A mutant of penicillin g acylase and its use in the synthesis of cefazolin

Abstract

The application belongs to the technical field of bioengineering, and particularly relates to a penicillin G acylase mutant and application thereof in synthesis of cefazolin. The amino acid sequence of the penicillin G acylase mutant is based on the sequence shown in SEQ ID NO. 1, wherein the alanine at the 123th position is mutated into other amino acids, and / or the glycine at the 565th position is mutated into other amino acids. The immobilized enzyme prepared by using the penicillin G acylase mutant of the application can maintain stable catalysis by using 10 batches of 30% supplement, and the reaction efficiency does not obviously attenuate. The penicillin G acylase mutant disclosed in the application has higher performance in catalyzing synthesis of cefazolin under low-temperature conditions, effectively inhibits a hydrolysis side reaction, reduces a side chain feeding ratio, and reduces production cost through recycling use of the immobilized enzyme, and is suitable for green industrialized production of cefazolin.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a penicillin G acylase mutant and its application in the synthesis of cefazolin. Background Technology

[0002] Penicillin G acylase (PGA) is a mature and widely used biocatalyst in the industrial field, extensively used in the enzymatic acylation synthesis of various semi-synthetic β-lactam antibiotics such as cephalexin, cefadroxil, and amoxicillin. The performance of the enzymatic synthesis of these antibiotics is highly dependent on the directional catalytic ability of PGA. The core evaluation indicators include two aspects: first, the S / H ratio, which is the ratio of the enzyme's synthetic capacity to its hydrolytic capacity, and this ratio directly determines the product yield and the level of hydrolysis byproducts; second, the molar ratio of the side chain to the parent core in the catalytic reaction, the lower the ratio, the less raw material waste and the stronger the process economy.

[0003] Currently, the mainstream industrial production route for cefazolin is chemical synthesis, using 7-aminocephalosporanic acid (7-ACA) as the starting core, and proceeding through multiple steps. For example, Chinese patent CN105541870B discloses a boron trifluoride catalytic mixed anhydride process, which can achieve high product purity, but requires a large amount of organic solvents and incurs high costs for waste treatment, which is inconsistent with the development direction of green and low-carbon manufacturing in the pharmaceutical industry.

[0004] Compared to chemical methods, enzymatic catalytic synthesis has natural advantages such as mild reaction conditions, high selectivity, and low emissions. There are numerous reports on protein engineering modifications of PGA. Chinese patent CN120699946A discloses a penicillin G acylase mutant derived from Kluyveromycin citrate, which, after site-directed mutagenesis, exhibits good catalytic performance against benzene ring side-chain substrates such as amoxicillin, cefaclor, and cefadroxil. However, all examples of this mutant focus on the aforementioned benzene ring side-chain substrates and do not involve the large-volume heterocyclic side chain of cefazolin, lacking measured activity and yield data for this specific side chain. In addition, Chinese patent CN115397979A discloses a penicillin G acylase mutant derived from Achromobacterium genus CCM4824, which is specifically used for targeted iterative screening of cefazolin synthesis. Under the condition of a core concentration of 200 mM 7-amino-3-[(5-methyl-1,3,4-thiadiazol-2-yl)thiomethyl]cephalosporanic acid (TDA), the molar ratio of acyl donor to core can be controlled to 1.2:1. However, the absolute value of the S / H ratio of its optimal mutant is only 1.7. The reaction is violent at high substrate concentrations, which will also aggravate the occurrence of hydrolysis side reactions. At the same time, this patent only completed the complete quantitative characterization of activity at 25°C, and did not disclose the enzyme activity retention rate data under lower temperature conditions, nor did it disclose the key parameters required for industrial scale-up, such as the immobilization preparation process and the batch reuse of enzyme. Summary of the Invention

[0005] The purpose of this invention is to provide a penicillin G acylase mutant and its application in the synthesis of cefazolin. Addressing the problems of low S / H ratio and insufficient catalytic activity at low temperatures in the synthesis of cefazolin using existing penicillin G acylases, a penicillin G acylase mutant containing novel mutation sites was obtained through random mutagenesis and high-throughput targeted screening. Unexpectedly, a dual-site mutation at A123αE and G565βA was found to simultaneously improve synthetic selectivity and catalytic performance at 10-15℃. By preparing an immobilized enzyme containing the above mutant, the aim is to effectively suppress substrate and product hydrolysis side reactions and reduce the proportion of side-chain feed.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a penicillin G acylase mutant (i.e., a PGA mutant), whose amino acid sequence is based on the sequence shown in SEQ ID NO. 1, with alanine at position 123 mutated to other amino acids, and / or glycine at position 565 mutated to other amino acids.

