Cefradine synthase mutant and coding gene thereof

A cephradine and encoding technology, applied in the field of biochemistry, can solve the problems of not synthesizing cephradine

Active Publication Date: 2017-08-29
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, only patents WO2005/003367, WO2008/110527 and WO2011/073166 have reported the use of penicillin G acylase single point mutant βF24A to synthesize cefradine. Un

Method used

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  • Cefradine synthase mutant and coding gene thereof
  • Cefradine synthase mutant and coding gene thereof
  • Cefradine synthase mutant and coding gene thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Embodiment 1, preparation and purification of penicillin G acylase mutant

[0060] 1. Construction of gene encoding penicillin G acylase mutant and recombinant expression vector

[0061] The wild Escherichia coli penicillin G acylase gene was obtained according to literature. Sequence The pET28a-PGA_M1 gene (sequence 1) and pET28a-PGA_M2 gene (sequence 3) in the present invention were amplified by Overlapping PCR. 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 plasmids pET28a-PGA_M1 and pET28a-PGA_M2 were obtained.

[0062] After sequencing, the sequences of pET28a-PGA_M1 and pET28a-PGA_M2 w...

Embodiment 2

[0076] Embodiment 2, the hydrolysis DHME catalytic activity assay of enzyme mutant

[0077] The DHME conversion product was analyzed by HPLC (LC-20AT, Shimadzu Corporation), so as to determine the catalytic activity and kinetic parameters of the hydrolysis of DHME by the two enzyme mutants PGA_M1 and PGA_M2 prepared in Example 1. 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: 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 gradient, 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 wavelengt...

Embodiment 3

[0082] Embodiment 3, the hydrolysis cephradine catalytic activity determination of enzyme mutant

[0083] 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 two enzyme mutants PGA_M1 and PGA_M2 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 solution (30mM, pH 4.5): 25% methanol (v / v), flow rat...

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Abstract

The invention discloses a cefradine synthetase mutant and a coding gene thereof. A protein shown as A) or B) or C) is provided, wherein A) is obtained by substitution of twenty-fourth-site phenylalanine of the beta chain of Escherichia coli natural penicillin G acylase into alanine and substitution of sixty-seventh-site serine of the beta chain of the Escherichia coli natural penicillin G acylase into the alanine; B) is obtained by substitution of 142nd-site methionine the alpha chain of the Escherichia coli natural penicillin G acylase into leucine, substitution of twenty-fourth-site phenylalanine of the beta chain into the alanine, and substitution of sixty-seventh-site serine of the beta chain into the alanine; and C) is a protein derived from the A) or B) by substitution and / or deletion and / or addition of one or a plurality of amino acid residue of the protein defined by the A) or B), and has the ability of synthesis of cefradine. The protein provided on has high activity for synthesis of the cefradine and Vs / Vh and lower alpha, and lays a foundation for the industrialization of the enzyme method synthesis of the cefradine.

Description

technical field [0001] The invention belongs to the field of biochemistry, and relates to a cephradine synthase 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 nuclei is also as high as 10 million to 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). Cefradine is the first generation of se...

Claims

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Application Information

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IPC IPC(8): C12N9/86C12N15/55C12P35/04
Inventor 朱玉山黄小强何金文
Owner TSINGHUA UNIV
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