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Immobilized recombinant penicillin G acylase and application thereof

A technology of penicillin and acylase, which is applied in the fields of immobilization on/in organic carriers, chemical industry, sustainable manufacturing/processing, etc., and can solve the problem of lack of immobilized carrier carrier, low immobilization efficiency, and poor stability and other issues, to achieve the effect of excellent operational stability

Active Publication Date: 2014-10-29
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 application thereof
  • Immobilized recombinant penicillin G acylase and application thereof
  • Immobilized recombinant penicillin G acylase and application thereof

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 Recombinant penicillin G acylase encoding gene

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

Embodiment 2

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

[0071] Slant culture: Inoculate the genetically engineered bacteria of Bacillus subtilis (Bacillus subtilisWB800 / pPZW103-PGA) constructed in Example 1 into a slant medium, culture at 37°C for 12h 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, natural pH, and water as the solvent.

[0072] Seed culture: Pick the slanted strains stored at 4°C and insert them into the seed medium (50mL seed liquid medium / 250mL conical flask) containing kanamycin with a final concentration of 50μg / mL, and culture at 37°C and 150rpm overnight (approximately 50 μg / mL). Cultivated 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, natural pH, and water as the solvent.

[0073] 5L fermenter cultu...

Embodiment 3

[0075] Example 3 Determination of recombinant penicillin G acylase activity

[0076] Take 0.2mL of the crude enzyme liquid of the recombinant penicillin G acylase obtained by culturing the 5L fermentor in Example 2, take 0.1mmol of N-phenylacetyl-(R,S)-o-chlorophenylglycine as a substrate, and take pH= 8.0 aqueous ammonia solution was used as the reaction medium to form a 5 mL reaction system, and the reaction was carried out in a water bath shaker at 30 ° C and 180 rpm for 6 min, and 1 mL of the reaction solution was placed in an EP tube to which 20 μL of 6M NaOH aqueous solution had been added to terminate the reaction. Centrifuged for 10 min under conditions, the supernatant was collected and detected by HPLC.

[0077]Definition of enzyme activity: The enzyme required to catalyze N-phenylacetic acid-(R,S)-2-aryl-amino acid to 1 μmol of (S)-2-aryl-amino acid per minute at 30°C and pH 8.0 The amount is one unit of enzyme activity, which is represented by U.

[0078] Under t...

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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 present invention relates to an immobilized enzyme and application thereof, in particular to a method for preparing an immobilized recombinant penicillin G acylase with high operational stability, and the catalyzed preparation of (S)-2- Methods of Aryl-Amino Acids and Cephalosporins. (2) Background technology [0002] Unnatural (S)-2-aryl-amino acids are an important class of pharmaceutical intermediates, and (S)-phenylglycine is used in the synthesis of 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 to synthesize a new type of anti-platelet aggregation drug, Clopidogrel. Clopidogrel was first developed by the French company Sanifi in 1986. It is generally used clinically as its sulfate salt, and its trade name is Plavix. [0003] The preparation method of (S)-o-chlor...

Claims

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Application Information

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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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