[0007] The sequence shown in SEQ ID NO. 1 is the wild-type penicillin G acylase, derived from Escherichia coli, with a total of 863 amino acids. It contains an α subunit and a β subunit, with the α subunit containing 306 amino acids and the β subunit containing 557 amino acids. The NCBI protein ID is AAY25991.1.

[0008] Secondly, the present invention also provides a penicillin G acylase mutant, the amino acid sequence of which is based on the sequence shown in SEQ ID NO. 1, with alanine at position 123 mutated to glutamic acid, i.e., A123αE. The amino acid sequence of this single mutant is shown in SEQ ID NO. 2.

[0009] Thirdly, the present invention also provides a penicillin G acylase mutant, whose amino acid sequence is based on the sequence shown in SEQ ID NO. 1, with glycine at position 565 mutated to alanine, G565βA.

[0010] Fourthly, the present invention also provides another penicillin G acylase mutant, the amino acid sequence of which is based on the sequence shown in SEQ ID NO. 1, with glycine at position 565 mutated to alanine and alanine at position 123 mutated to glutamic acid, i.e., A123αE+G565βA. The amino acid sequence of this double mutant is shown in SEQ ID NO. 3.

[0011] Furthermore, in addition to the aforementioned mutation sites, the penicillin G acylase mutant of the present invention may also include conventional sequence modifications in the art, such as adding a tag sequence to the amino or carboxyl terminus of the amino acid sequence, introducing conserved amino acid substitutions at other positions, or truncating the amino acid sequence. The conserved amino acid substitution refers to replacing a certain amino acid residue with an amino acid residue with similar side chain properties. As long as the polypeptide modified in the above way still retains catalytic activity higher than that of the wild type, it belongs to the penicillin G acylase mutant of the present invention.

[0012] Fifthly, the present invention provides a plasmid carrying the coding gene of any of the above-mentioned penicillin G acylase mutants.

[0013] The expression hosts of the plasmids are not limited to *Escherichia coli*, but also include common expression hosts in the field, such as *Escherichia coli* BL21(DE3), BL21, and M15; and also include *Bacillus subtilis*, yeast, *Aspergillus*, *Streptomyces*, etc. The expression vectors, in addition to the pET vectors commonly used in prokaryotes, also include vectors compatible with various host systems. The integration of the vector with the host includes not only free existence within the host, but also integration at specific locations in the genome and integration at random locations. The expression methods are not limited to expression within the host or secretory expression.

[0014] In a sixth aspect, the present invention provides the application of penicillin G acylase mutants in catalyzing the synthesis of cefazolin from the cefazolin core and acyl donor side chain.

[0015] As a limitation of the present invention, the cefazolin core is 7-amino-3-[(5-methyl-1,3,4-thiadiazol-2-yl)thiomethyl]cephalosporanic acid, the acyl donor side chain is methyl tetrazolium acetate, and the molar ratio of the cefazolin core to the acyl donor side chain is (1.7~2):1.

[0016] As another limitation of the present invention, the application of the penicillin G acylase mutant includes the following steps: S1. Prepare a reaction system containing the following components: 30-45 g / L of cefazolin core, add acyl donor side chain according to the molar ratio, bring to volume with 0.1-0.3 M PBK buffer, and adjust pH to 7.0-7.5; S2. Adjust the temperature of the reaction system to 10~30℃, add the immobilized enzyme obtained from the penicillin G acylase mutant, so that the total enzyme activity of the reaction system is 10000~16000U / L, stir the reaction for 0.15~5h, and obtain cefazolin.

[0017] Calculations show that when using the above reaction system, the conversion rate of the parent nucleus substrate is greater than 98%.

