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A novel lactam antibiotic synthetase and its coding gene and application

A technology of lactams and antibiotics, applied in the fields of genetic engineering and enzyme engineering, can solve the problems such as the corresponding conversion rate is not mentioned.

Active Publication Date: 2015-10-14
NORTH CHINA PHARMA HEBEI HUAMIN PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The later patent CN101802212 provides a method for engineering bacteria to prepare penicillin acylase, which can be used in the synthesis of amoxicillin and cephalexin, and the molar ratio of 6-APA and HPGME in the synthesis of amoxicillin can reach 1.5- Between 1.8, the molar ratio of 7-ADCA and PGME in the synthesis of cephalexin is better between 1.5-1.7; the corresponding conversion rate is not mentioned, and there is a certain gap between it and the Escherichia coli penicillin acylase mentioned in CN102264904, and At present, no relevant reports have been seen on the transformation of Achromobacter penicillin acylase. People hope to carry out molecular transformation and optimization of the original Achromobacter penicillin acylase to construct a better performance and good industrial application. Promising Enzymes for the Synthesis of Lactam Antibiotics

Method used

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  • A novel lactam antibiotic synthetase and its coding gene and application
  • A novel lactam antibiotic synthetase and its coding gene and application
  • A novel lactam antibiotic synthetase and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Gene Design and Gene Synthesis

[0052] (1) Genetic design:

[0053] (1.1) According to the amino acid sequence of penicillin acylase of Achromobacter genus CCM4824 in Genebank (SEQ ID NO: 1, Genebank: AY919310.1), the 330th phenylalanine was replaced with alanine, and the 415th lysine Amino acid was replaced by arginine, and the 770th leucine was replaced by phenylalanine. After these sites were introduced into mutation sites, the amino acid sequence as shown in SEQ ID NO:2 was obtained, and then the online design tool Jcat (http : / / www.jcat.de / ) to reverse design the nucleotide sequence;

[0054] (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 acyla...

Embodiment 2

[0056] Example 2: Construction of expression vector PET28-ASPGA (i.e. recombinant vector)

[0057] (1) The ASPGA gene obtained in Example 1 was double digested with BamHI and HindIII, and the enzyme digestion system was as follows:

[0058]

[0059] The concentration of the ASPGA gene is 2 μg / 5 μL;

[0060] 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.

[0061] (2) Use BamHI and HindIII double enzymes to digest the PET28 plasmid (Novagen company), and the enzyme digestion system is as follows:

[0062]

[0063] The concentration of the PET28 plasmid is 2 μg / 5 μL;

[0064] 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.

[0065] (3) Use T4 ligase to ligate the...

Embodiment 3

[0072] Embodiment 3: Construction of transformant BL21(DE3) / PET28-ASPGA (i.e. recombinant strain)

[0073] (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 with vigorous shaking at 37°C for about 2 hours. Bacteria were grown to pre-logarithmic phase.

[0074] (2) Transfer the Escherichia 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...

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Abstract

The present invention discloses a novel lactam antibiotic synthetase. According to the Achromobacter CCM4824 penicillin acylase amino acid sequence (Genebank: AY919310.1) in the Genebank, at least one of the following site mutations exists, wherein the site mutations comprise: replacement of asparagine on site 184 into tyrosine, replacement of phenylalanine on site 330 into alanine, replacement of lysine on site 415 into arginine, replacement of leucine on site 454 into alanine, replacement of aspartic acid on site 623 into asparagine, replacement of leucine on site 770 into phenylalanine, and replacement of glycine on site 790 into alanine. The present invention further discloses an optimized coding gene of the novel lactam antibiotic synthetase, a recombinant vector containing the coding gene, a recombinant strain containing the coding gene, and applications of the novel lactam antibiotic synthetase in synthesis of amoxicillin, cephalexin and cefaclor. With application of the novel lactam antibiotic synthetase as the enzyme for synthesizing amoxicillin, cephalexin, cefaclor and other lactam antibiotics, advantages of high synthesis activity, high substrate conversion rate, lower side chain ratio, wide application and the like are provided, and good industrial application prospects are provided.

Description

technical field [0001] The invention belongs to genetic engineering and enzyme engineering technology, and specifically relates to a novel lactam antibiotic synthetase, its coding gene and application. Background technique [0002] β-lactam antibiotics mainly include antibiotics such as penicillins and cephalosporins, which are currently the main antibiotics used at home and abroad. Such antibiotics occupy an important share in the pharmaceutical industry, accounting for more than 65% of the global annual sales of antibiotics. In May 2012, the China Market Research Network predicted the consumption of cephalosporin antibiotics in hospitals: China's current pharmaceutical market capacity is about 120 billion yuan, of which cephalosporin antibiotics are about 13 billion yuan. The cost of cephalosporin antibiotics abroad accounts for 40% of the total cost of anti-infective drugs (Xue Yu et al., Chinese Journal of Antibiotics, 36(2):86-92, 2011.). [0003] Compared with develo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/55C12N9/84C12N15/70C12N1/21C12P37/04C12P35/04
Inventor 刘力强牛昆吕娜贾娟娟范俊辉石晨光王召业武芳王欢杨丽萍邸胜苗刘东
Owner NORTH CHINA PHARMA HEBEI HUAMIN PHARMA
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