Penicillin G acylase mutant for synthesis and application thereof in preparation of amoxicillin

A penicillin and mutant technology, applied in the field of genetic engineering and enzyme engineering, can solve the problems of weak acid resistance and low synthetic activity of mutant enzymes, and achieve the effects of low hydrolytic activity, good synthetic activity and high hydrolysis ratio

Active Publication Date: 2016-01-27
HUNAN FLAG BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Although the use of genetic engineering and protein engineering technology at home and abroad has modified penicillin G acylase, making its synthetic performance and synthetic activity greatly

Method used

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  • Penicillin G acylase mutant for synthesis and application thereof in preparation of amoxicillin
  • Penicillin G acylase mutant for synthesis and application thereof in preparation of amoxicillin
  • Penicillin G acylase mutant for synthesis and application thereof in preparation of amoxicillin

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Embodiment 1: Preparation of SPGA mutant with low hydrolytic activity and high synthetic activity

[0077] Depend on figure 1The reaction formula shows that SPGA can not only catalyze the synthetic product amoxicillin from the parent nucleus 6-aminopenicillanic acid (6-APA) and the side chain D-hydroxyphenylglycine methyl ester (D-HPM), but also hydrolyze the product to generate parent Core and side chain, the reversibility of the reaction is determined by the equilibrium constant of the reaction; at the same time, p-hydroxyphenylglycine methyl ester can be hydrolyzed into p-hydroxyphenylglycine by SPGA, therefore, the hydrolysis activity of SPGA is reduced and its synthesis activity is improved is of great significance.

[0078] 1-1 Determine the mutation site by computer simulation

[0079] 1-1-1 SPGA 3D structure modeling

[0080] Using penicillin G acylase derived from Escherichia coli (E.coli) and Alcaligenes faecalis (A.faecalis) whose three-dimensional struct...

Embodiment 2

[0109] Embodiment 2: the preparation of acid-resistant SPGA mutant

[0110] Because the enzymatic synthesis of amoxicillin is carried out under acidic pH conditions, and because the penicillin G acylase needs to be used continuously for 5 to 10 days in the industrial production process, this requires the enzyme to be stable and not easy to produce under acidic pH conditions. Inactivation, and the acid resistance of wild-type penicillin G acylase is poor, so it is urgent to develop a new type of PGA resistant to acidic pH. In order to solve this difficult problem in industrial production, the inventors set up a new development route to further modify the above-mentioned SPGA-2 mutant with the best effect.

[0111] 2-1 Construction of acid-resistant SPGA random mutant library

[0112] In order to improve the acid resistance of SPGA, the inventor used the SPGA-2 template, wherein the primers are T7 universal primers (SEQ ID NO: 13 and 14), to construct a random mutant library by...

Embodiment 3

[0123] Example 3 Separation, purification and immobilization of recombinant SPGA protein

[0124] 3-1 Separation and purification of recombinant SPGA protein

[0125] Since the two His-tags at the N-terminal and C-terminal in the prokaryotic expression vector pET28b(+) were introduced during the construction of the expression vector, the inventors used the histidine tag to carry out Immobilized Metal Chelate Affinity Chromatography (IMAC) To purify the recombinant protein, the specific method is as follows.

[0126] Take 100mL of the SPGA fermentation broth after overnight induction, centrifuge and discard the supernatant to collect the bacteria (10000rpm, 4°C, 10min), wash the bacteria twice with phosphate buffer (pH8.0, 0.1mol / L), centrifuge The bacteria were collected, concentrated 5 times and resuspended in 20ml of phosphate buffer (pH8.0, 0.1mol / L). Put the above-mentioned treated bacterial solution in ice water and perform ultrasonic crushing until clarification, wher...

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Abstract

The invention provides a penicillin G acylase mutant for synthesis and an application thereof in the preparation of amoxicillin. Penicillin G acylase of Achromobacter xylosoxidans origin is mutated by computer aided design in connection with semi-rational design of site-saturation mutagenesis technique and enzyme engineering modification of orthogenesis, thus acquiring the penicillin G acylase mutant lower in hydrolytic activity, better in synthetic activity, higher in synthesis-hydrolysis ratio (S/H), higher in acid resistance and better in stability, and amoxicillin can be catalytically synthesized more effectively and quickly. Immobilized enzyme hydrolytic activity of the mutant SPGA-4 obtained is decreased by 8.7 times, synthetic activity is increased by 5.6 times, the S/H ratio is increased by 8 times, the activity remains at 79% for 60 min under the condition of pH 2.0, amoxicillin is catalytically synthesized by a solid method at 10 DEG C or 20 DEG C, substrates 6-APA and D-HPM are directly charged in a solid form without dissolving, reaction pH need not be controlled, substrate conversion rate is higher than 99%, continuous use is available in more than 300 batches, and good operational stability is given.

Description

technical field [0001] The invention belongs to the fields of genetic engineering and enzyme engineering, and specifically relates to a synthetic penicillin G acylase mutant and the application of the mutant immobilized enzyme in the production of amoxicillin. Background technique [0002] Amoxicillin is the most commonly used penicillin-like broad-spectrum β-lactam antibiotic. At present, there are two methods for industrially producing amoxicillin: chemical synthesis and enzymatic method. Enzymatic method has the advantages of low cost, simple process, green and pollution-free, and mild reaction conditions. Therefore, it has been paid more and more attention. [0003] Penicillin Gacylase (penicillinGacylase, PGA, EC3.5.1.11), under acidic conditions, catalyzes the synthesis of β-lactam antibiotics (such as amoxicillin, Ampicillin, cephalexin and cefadroxil, etc.); hydrolysis of penicillin G under alkaline conditions to prepare 6-APA or hydrolysis of cephalosporin G to pre...

Claims

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Application Information

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IPC IPC(8): C12N9/84C12N15/55C12P37/04
CPCC12N9/84C12P37/04C12Y305/01011
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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