[0018] As a further limitation of the present invention, the temperature of the reaction system is adjusted to 10~15°C; When the concentration of the parent nucleus substrate is ≥40g / L, 15%~30% of the total enzyme activity should be added after 1 hour of reaction, and the total reaction time is 4~5 hours.

[0019] Among them, the penicillin G acylase mutant obtained by the mutation site discovered in this invention has a substrate conversion rate of more than 98% when it is used to catalyze the synthesis of cefazolin at a low temperature of 10~15℃.

[0020] In a seventh aspect, the present invention provides a method for preparing an immobilized enzyme of the penicillin G acylase mutant, comprising mixing a resin for immobilizing the enzyme with an enzyme solution of the purified and concentrated penicillin G acylase mutant at a ratio of 600-1500 U enzyme activity per gram of epoxy resin, adjusting the pH to 7.0-7.5, stirring at room temperature for 24-48 hours in the presence of 200-500 mM phosphate, washing away the enzyme adsorbed on the surface with 20-50 mM phosphate buffer, and obtaining the immobilized enzyme by filtration.

[0021] The method for preparing the enzyme solution of the penicillin G acylase mutant is as follows: the expression host strain containing the above-mentioned mutation site is inoculated and cultured to induce the expression of penicillin G acylase, and the enzyme solution is obtained by cell disruption, protein extraction and ultrafiltration concentration.

[0022] The resin used for immobilizing the enzyme includes various types of amino resins, epoxy resins, etc., used for preparing immobilized enzymes, preferably epoxy resin LX2000EP. The preferred method is to use a Buchner funnel for vacuum filtration.

[0023] The immobilized enzyme collected after the reaction is complete is washed with buffer and then used in the next batch reaction, which can be used for about 30 batches.

[0024] By adopting the above technical solution, the technical progress achieved by this invention compared with the prior art is as follows: This invention uses wild-type penicillin G acylase as the starting sequence and obtains a double mutant M3 containing two amino acid substitutions, A123αE and G565βA, through error-prone PCR random mutagenesis and targeted screening. The A123αE mutation, located in the α-subunit surface charge distribution region, significantly improves the conformational stability of the enzyme protein at 10–15°C, increasing the relative activity retention to over 70%. The G565βA mutation, located near the substrate binding pocket, moderately reduces the activity of hydrolysis side reactions. The two mutations act on the conformational stability region and the substrate binding functional region, respectively, exhibiting a synergistic effect after combined mutation. The relative activity retention at 10–15°C is further improved, and the synthesis / hydrolysis ratio is 33.3 times higher than that of the wild-type. This performance improvement is superior to the simple summation of the effects of the two individual mutations, achieving simultaneous optimization of low-temperature stability and synthetic selectivity.

[0025] The penicillin G acylase mutant of the present invention still maintains a substrate conversion rate of 98% at 10°C and a substrate concentration of 30 g / L. These results indicate that the mutant of the present invention can maintain high catalytic activity and synthetic selectivity under low-temperature conditions of 10–15°C, effectively suppressing hydrolysis side reactions of substrates and products under low-temperature conditions, improving the core conversion utilization rate, and maintaining a high conversion level even when the side chain molar ratio is reduced to 1.7:1, significantly reducing the side chain feed cost.

[0026] The penicillin G acylase mutant of this invention can be directly adapted to the existing PBK buffer system and epoxy resin immobilization process used in the synthesis of cefazolin, without requiring modifications to existing production equipment and processes. No significant reaction attenuation was observed after 30 consecutive batches of immobilized enzyme application; adding 10%–30% of the immobilized enzyme every 10 batches maintained stable catalysis, demonstrating good operational stability and industrial compatibility, aligning with the development direction of green manufacturing in the pharmaceutical industry.

[0027] Therefore, the mutant of this invention can achieve efficient enzymatic synthesis of cefazolin with high substrate concentration under low temperature conditions of 10~15℃, reduce the proportion of hydrolysis by-products and side chain raw material consumption, and further reduce the industrial production cost of cefazolin by recycling immobilized enzymes.

