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CN-121975780-A - Penicillin G acylase mutant and polynucleotide, expression vector and application

CN121975780ACN 121975780 ACN121975780 ACN 121975780ACN-121975780-A

Abstract

The invention relates to the technical field of bioengineering, in particular to penicillin G acylase mutant, polynucleotide, expression vector and application. The penicillin G acylase mutant is obtained by carrying out site-directed mutagenesis on a wild penicillin G acylase gene of a parent escherichia coli (ESCHERICHIA COLI), and introducing mutation into phenylalanine (F) at 24 th site of an alpha chain, proline (P) at 383 th site of a beta chain, threonine (T) at 384 th site of the beta chain, glutamic acid (G) at 385 th site of the beta chain and serine (S) at 386 th site of the beta chain of the penicillin G acylase by a full plasmid PCR technology. The penicillin G acylase mutant disclosed by the invention has the advantages that when catalyzing methyl mandelate and 3- (1-methyl-1H-tetrazole-5-yl) thiomethyl-7-aminocephalosporanic acid to synthesize cefamandole, the synthesis activity is improved, and the side reaction rate is greatly reduced.

Inventors

  • LIU HAOYU
  • XU YI
  • WU XIAOMEI
  • MA BAODI

Assignees

  • 上海应用技术大学

Dates

Publication Date
20260505
Application Date
20251201

Claims (10)

  1. 1. A penicillin G acylase mutant with high synthesis activity and low hydrolysis activity is characterized in that the penicillin G acylase mutant is a wild type penicillin G acylase mutant composed of an alpha chain and a beta chain, wherein the amino acid sequence of the alpha chain is shown as SEQ ID NO.1, and the amino acid sequence of the beta chain is shown as SEQ ID NO. 2; the penicillin G acylase mutant is selected from one of the following mutations: (1) Mutating phenylalanine at position 24 of alpha chain into threonine or alanine; (2) Mutating proline at position 383 of the beta chain to leucine or phenylalanine; (3) Mutation of glutamic acid at position 385 of the beta chain to leucine, alanine or phenylalanine; (4) Mutating serine at 386 of the beta chain into threonine; (5) Mutating phenylalanine at position 24 of alpha chain to alanine and mutating serine at position 386 of beta chain to threonine; (6) Serine at position 386 of the beta chain is mutated to threonine and threonine at position 384 of the beta chain is mutated to asparagine, serine or cysteine.
  2. 2. The penicillin G acylase mutant with high synthesis activity and low hydrolysis activity according to claim 1, wherein the penicillin G acylase mutant is a penicillin G acylase mutant obtained by mutating serine at position 386 of the beta-chain to threonine and mutating threonine at position 384 of the beta-chain to asparagine.
  3. 3. A polynucleotide encoding the penicillin G acylase mutant of claim 1.
  4. 4. An expression vector comprising the polynucleotide of claim 3, wherein said expression vector is used to encode in an expression host one or more penicillin G acylase mutants of claim 1.
  5. 5. A recombinant cell comprising the expression vector of claim 4.
  6. 6. A process for producing a penicillin G acylase mutant as defined in claim 1, characterized in that the penicillin G acylase mutant is recovered after culturing the recombinant cell as defined in claim 5.
  7. 7. A penicillin G acylase catalyst, characterized in that the penicillin G acylase is in any of the following forms: (i) Culturing the recombinant cell of claim 5, and isolating the recombinant cell comprising the penicillin G acylase mutant; (ii) Freeze-dried cells obtained by freeze-drying the recombinant cells of (i); (iii) Disrupting the recombinant cells of (i) to obtain a crude enzyme solution; (iv) Purifying the crude enzyme solution obtained in step (iii).
  8. 8. Use of a penicillin G acylase mutant according to claim 1 or a penicillin G acylase catalyst according to claim 7 for the synthesis of cefamandole.
  9. 9. A method for synthesizing cefamandole, comprising the steps of: taking methyl mandelate and 7-TMCA as substrates, and reacting under the catalysis of penicillin G acylase mutant according to claim 1 or penicillin G acylase catalyst according to claim 7 to obtain cefamandole.
  10. 10. The method for synthesizing cefamandole according to claim 9, wherein the temperature is 5-30 ℃ and the pH is 5.5-9.0 during the reaction.

