Immobilized recombinant penicillin g acylase and its application

A penicillin and acylase technology, applied in the direction of immobilization on/in organic carriers, chemical industry, sustainable manufacturing/processing, etc., can solve the problem of no immobilized carrier carrier, low immobilization efficiency and poor stability. and other problems, to achieve the effect of excellent operating stability

Active Publication Date: 2016-08-17
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

my country's immobilization technology research started relatively late. After years of research, a variety of carriers and methods have been developed at will, but there is still no carrier comparable to foreign first-class immobilization carriers, and there are not many that can be put into industrial applications. , the main reason is that the cost of reagents and carriers used for immobilization is high, the immobilization efficiency is low, and the stability is poor. In addition, due to commercial confidentiality, many process details are not disclosed. Therefore, it is of great significance to study efficient immobilized penicillin acylase

Method used

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  • Immobilized recombinant penicillin g acylase and its application
  • Immobilized recombinant penicillin g acylase and its application
  • Immobilized recombinant penicillin g acylase and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Construction of genetically engineered bacteria Bacillus subtilis WB800 / pPZW103-PGA

[0043] 1.1 Gene encoding recombinant penicillin G acylase

[0044] Synthesize the gene sequence of penicillin G acylase (PGA) (GenBank: AF161313.1) derived from B. megaterium CA4098 (synthesis method reference: Nucleic.Acids.Res.2005,33,W521-W525), the full length is 2046bp (shown in SEQ ID NO.2), encoding 802 amino acids, including 26 amino acid residues encoding signal peptide, 208 amino acid residues encoding α subunit, 31 amino acid residues encoding spacer peptide, and 537 encoding β subunit amino acid residues (shown in SEQ ID NO.1). The synthesized gene was connected into PES plasmid (provided by Shanghai Xuguan Biotechnology Development Co., Ltd.), and the recombinant plasmid PES-PGA was obtained, which was transformed into the cloning host Escherichia coli JM109 (provided by Beijing Tiangen Biochemical Technology Co., Ltd.), and the recombinant strain JM109 / PES was ...

Embodiment 2

[0070] Example 2 Fermentation of genetically engineered bacteria Bacillus subtilis WB800 / pPZW103-PGA

[0071] Slant culture: inoculate the Bacillus subtilis genetically engineered bacterium (Bacillus subtilis WB800 / pPZW103-PGA) constructed in Example 1 into the slant medium, culture at 37°C for 12 hours to obtain slant cells, and store at 4°C. The final concentration of the slant medium is composed of: peptone 10g / L; yeast powder 5g / L; NaCl 10g / L; agar 20g / L, pH natural, solvent is water.

[0072] Seed culture: Pick the slant strains stored at 4°C and insert them into the seed medium (50mL seed liquid medium / 250mL Erlenmeyer flask) containing kanamycin at a final concentration of 50 μg / mL, and culture overnight at 37°C and 150rpm (approx. Cultivate for 10-12 hours) to obtain seed liquid. The final concentration of the seed medium is composed of: peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, pH natural, solvent is water.

[0073] 5L fermenter cultivation: inoculate the seed s...

Embodiment 3

[0075] Example 3 Determination of Recombinant Penicillin G Acylase Activity

[0076] Get the crude enzyme liquid of the recombinant penicillin G acylase obtained by cultivating 5L fermentor in 0.2mL embodiment 2, with the N-phenylacetyl-(R, S)-o-chlorophenylglycine of 0.1mmol as substrate, with pH= 8.0 Ammonia solution was used as the reaction medium to form a 5mL reaction system, reacted in a water-bath shaker at 30°C and 180rpm for 6min, took 1mL of the reaction solution and placed it in an EP tube that had been added with 20μL of 6M NaOH aqueous solution to terminate the reaction, and placed it at 12000rpm at 4°C Centrifuge for 10 min under the conditions, take the supernatant, and detect it by HPLC.

[0077]Enzyme activity definition: the enzyme required to catalyze N-phenylacetic acid-(R,S)-2-aryl-amino acid to generate 1 μmol of (S)-2-aryl-amino acid per minute at 30°C and pH 8.0 The amount is 1 enzyme activity unit, expressed in U.

[0078] Under the above measurement...

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Abstract

The invention discloses an immobilized recombinant penicillin G acylase and an application thereof to preparation of (S)-2-aryl-amino acid and cephalosporin antibiotics. The immobilized recombinant penicillin G acylase disclosed by the invention is capable of converting an S-type substrate into an S-type product within shorter time, high in substrate tolerance and strict in S selectivity for the substrate and structural analogues thereof; due to the addition of cobalt ions as well as a protective agent with phenylacetic acid and glycerin as enzyme active centers, the immobilized recombinant penicillin G acylase disclosed by the invention is prevented from being successfully subjected to multipoint covalent immobilization on the surface of an epoxy resin carrier under the condition that enzyme molecules are seriously inactivated in an immobilization process, and the immobilization method is simple and feasible, cheap in raw materials and suitable for large-scale operation; the immobilized recombinant penicillin G acylase can be used for synthesizing cephalosporin antibiotics and splitting the prepared (S)-2-aryl-amino acid, and can be continuously used for more than 50 batches before the activity of the immobilized enzyme is not remarkably reduced.

Description

(1) Technical field [0001] The invention relates to an immobilized enzyme and its application, in particular to a method for preparing an immobilized recombinant penicillin G acylase with high operational stability, and the preparation of (S)-2- Aryl-amino acid and cephalosporin antibiotic methods. (2) Background technology [0002] Unnatural (S)-2-aryl-amino acids are important pharmaceutical intermediates, and (S)-phenylglycine is used to synthesize prolactin analogs, AIDS protease inhibitors and taxol. (S)-o-chlorophenylglycine, its chemical name is 2-amino-(2-chlorophenyl)acetic acid, and its molecular formula is C 8 h 8 NO 2 One of the most important uses of Cl is the synthesis of a novel anti-platelet aggregation drug, clopidogrel. Clopidogrel was first developed by the French company Sanifi in 1986. Its sulfate salt is generally used clinically, and its trade name is Plavix. [0003] The preparation method of (S)-o-chlorophenylglycine comprises: 1) chemical resol...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/08C12P13/04C12P35/04
CPCY02P20/50
Inventor 薛亚平郑裕国柳志强侯鹏云刘学
Owner ZHEJIANG UNIV OF TECH
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