[0028] The present invention will be further described in detail below with reference to specific embodiments. Detailed Implementation

[0029] The present invention will be further described in detail below through specific embodiments. It should be understood that the described embodiments are only for explaining the present invention and do not limit the present invention.

[0030] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available. Experimental methods not specifically described in the embodiments are generally performed under standard conditions or as recommended by the manufacturer.

[0031] The molecular biology experimental procedures in the examples, including plasmid construction and culture medium preparation, were mainly performed in accordance with "Molecular Cloning: A Laboratory Manual" (3rd edition), edited by J. Sambrook and DW Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002. Example 1

[0032] This embodiment describes the A123αE, G565βA, and A123αE+G565βA mutants and their acquisition methods, as detailed below: A penicillin G acylase mutant A123αE, with the amino acid sequence shown in SEQ ID NO.2, has a mutation at amino acid position 123 from alanine to glutamic acid compared to the wild-type penicillin G acylase shown in SEQ ID NO.1.

[0033] A penicillin G acylase mutant, G565βA. Compared with the wild-type penicillin G acylase shown in SEQ ID NO.1, this mutant has an amino acid mutation at position 565, where glycine is replaced by alanine.

[0034] A penicillin G acylase mutant A123αE+G565βA, with the amino acid sequence shown in SEQ ID NO.2, has a mutation at amino acid position 123 (alanine) to glutamic acid and amino acid position 565 (glycine) to alanine compared to the wild-type penicillin G acylase shown in SEQ ID NO.1.

[0035] The preparation process of the above mutants is as follows: Using the penicillin-G acylase gene (PGA) sequence from Escherichia coli as a template, the full-length coding gene of wild-type PGA was synthesized. This wild-type penicillin-G acylase has a total of 863 amino acids, with 306 amino acids in the α subunit and 557 amino acids in the β subunit. Its amino acid sequence is shown in SEQ ID NO.1.

[0036] Primers Pga1 and Pga2 were designed for the full-length amplification of the wild-type penicillin G acylase gene. The primer sequences are shown in Table 1. High-fidelity DNA polymerase was used for PCR amplification to obtain the target gene fragment.

[0037] Table 1 Primer sequence information

[0038] The PCR amplification system used was 50 μL: 5 μL 10×PCR buffer, 4 μL 2.5 mM dNTP mixture, 1 μL 10 μM upstream primer Pga1, 1 μL 10 μM downstream primer Pga2, 1 μL template plasmid, 1 μL high-fidelity DNA polymerase, and sterile ultrapure water to a final volume of 50 μL. The PCR program was as follows: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 2.5 min, for a total of 30 cycles, and a final extension at 72℃ for 10 min.

[0039] After verification by agarose gel electrophoresis, the PCR products were double-digested with NdeI and HindIII restriction endonucleases. The digested products were then recovered and ligated into the pET-24a expression vector, which had been digested with the same enzymes. The ligation products were transformed into E. coli BL21(DE3) competent cells, plated on LB agar plates containing 50 mg / L kanamycin, and incubated at 37°C for 12–16 h. Single colonies were picked and cultured in shake flasks, and plasmids were extracted and sequenced for verification. The sequence was completely consistent with the design, yielding a wild-type PGA expression strain.

[0040] This embodiment obtained a PGA mutant adapted for cefazolin synthesis through high-throughput screening. Specifically, a random mutant gene library was constructed using error-prone PCR. By adjusting the final Mn2+ concentration in the reaction system to 0.1 mM and changing the dNTP substrate ratio, random point mutations were introduced into the PGA-encoding gene. Using the wild-type recombinant plasmid as a template, error-prone PCR amplification was performed. The amplification product was recovered after double enzyme digestion and ligated into the pET-24a expression vector, which was then transformed into E. coli BL21(DE3) competent cells to construct a random mutant expression library with a capacity of approximately 10⁴ cells.

[0041] Using the cefazolin synthesis reaction as a screening model, a 96-well plate system was employed for high-throughput primary screening. Synthetic activity and S / H ratio were used as primary screening indicators, yielding several positive clones with enhanced synthetic selectivity. Further screening under low-temperature conditions revealed that some mutant strains exhibited significantly superior low-temperature catalytic activity compared to the wild type. Ultimately, one mutant strain with the best overall performance was selected.

