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Novel beta-lactam antibiotic synthetase production method

A technology for lactams and production methods, applied in the field of production of novel β-lactam antibiotics synthetases, can solve the problems of low activity, unsuitable for industrial production, weak synthetic activity, etc., and achieve high enzyme activity and good industrial application The effect of stabilizing the value and substrate conversion rate

Active Publication Date: 2014-04-02
NORTH CHINA PHARMA HEBEI HUAMIN PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] As mentioned above, although people have obtained various engineering bacteria with high expression levels through genetic engineering and large-scale culture technology, the enzymes produced cannot meet the needs of industrial production due to low activity or weak synthetic activity.

Method used

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  • Novel beta-lactam antibiotic synthetase production method
  • Novel beta-lactam antibiotic synthetase production method
  • Novel beta-lactam antibiotic synthetase production method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Gene Design and Gene Synthesis

[0043] (1) Genetic design:

[0044] (1.1) According to the amino acid sequence of penicillin acylase of Achromobacter sp. Amino acid was replaced by arginine, and the 770th leucine was replaced by phenylalanine. These sites were introduced into mutation sites to obtain the amino acid sequence shown in SEQ ID NO: 2, and then use the online design tool Jcat (http: / / www.jcat.de / ) reverse design the nucleotide sequence after introducing the mutation site;

[0045] (1.2) According to the preferred codons required for host Escherichia coli gene expression and the G+C base content required for high-efficiency gene expression, optimize the above reverse-designed nucleotide sequence. The optimized nucleotide sequence is shown as SEQ ID NO : 3, compared with the nucleotide sequence of Achromobacter genus CCM4824 penicillin acylase (as shown in SEQ ID NO: 4): ① The rare codons of Escherichia coli are reduced from 55 to 2; ② G+C base ...

Embodiment 2

[0047] Example 2: Construction of expression vector PET28-ASPGA (i.e. recombinant vector)

[0048] (1) The ASPGA gene obtained in Example 1 was double digested with BamHI and HindIII, and the enzyme digestion system was as follows:

[0049]

[0050] The concentration of the ASPGA gene is 2 μg / 5 μL;

[0051] The above enzyme digestion system was incubated at 37°C for 4 hours, and then purified using a DNA gel recovery kit (Sangon Bioengineering (Shanghai) Co., Ltd.) to obtain the target ASPGA gene fragment.

[0052] (2) Use BamHI and HindIII double enzymes to digest the PET28 plasmid (Novagen company), and the enzyme digestion system is as follows:

[0053]

[0054] The concentration of the PET28 plasmid is 2 μg / 5 μL;

[0055] The above enzyme digestion system was incubated at 37°C for 4 hours, and then purified using a DNA gel recovery kit (Sangon Bioengineering (Shanghai) Co., Ltd.) to obtain the target PET28 plasmid fragment.

[0056] (3) Use T4 ligase to ligate the...

Embodiment 3

[0063] Embodiment 3: Construction of transformant BL21(DE3) / PET28-ASPGA (i.e. recombinant strain)

[0064] (1) Pick a single colony of Escherichia coli BL21 (DE3) and inoculate it into an LB test tube. After shaking and culturing at 37°C for 8-10 hours, take 0.5ml of the culture solution and add it to a Erlenmeyer flask containing 50ml of LB, and culture it at 37°C for about 2 hours with vigorous shaking. Bacteria were grown to pre-logarithmic phase.

[0065] (2) Transfer the E. coli culture solution in the early logarithmic phase of growth to an ice-precooled polypropylene tube (capacity 50ml), place it on ice for 10 minutes, centrifuge at 4°C and 4000rpm at low temperature, and discard the supernatant. Add 6ml of ice-cooled CaCl 2 Solution (concentration 0.1mol / L) to resuspend the bacterial pellet, then place it on ice for 30min, centrifuge again at 4°C and 4000rpm at low temperature, then discard the supernatant, add 1.2ml ice-cooled CaCl 2 solution (concentration 0.1mol / ...

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Abstract

The present invention discloses a method for producing novel lactam antibiotics by using specific engineered bacteria transformant BL21(DE3) / PET28-ASPGA. According to the method, a penicillin acylase amino acid sequence represented by SEQIDNO:1 is adopted as basis, a mutation site is introduced, reverse design is performed to obtain a nucleotide sequence, optimization is performed, complete gene synthesis and recombinant vector construction are sequentially performed to construct a recombinant strain, and the constructed recombinant strain is treated through steps of fermentation, cell disruption, separation, purification, decoloration and immobilization to finally obtain the novel lactam antibiotic immobilized enzyme finished product capable of being directly used for industrial production. According to the present invention, the immobilized enzyme prepared by using the method has characteristics of high enzyme activity, good stability and excellent repeated use effect, and can be provided for catalyzing synthesis of a variety of lactam antibiotics such as amoxicillin, cephalexin and cefaclor.

Description

technical field [0001] The invention relates to microbial fermentation and enzyme purification and immobilization technology, in particular to a production method of novel beta-lactam antibiotic synthetase. Background technique [0002] β-lactam antibiotics are a very important and commonly used class of antibiotics, with high curative effect and low toxicity. They are currently the main drugs for the treatment of infectious diseases. They account for a large proportion in the use of antibiotics, and they are also developing varieties in antibacterial drugs. The most common class of antibiotics mainly includes penicillins, cephalosporins, penicillins, monobactams and β-lactamase inhibitors, among which penicillins and cephalosporins are typical representatives. [0003] Traditional semi-synthetic antibiotics are mainly synthesized by chemical methods. Penicillins such as amoxicillin and cephalosporins such as cephalexin, etc., undergo mixed anhydride, condensation, hydrolysi...

Claims

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Application Information

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IPC IPC(8): C12N9/84
CPCC12N9/84C12N15/70C12Y305/01011
Inventor 刘力强石晨光范俊辉王召业王欢武芳贾娟娟牛昆吕娜杨丽萍邸胜苗刘东
Owner NORTH CHINA PHARMA HEBEI HUAMIN PHARMA
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