNYWPDAIRAQIAALSPEDMSILQGYADGMNAWIDKVNTNPETLLPKQFNTFGFTPKRWEPFDVAMIFVGTMANRFSDSTSEIDNLALLTALKDKYGVSQGMAVFNQLKWLVNPSAPTTIAVQESNYPLKFNQQNSQTA

[0120] SEQ ID NO.2: Amino acid sequence of the β-chain of wild-type penicillin G acylase

[0121] SNMWVIGKSKAQDAKAIMVNGPQFGWYAPAYTYGIGLHGAGYDVTGNTPFAYPGLVFGHNGVISWGSTAGFGDDVDIFAERLSAEKPGYYLHNGKWVKMLSREETITVKNGQAETFTVWRTVHGNILQTDQTTQTAYAKSRAWDGKEVASLLAWTHQMKAKNWQEWTQQAAKQALTINWYYADVNGNIGYVHTGAYPDRQSGHDPRLPVPGTGKWDWKGLLPFEMNPKVYNPQSGYIANWNNSPQKDYPASDLFAFLWGGADRVTEIDRLLEQKPRLTADQAWDVIRQTSRQDLNLRLFLPTLQAATSGLTQSDPRRQLVETLTRWDGINLLNDDGKTWQQPGSAILNVWLTSMLKRTVVAAVPMPFDKWYSASGYETTQDGPTGSLNISVGAKILYEAVQGDKSPIPQAVDLFAGKPQQEVVLAALEDTWETLSKRYGNNVSNWKTPAMALTFRANNFFGVPQAAAEETRHQAEYQNRGTENDMIVFSPTTSDRPVLAWDVVAPGQSGFIAPDGTVDKHYEDQLKMYENFGRKSLWLTKQDVEAHKESQEVLHVQR

[0122] SEQ ID NO.3: Wild - type penicillin G acylase gene

[0123]

[0124] SEQ ID NO.4: Primer αF24A primer-F

[0125] 5’-GCGCATACCAGCC CGC CTGCGGACCA-3’

[0126] SEQ ID NO.5: Primer αF24A primer-R

[0127] 5’-TGGTCCGCAG GCG GGCTGGTATGCGC-3’

[0128] SEQ ID NO.6: Primer βS386A primer-F

[0129] 5’-TTATATTCAG TGC ACCAGTTGGGCC-3’

[0130] SEQ ID NO.7: Primer βS386A primer-R

[0131] 5’-GGCCCAACTGGT GCA CTGAATATAA-3’

[0132] SEQ ID NO.8: Primer 68℃primer-F

[0133] 5’-TTGAGTGAGCTGATACCGCTCGCCG-3’

[0134] SEQ ID NO.9: Primer 68℃primer-R

[0135] 5’-CGGCGAGCGGTATCAGCTCACTCAA-3’

Claims

1. A penicillin G acylase mutant with high synthetic activity and low hydrolysis activity, characterized in that, It is a mutant of wild-type penicillin G acylase, and the wild-type penicillin G acylase is composed of an α-chain and a β-chain. The amino acid sequence of the α-chain of the wild-type penicillin G acylase is shown as SEQ ID NO.1, and the amino acid sequence of the β-chain of the wild-type penicillin G acylase is shown as SEQ ID NO.

2. The penicillin G acylase mutant with high synthetic activity and low hydrolysis activity is selected from one of the following: (1) The phenylalanine F at position 24 of the α-chain of the amino acid sequence shown as SEQ ID NO.1 is mutated to alanine A; (2) The phenylalanine F at position 146 of the α-chain of the amino acid sequence shown as SEQ ID NO.1 is mutated to valine V; (3) The serine S at position 386 of the β-chain of the amino acid sequence shown as SEQ ID NO.2 is mutated to alanine A; (4) The phenylalanine F at position 24 of the α-chain of the amino acid sequence shown as SEQ ID NO.1 is mutated to alanine A, and the serine S at position 386 of the β-chain of the amino acid sequence shown as SEQ ID NO.2 is mutated to alanine A; (5) The phenylalanine F at position 146 of the α-chain of the amino acid sequence shown as SEQ ID NO.1 is mutated to valine V, and the serine S at position 386 of the β-chain of the amino acid sequence shown as SEQ ID NO.2 is mutated to alanine A; (6) The phenylalanine F at position 24 of the α-chain of the amino acid sequence shown as SEQ ID NO.1 is mutated to alanine A, and the phenylalanine F at position 146 of the α-chain of the amino acid sequence shown as SEQ ID NO.1 is mutated to valine V; (7) The phenylalanine F at position 24 of the α-chain of the amino acid sequence shown as SEQ ID NO.1 is mutated to alanine A, the phenylalanine F at position 146 of the α-chain of the amino acid sequence shown as SEQ ID NO.1 is mutated to valine V, and the serine S at position 386 of the β-chain of the amino acid sequence shown as SEQ ID NO.2 is mutated to alanine A.

2. A penicillin G acylase mutant with high synthetic activity and low hydrolysis activity according to claim 1, characterized in that The penicillin G acylase mutant with high synthetic activity and low hydrolysis activity is the one in which the phenylalanine F at position 24 of the α-chain of the amino acid sequence shown as SEQ ID NO.1 is mutated to alanine A, and the serine S at position 386 of the β-chain of the amino acid sequence shown as SEQ ID NO.2 is mutated to alanine A.

3. An isolated nucleic acid, characterized in that, The nucleic acid is a nucleic acid encoding the penicillin G acylase mutant with high synthetic activity and low hydrolysis activity as described in claim 1 or 2.

4. A recombinant expression vector comprising the nucleic acid according to claim 3.

5. A recombinant expression transformant comprising the recombinant expression vector according to claim 4.

6. A recombinant penicillin G acylase mutant catalyst, characterized in that, The recombinant penicillin G acylase mutant catalyst is any one of the following forms: (1) Culturing the recombinant expression transformant according to claim 5, and separating the transformed cells containing the penicillin G acylase mutant as described in claim 1 or 2; (2) Lyophilized stem cells obtained by lyophilizing the transformed cells as described in (1). (3) The transformed somatic cells containing the penicillin G acylase mutant are disrupted to obtain a crude enzyme solution; (4) A pure enzyme solution obtained by purifying the cell lysate containing the penicillin G acylase mutant.

7. Method for obtaining penicillin G acylase mutant with high synthetic activity and low hydrolysis activity according to claim 1 or 2, characterized in that, The penicillin G acylase mutant is expressed by using the recombinant expression transformant described in claim 5.

8. Use of the penicillin G acylase mutant with high synthetic activity and low hydrolysis activity described in claim 1 or 2 or the recombinant penicillin G acylase mutant catalyst described in claim 6 in the synthesis of cefamandole.

9. The application according to claim 8, wherein Using methyl mandelate and 7-TMCA as substrates to produce cefamandole and the side reaction product mandelic acid.

10. The application according to claim 8, wherein The reaction conditions are: the reaction temperature is 20 - 30 °C and the pH is 7.5.

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