[0042] Sequencing analysis of the plasmid extracted from the positive strain confirmed the presence of two amino acid mutations at A123αE and G565βA. To verify the independent function and synergistic effect of these two sites, site-directed mutagenesis primers were designed. Using the wild-type plasmid as a template, whole-plasmid PCR was used to construct single-site mutants of A123αE and G565βA, named M1 and M2, respectively, as well as a double-site mutant of A123αE / G565βA (named M3).

[0043] The PCR product was digested with DpnI enzyme to remove the methylated original template, transformed into E. coli BL21(DE3) competent cells, and single clones were selected to extract plasmids for sequencing verification. After confirming that only the expected mutation occurred at the target site and there were no additional random mutations, the strain was preserved for subsequent effect experiments.

[0044] The expression host strain containing the above-mentioned mutation site plasmid was inoculated and cultured to induce the expression of penicillin G acylase. The enzyme solution of the corresponding mutant was obtained by cell disruption, protein extraction and ultrafiltration concentration.

[0045] The A123αE, G565βA, and A123αE+G565βA mutants have been verified to have superior catalytic performance in the synthesis of cefazolin. Example 2

[0046] This example demonstrates the application of the penicillin G acylase mutant obtained in Example 1 in the synthesis of cefazolin at 30°C. It is used to evaluate the suitability and catalytic efficiency of each mutant in a room-temperature reaction system, with catalytic data from the wild type under the same conditions used as a reference. The specific application steps are as follows: S0. Preparation of immobilized enzyme: Take the enzyme solution of the mutant obtained in Example 1, and mix the epoxy resin LX2000EP used for immobilization with the purified and concentrated enzyme solution of the penicillin G acylase mutant at a ratio of 600-1500 U enzyme activity per gram of epoxy resin. Adjust the pH to 7.0-7.5, and stir at room temperature for 24 hours in the presence of 200 mM phosphate. Wash off the enzyme adsorbed on the surface with 20-50 mM phosphate buffer, and obtain the immobilized enzyme by filtration.

[0047] In this embodiment, enzyme solutions containing penicillin G acylase mutants M1, M2 and M3 were mixed at a rate of 600 U enzyme activity per gram of epoxy resin, the pH was adjusted to 7.0, and the mixture was stirred at room temperature for 24 h in the presence of 200 mM phosphate. The surface-adsorbed enzymes were washed away with 20 mM phosphate buffer, and the three immobilized enzymes were obtained by filtration.

[0048] S1. Preparation of the cefazolin synthesis reaction system: final concentration of cefazolin core (TDA) 20 g / L, final concentration of tetrazolium acetate side chain 12.8 g / L, volume adjusted with 0.1 M PBK buffer, and pH adjusted to 7.0.

[0049] S2. Control the temperature of the reaction system to 30℃, add the immobilized enzymes prepared from each mutant, stir at 220 rpm for 10 min, take the reaction solution, dilute with mobile phase A and boil for 2 min to terminate the reaction.

[0050] The product concentration was quantitatively determined by HPLC, and the synthetic activity was calculated. For the hydrolysis activity determination, 5 g / L cefazolin was used as the substrate, diluted to volume with 0.1 M PBK buffer, pH 7.0, and reacted at 30℃ for 30 min. The remaining substrate was detected by HPLC using the same method, and the hydrolysis activity was calculated.

[0051] The HPLC detection conditions were as follows: mobile phase A consisted of 3.35 g / L Na2HPO4•12H2O and 1.12 g / L citric acid aqueous solution, mobile phase B consisted of acetonitrile, with a volume ratio of 92:8, detection wavelength of 210 nm, flow rate of 1 mL / min, injection volume of 10 μL, and external standard method for quantification.

[0052] Enzyme activity unit is defined as the amount of enzyme required to catalyze the production of 1 μmol of cefazolin per minute under the above conditions. The S / H ratio is calculated as the ratio of synthetic activity to hydrolytic activity.

[0053] The results of the application performance test of each mutant in the synthesis of cefazolin at 30℃ are shown in Table 2.

