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High-selectivity mutant of cefradine synthesizing enzyme and coding gene thereof

A cephradine and encoding technology, applied in the field of biochemistry, to achieve high activity and high expression level

Active Publication Date: 2018-12-07
TSINGHUA UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Currently there is a patent WO98 / 20120 reporting the use of penicillin G acylase single-point mutant F24βA to synthesize cefradine. Under the same reaction conditions, its V s / V h It is higher than the wild-type enzyme. In addition, the patent 201710451848.X reports the F24βA-based two-point mutant F24βA / S67βA and the three-point mutant F24βA / M142αL / S67βA in V s / V h It has been further improved, but there is no industrialization report based on related mutations to realize the enzymatic synthesis of cephradine

Method used

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  • High-selectivity mutant of cefradine synthesizing enzyme and coding gene thereof
  • High-selectivity mutant of cefradine synthesizing enzyme and coding gene thereof
  • High-selectivity mutant of cefradine synthesizing enzyme and coding gene thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1, preparation and purification of penicillin G acylase mutant

[0053] 1. Construction of gene encoding penicillin G acylase mutant and recombinant expression vector

[0054] The wild Escherichia coli penicillin G acylase gene was obtained according to literature. Sequence The pET28a-PGA_M3 gene of the present invention was amplified by Overlapping PCR (see SEQ ID No.1 for the sequence). Histidine tag His 6 -tag is added at the end of the carbon-terminus of the gene sequence to facilitate subsequent purification steps. After being double-digested and purified with NcoI and XhoI, it was ligated overnight with the expression vector pET28a(+) (containing the kanamycin resistance gene) that was double-cut with the same endonuclease, and then transformed into competent cells BL21( DE3), the recombinant expression plasmid pET28a-PGA_M3 was obtained.

[0055] After sequencing, the sequence of pET28a-PGA_M3 was correct.

[0056] Description of the structure of ...

Embodiment 2

[0070] Embodiment 2, the hydrolysis DHME catalytic activity assay of enzyme mutant

[0071] The DHME conversion product was analyzed by HPLC (LC-20AT, Shimadzu Corporation), so as to determine the catalytic activity and kinetic parameters of the enzyme mutant PGA_M3 prepared in Example 1 to hydrolyze DHME. The chromatographic column selected was an Inertsil C18 reverse-phase column (GL Sciences, 5 μm, 150×4.6 mm). During the reaction, the ratio of the enzyme mutant to the substrate was as follows: 0.5mL of the purified target protein (sufficient, stored in 100mM potassium phosphate buffer, pH 7.0) and 0.5mL of DHME solution (pH 7.0, the concentration was a gradient change, the highest Concentration is 1g / 100mL), react at 22°C for 8min. After the reaction was completed, 1 mL of methanol was added to terminate the reaction. The mobile phase ratio of HPLC is: 75% potassium phosphate buffer solution (30mM, pH 4.5): 25% methanol (v / v), flow rate 0.8mL / min, detection wavelength is...

Embodiment 3

[0076] Embodiment 3, the hydrolysis cephradine catalytic activity determination of enzyme mutant

[0077] The conversion product of cephradine was analyzed by HPLC (LC-20AT, Shimadzu Corporation), thereby determining the catalytic activity and kinetic parameters of the hydrolysis of cephradine by the enzyme mutant PGA_M3 prepared in Example 1. The selected chromatographic column is an Inertsil C18 reverse-phase column (GL Sciences, 5 μm, 150×4.6 mm). During the reaction process, the ratio of enzyme mutant to substrate is: 0.5mL purified target protein (sufficient, stored in 100mM potassium phosphate buffer, pH 7.0) and 0.5mL cephradine solution (pH 7.0, the concentration is a gradient change, The highest concentration is 2g / 100mL), react at 22°C for 8min. After the reaction was completed, 1 mL of methanol was added to terminate the reaction. The mobile phase ratio of HPLC is: 75% potassium phosphate buffer (30mM, pH 4.5), 25% methanol (v / v), flow rate 0.8mL / min, detection wa...

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Abstract

The invention discloses high-selectivity mutant of cefradine synthesizing enzyme and a coding gene thereof. The invention provides the following protein obtained by the steps of substituting 142-sitemethionine of an alpha chain of natural penicillin G acylase of escherichia coli with phenylalanine, substituting 24-site phenylalanine of a beta chain with alanine and substituting 67-site serine ofthe beta chain with alanine. The protein provided by the invention has higher activity and Vs / Vh for synthesizing the cefradine and lower alpha and lays foundation for industrialization of enzymatic synthesis of the cefradine.

Description

technical field [0001] The invention belongs to the field of biochemistry, and relates to a highly selective cephradine synthetase mutant and its encoding gene, in particular to an Escherichia coli penicillin G acylase combination mutant, an encoding gene and its application in synthesizing cephradine. Background technique [0002] Semi-synthetic β-lactam antibiotics are the most widely used antibiotics in the pharmaceutical industry, with an annual output of 30,000 tons and annual sales of more than 15 billion US dollars, accounting for 65% of the entire antibiotic market, of which cephalosporins account for about 2 / 3. At the same time, the amount of penicillin G acylase used to synthesize β-lactam antibiotics and β-lactam mother nucleus is as high as 10-30 million tons. ( H, M, Grulich M, et al.Current state and perspectives of penicillin G acylase-based biocatalyses.Applied Microbiology and Biotechnology, 2014,98(7):2867-2879) Cephradine is the first generation and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/84C12N15/55C12P35/04
CPCC12N9/84C12P35/04C12Y305/01011
Inventor 朱玉山何金文
Owner TSINGHUA UNIV
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