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Mutant of penicillin G acylase (PGA) and preparation method and application of mutant

A technology of penicillin and acylase, which is applied in the field of penicillin G acylase mutants and its preparation, which can solve the problems of low catalytic activity, inability to increase 6-APA production rapidly, long reaction time, etc., and achieve good conversion rate, The effect of fast reaction rate and low substrate residue

Active Publication Date: 2015-11-25
HUNAN FLAG BIOTECHNOLOGY CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, in the process of enzymatic production of 6-APA by raw material pharmaceutical companies, it is found that the production of 6-APA cannot be produced rapidly due to the disadvantages of low catalytic activity of penicillin G acylase, strong inhibition of phenylacetic acid, long reaction time, and substrate inhibition. Therefore, the development of new penicillin G acylases with high activity, high reaction rate, high conversion rate, high concentration substrate tolerance, high concentration phenylacetic acid tolerance, and low substrate residue "five high and one low" has been imminent

Method used

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  • Mutant of penicillin G acylase (PGA) and preparation method and application of mutant
  • Mutant of penicillin G acylase (PGA) and preparation method and application of mutant
  • Mutant of penicillin G acylase (PGA) and preparation method and application of mutant

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Comparison scheme
Effect test

Embodiment 1

[0063] Example 1: Cloning and expression of wild-type PGA gene and purification and immobilization of recombinant protein

[0064] 1-1 Obtaining of wild-type PGA gene

[0065] According to the amino acid and nucleotide sequence of Escherichia coli ATCC11105 penicillin G acylase provided by GeneBank (GeneBank accession number: X04114.1), the inventors obtained a complete intracellular The mature peptide of PGA (SEQ ID NO: 2) was expressed, and the optimized amino acid was subjected to whole gene synthesis (SEQ ID NO: 1).

[0066] 1-2 Construction of wild-type PGA prokaryotic expression vector

[0067] Primers were designed according to the prokaryotic expression vector pET30a(+) and the wild-type PGA gene synthesized by the whole gene. The specific primer sequences are as follows:

[0068] P1: 5'-CCC AAGCTT ATGGAGCAGTCGTCAAGT-3' (where the base in the underline is the HindIII restriction site)

[0069] P2: 5'-CCG CTCGAG TTATCTCTGAACGTGCAA-3' (the underlined base is the X...

Embodiment 2

[0084] Embodiment 2: the preparation of PGA mutant

[0085] 2-1 Preparation of highly active PGA mutants

[0086] 2-1-1 Construction of PGA Random Mutant Library

[0087] In order to improve the activity of wild-type PGA to penicillin G, the inventors used PGA-WT as a template, wherein the primers were T7 universal primers (SEQ ID NO: 23 and 24), constructed a random mutant library by error-prone PCR, and passed Adjusting Mg in error-prone PCR reaction system 2+ and Mn 2+ concentration and dCTP and dTTP oligonucleotide concentrations, the base mismatch rate of the mutant library is only 2 / 1000, that is, it is guaranteed that only 1 to 2 amino acids are mutated in a mutant. The specific process of constructing the mutant library is as follows.

[0088] Error-prone PCR reaction system:

[0089]

[0090] The error-prone PCR reaction conditions are: 95°C pre-denaturation for 5 minutes; then 94°C denaturation for 30 seconds, 56°C annealing for 1 minute, 72°C for 1.5 minutes...

Embodiment 3

[0154] Application of embodiment 3 mutant PGA-6 immobilized enzyme

[0155] 3-1 Wild-type PGA and PGA-6 immobilized enzyme cleave penicillin G to prepare 6-APA experiment

[0156] (1) Cleavage reaction

[0157] The above-mentioned wild-type PGA and PGA-6 immobilized enzymes were respectively placed in penicillin G solutions of certain concentrations (8%, 15%, 20%, 25% and 30%) with the same enzyme amount (8000U), at 25°C , react under the condition of pH8.00, in the reaction process, continuously drip 3mol / L of concentrated ammonia water to neutralize the acid produced in the reaction process, make the pH constant at 8.00, and record the amount of ammonia water added, when the reaction reaches the end point (judgment end point The best method is: automatically stop adding ammonia water, the pH value remains unchanged for more than 3 minutes), and record the total reaction time. Filter the above lysate, rinse the immobilized enzyme with sterile deionized water 4-5 times repea...

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Abstract

The invention provides a mutant of penicillin G acylase (PGA) and a preparation method and application of the mutant. The non-rationally and semi-rationally designed enzyme engineering reconstruction technology is adopted for mutation of penicillin G acylase obtained from Escherichia coli ATCC 11105, so that a PGA mutant with higher reactivity, higher reaction rate, better conversion rate, stronger in penicillihe concentration tolerance, less substrate residue, and higher in phenyl acetic acid concentration tolerance; meanwhile, the mutant is subjected to recombinant expression, bacteria strain construction, fermenting cultivation, immobilization and application to prepare 6-amino-penicillanic acid (6-APA). The activity of the PGA-6 mutant prepared by the invention is increased by 102 times, the substrate penicillihe concentration tolerance is increased to 30%, and the phenyl acetic acid concentration tolerance is increased to 20 mmol / L; meanwhile, the immobilized PGA-6 mutant is used to decompose penicillihe with a concentration of 25% under the condition of pH 8.0 and 25 DEG C so as to prepare 6-APA, and the reaction time is shortened to 55 minutes, the substrate conversion rate is 98% or above, and after being used for 600 batches and above, the activity is not lost obviously, therefore, good operation stability is achieved.

Description

technical field [0001] The invention belongs to the fields of genetic engineering and enzyme engineering, and specifically relates to a mutant of penicillin G acylase and a preparation method and application thereof. Background technique [0002] Penicillin is the first anti-infective drug used clinically in the world. For a long time, penicillin has been prosperous in the antibiotic market and has become the first-line antibacterial drug widely used in the world. 6-amino-penicillanic acid (6-APA) is an important intermediate in the synthesis of ampicillin and amoxicillin. There are two methods of industrially producing 6-APA, chemical cleavage and enzymatic method, among which the chemical cleavage method has been gradually eliminated due to the disadvantages of high cost, many process steps, serious wastes and harsh conditions; and the enzymatic method has a relatively low cost. Low cost, simple process, green and non-polluting, and mild reaction conditions, etc., so it ...

Claims

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Application Information

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IPC IPC(8): C12N9/84C12N15/55C12N15/70C12P37/06C12P7/40
CPCY02P20/52C12N9/84C12P7/40C12P37/06C12Y305/01011
Inventor 许岗黄斌郭宁周晶辉赵强曾红宇
Owner HUNAN FLAG BIOTECHNOLOGY CO LTD
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