[0054] Table 2. Results of enzyme activity and synthetic selectivity assays for wild-type and mutant strains at 30℃

[0055] Table 2 shows that all three mutants can effectively catalyze the synthesis of cefazolin at 30℃, with significantly improved S / H ratios compared to the wild type and a significant reduction in hydrolysis side reactions. The double mutant M3 exhibits the best synthetic selectivity, with an S / H ratio more than 33.3 times that of the wild type, and the improvement is greater than the simple sum of the effects of the two single mutants, demonstrating a synergistic gain effect. These results indicate that the mutants of this invention can be directly used for the enzymatic synthesis of cefazolin at room temperature (30℃). The improved synthetic selectivity can effectively reduce the excess feed ratio of side-chain substrates, improve the utilization rate of the parent nucleus, and improve process economy. Other mutants or combinations of mutant points can also be applied to the catalytic synthesis of cefazolin when the enzyme activity meets the corresponding requirements and at appropriate substrate concentrations. Example 3

[0056] This embodiment provides the coding gene of a penicillin G acylase mutant and a plasmid carrying the coding gene. The coding gene is based on the wild-type penicillin G acylase gene sequence shown in SEQ ID NO.1, with a mutation containing the amino acid substitutions described in Example 1. The plasmid is a recombinant plasmid obtained by inserting the above coding gene into the pET-24a expression vector, constructed according to the method described in Example 1, except that the wild-type gene is replaced with the corresponding mutant coding gene. Example 4

[0057] This embodiment verifies the catalytic performance of the M3 double mutant obtained in Example 1 under low-temperature conditions. The reaction temperature was controlled at 10℃, 12℃, and 15℃, respectively, with the wild type as a control. The remaining operations were the same as the synthetic activity determination method in Example 2. The synthetic activity at 30℃ was taken as 100%, and the low-temperature relative synthetic activity retention rate was calculated. The results are shown in Table 3.

[0058] Table 3 Enzyme activity retention at different temperatures

[0059] The results showed that wild-type PGA exhibited significant activity degradation at low temperatures, with less than 40% of its synthetic activity retained at 10℃, resulting in a substantial decrease in catalytic efficiency. The M3 mutant, however, maintained over 70% of its synthetic activity retention within the 10–15℃ range, demonstrating significantly improved low-temperature conformational stability. Considering both the S / H ratio and low-temperature retention rate as key indicators, the M3 mutant demonstrated the best overall performance. Example 5

[0060] This example demonstrates the application of M3 double mutant immobilized enzyme in the catalytic synthesis of cefazolin at 15°C. The procedure is as follows: S1. Preparation of reaction system: The final concentration of TDA in cefazolin core is 30 g / L. The side chain of tetrazolium acetate methyl ester acyl donor is added at a molar ratio of side chain to core of 2:1. The volume is adjusted with 0.3 M PBK buffer, and the pH of the system is adjusted to 7.0 with ammonia.

[0061] S2. Pre-cool the reaction system to 15℃, add the M3 double mutant immobilized enzyme (with a specific activity of 600 U / g), and the total enzyme activity of the added system is 10000 U / L. Stir the mixture at a constant speed for 2.5 hours. Samples were taken periodically during the reaction. After dilution with purified water and inactivation by boiling for 2 minutes, the substrate conversion and product concentration were determined by HPLC. After the reaction, the immobilized enzyme was filtered and recovered.

[0062] At the end of the reaction, the conversion rate of the parent substrate reached 99.5%, the proportion of hydrolysis byproducts was extremely low, and the product had few impurities and high purity, demonstrating the excellent low-temperature catalytic activity and synthetic selectivity of the mutant. Under these conditions, after the first 10 batches of immobilized enzyme were added, 10% immobilized enzyme was added, and no significant reaction attenuation was observed in a total of 30 replicate batches. Example 6