Description

Penicillin G acylase mutant and polynucleotide, expression vector and application Technical Field The invention relates to the technical field of bioengineering, in particular to penicillin G acylase mutant, polynucleotide, expression vector and application. Background Penicillin G acylase (PGA, EC 3.5.1.11) is an enzyme that plays a key role in the production of semisynthetic β -lactam antibiotics and is classified as an amide bond hydrolase. The enzyme consists of alpha subunit and beta subunit, has catalytic activity after being folded in a specific mode, has various functions of PGA, and can catalyze the hydrolysis reaction of penicillin and its ring-expanding acid to remove side chains, and can catalyze the condensation reaction of 6-aminopenicillanic acid (6-APA) or 7-amino deacetylated oxo-cephalosporanic acid (7-ADCA) and corresponding side chains to generate semisynthetic beta-lactam antibiotics. In addition, the enzyme is involved in the hydrolysis and synthesis of various beta-lactam antibiotics including penicillin and part of first and second generation cephalosporins .（ Kallenberg A , Van Rantwijk F , Sheldon R .Immobilization of Penicillin G Acylase: The Key to Optimum Performance[J]. Advanced Synthesis&Catalysis, 2005, 347(7-8): 905-926. DOI:10.1002/adsc.200505042.）. Cefamandole is the free acid of cefamandole nafate, also known as hydroxybenzyltetrazole cephalosporin, pioneer's mandolin, etc., the target product in the synthetic drug is cefamandole nafate, cefamandole (Cefamandole) is the second generation cephalosporin approved in japan by salt field company under the trade name Kefdole in 1983, produced and marketed exclusively by new pioneer in Shanghai in China. The traditional process method for synthesizing cefamandole nafate mainly comprises an active ester method and an amide method, but the method has the advantages that multi-step group protection and deprotection exist, the reaction condition is extremely harsh, the post-reaction treatment is extremely difficult, challenges .(MOODY H,MAAS P,JOHANNES D,et al. Prcess For the Preparation of Cefamandole Nafate: wO, 2013057196 [P.2013-03-07]). are generated in industrial production compared with a chemical method, penicillin G acylase is adopted as a biocatalyst to synthesize cefamandole, the difficulty of post-reaction treatment can be greatly reduced while the yield is ensured, the industrial environmental friendliness is improved, the PGA variety which can be used for cefamandole nafate synthesis is extremely few, and the enzyme has higher hydrolytic activity on acyl donors and cefamandole products, and excessive acyl donors and water-soluble organic solvents are needed to be added in practical application to improve the yield, so that the raw material consumption is increased, the downstream purification cost is also improved, and compared with the penicillin G acylase mutant alpha F24A/beta S386 developed by the patent CN120384068A, the yield of the penicillin G acylase mutant is successfully improved to 80.11%, the yield of cefamandole has the industrial yield which is greatly improved, and the optimal catalytic efficiency is not improved until the industrial production efficiency is greatly improved, but the optimal by 82% is achieved, and the industrial production efficiency is still not improved. At present, the rational design of proteins is based on the deep understanding of the sequence, structure, catalytic mechanism and other enzymatic properties of target proteins, and aims to predict the influence of specific site mutation on enzyme stability and catalytic performance through bioinformatics tools. In various site-directed mutagenesis technologies, the whole plasmid PCR method is the most widely applied method at present due to simple and convenient operation, high efficiency and high speed, and the research on penicillin G acylase is mainly focused on the aspects of immobilization, enzymatic property analysis, catalytic property modification and the like, for example Mohammad Karim Emadzadeh, ferroferric oxide magnetic nanoparticles are subjected to functional modification through polyethyleneimine, PGA is fixed on the nanoparticles by means of glutaraldehyde as a cross-linking agent, design Expert 10.0 software is utilized, immobilization conditions are optimized based on a Response Surface Method (RSM) and a Central Composite Design (CCD), and finally intermediate 6-APA with the maximum yield can be obtained after a reaction at 10 ℃ for 336 minutes. In addition, the V max and K m of the free PGA are 0.024 mM and 1.04 mM（Emadzadeh K M ,Pourzamani H ,Hekmat A , et al.An optimized and efficient method for immobilization and stabilization of penicillin G acylase onto PEI-coated magnetic Fe3O4 nanoparticles.[J].Drug development and in dustrial pharmacy,2025,1-17.DOI: 10.1080 /03639045.2025.2567548.); respectively, the EcPGA mutant of the escherichia coli achieves significant improvement （Cecchini D A, Pavesi R, Sanna S, e