[0063] This embodiment is basically the same as Example 5, except that the reaction system was pre-cooled to 10°C in step S2, and the reaction time was extended to 5 hours. At the end of the reaction, the conversion rate of the parent substrate reached 98%, maintaining a high level of conversion efficiency. Under the same reaction time, the conversion rate of the low-temperature group decreased slightly, demonstrating the effect of temperature on the catalytic rate. Meanwhile, the mutant still maintained excellent low-temperature catalytic activity. Under these conditions, after the first 10 batches of immobilized enzyme were added, 10% of the immobilized enzyme was added, and no significant reaction attenuation was observed in a total of 30 replicate batches. Example 7

[0064] This embodiment is basically the same as Example 5, except that the molar ratio of side chain to parent core is adjusted to 1.8:1 in step S1, the total enzyme activity of the system is 11000 U / L in step S2, and the reaction time is 2.5~3 h. At the end of the reaction, the TDA parent core conversion rate reached 99.1%, maintaining a high conversion level even with a reduced side chain dosage. Under these conditions, after the first 10 batches of immobilized enzyme were added, 15% of the immobilized enzyme was added, and no significant reaction attenuation was observed in a total of 30 replicate batches. Example 8

[0065] This embodiment is basically the same as Example 7, except that the final concentration of cefazolin nucleus is increased to 40 g / L in step S1, the total enzyme activity of the system is 12000 U / L in step S2, 10% of the total enzyme activity is added after 1 hour of reaction, and the total reaction time is extended to 4.5-5 hours. At the end of the reaction, the substrate nucleus conversion rate reached 98.7%. After increasing the substrate concentration, near-complete conversion can still be achieved by extending the reaction time and supplementing with a small amount of enzyme. Under these conditions, after the first 10 batches of immobilized enzyme were added, 15% of the immobilized enzyme was added, and no significant reaction attenuation was observed in a total of 30 replicate batches. Example 9

[0066] This embodiment is basically the same as Example 7, except that the final concentration of cefazolin nucleus is increased to 45 g / L in step S1, the total enzyme activity of the system is 16000 U / L in step S2, 15% of the total enzyme activity is added after 1 hour of reaction, and the total reaction time is extended to 4.5 hours. At the end of the reaction, the substrate nucleus conversion rate reached 98.5%. Under high substrate loading, by further extending the reaction time and increasing the amount of enzyme added, a high conversion rate can be stably maintained, adapting to high-capacity production needs. Under these conditions, after the first 10 batches of immobilized enzyme were added, 30% of the immobilized enzyme was added, and no significant reaction attenuation was observed in a total of 30 replicate batches. Example 10

[0067] This embodiment is basically the same as Example 5, except that in step S1, the molar ratio of side chain to parent core is further reduced to 1.7:1, and in step S2, the total enzyme activity of the system is 14000 U / L, the immobilized enzyme is added in 4 portions, and the total reaction time is 3.5 h. At the end of the reaction, the substrate-parent core conversion rate reached 98.3%. Under these conditions, no significant reaction attenuation was observed in the first 10 batches of immobilized enzyme. Even with a further reduction in the excess proportion of side chain, a high conversion rate acceptable for industrial production can still be achieved by appropriately extending the reaction time, further improving the raw material utilization rate.

[0068] In other embodiments, different implementation parameters were used based on Example 10. Specifically, in step S2, the total enzyme activity of the system was 16000 U / L or 18000 U / L, the immobilized enzyme was added in 3 or 5 portions, and the total reaction time was 3.5 h. At the end of the reaction, the substrate core conversion rate was measured to be 98.3%.

[0069] In other embodiments, the concentration of PBK buffer in step S1 can be adjusted within the range of 0.3~0.45M, and the pH of the reaction system can be adjusted within the range of 7.0~7.5. For example, the concentration of PBK buffer can be 0.35M, 0.4M, or 0.45M; the pH of the reaction system can be 7.2, 7.3, or 7.5. In step S2, the reaction temperature can be adjusted within the range of 10~30℃, and the stirring reaction time can be adjusted within the range of 0.15~5h. The amount of immobilized enzyme added should be such that the total enzyme activity of the system reaches 10000~16000U / L. For example, the reaction temperature can be 20℃ or 30℃; the stirring reaction time can be 0.15h (i.e., 10min) for rapid synthesis at room temperature, or 1h, 2h, or 4h for reaction conditions under different substrate concentrations and temperature combinations. When the concentration of the parent substrate is ≥40g / L, the percentage of total enzyme activity added can be adjusted within the range of 10%~15%. For example, the percentage of enzyme added can be 12.5%.

[0070] During the preparation of the immobilized enzyme, the enzyme activity added per gram of epoxy resin can be adjusted within the range of 600-1500 U, the immobilization pH can be adjusted within the range of 7.0-7.5, the phosphate concentration can be adjusted within the range of 200-500 mM, the room temperature stirring immobilization time can be adjusted within the range of 24-48 h, and the washing phosphate buffer concentration can be adjusted within the range of 20-50 mM. For example, the enzyme activity added per gram of epoxy resin can be 600 U, 500 U, 700 U, or 1500 U; the immobilization pH can be 7.0, 7.2, or 7.5; the phosphate concentration can be 200 mM, 300 mM, or 500 mM; the stirring immobilization time can be 24 h, 36 h, or 48 h; and the washing buffer concentration can be 20 mM, 35 mM, or 50 mM. All of the above embodiments can realize the application of mutant immobilized enzyme catalyzing the synthesis of cefazolin.

[0071] It should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still modify the technical solutions described in the above embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A penicillin G acylase mutant, characterized in that, Its amino acid sequence is based on the sequence shown in SEQ ID NO. 1, with alanine at position 123 mutated to other amino acids, and / or glycine at position 565 mutated to other amino acids.

2. A penicillin G acylase mutant, characterized in that, Its amino acid sequence is based on the sequence shown in SEQ ID NO. 1, with alanine at position 123 mutated to glutamic acid.

3. A penicillin G acylase mutant, characterized in that, Its amino acid sequence is based on the sequence shown in SEQ ID NO. 1, with glycine at position 565 mutated to alanine.

4. A penicillin G acylase mutant, characterized in that, Its amino acid sequence is based on the sequence shown in SEQ ID NO. 1, with glycine at position 565 mutated to alanine and alanine at position 123 mutated to glutamic acid.

5. A plasmid, characterized in that, The plasmid carries the encoding gene of the penicillin G acylase mutant according to any one of claims 1 to 4.

6. The use of the penicillin G acylase mutant according to any one of claims 1 to 4 in the synthesis of cefazolin by catalyzing the cefazolin core and acyl donor side chain.

7. The application of the penicillin G acylase mutant according to claim 6, characterized in that, The cefazolin core is 7-amino-3-[(5-methyl-1,3,4-thiadiazol-2-yl)thiomethyl]cephalosporanic acid, the acyl donor side chain is methyl tetrazolium acetate, and the molar ratio of the cefazolin core to the acyl donor side chain is (1.7~2):

1.

8. The application of the penicillin G acylase mutant according to claim 7, characterized in that, Includes the following steps: S1. Prepare a reaction system containing the following components: 30-45 g / L of cefazolin core, add acyl donor side chain according to the molar ratio, bring to volume with 0.1-0.3 M PBK buffer, and adjust pH to 7.0-7.5; S2. Adjust the temperature of the reaction system to 10~30℃, add the immobilized enzyme obtained from the penicillin G acylase mutant, so that the total enzyme activity of the reaction system is 10000~16000U / L, stir the reaction for 0.15~5h, and obtain cefazolin.

9. The application of the penicillin G acylase mutant according to claim 8, characterized in that, Adjust the temperature of the reaction system to 10~15℃; When the concentration of the parent nucleus substrate is ≥40g / L, 15%~30% of the total enzyme activity should be added after 1 hour of reaction, and the total reaction time is 4~5 hours.

10. A method for preparing an immobilized enzyme based on the penicillin G acylase mutant according to any one of claims 1 to 4, characterized in that, The resin used for immobilizing the enzyme was mixed with the purified and concentrated enzyme solution of the penicillin G acylase mutant at a ratio of 600-1500 U enzyme activity per gram of epoxy resin. The pH was adjusted to 7.0-7.5, and the mixture was stirred at room temperature for 24-48 hours in the presence of 200-500 mM phosphate. The surface-adsorbed enzyme was washed away with 20-50 mM phosphate buffer, and the immobilized enzyme was obtained by filtration.

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