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Gamma-lactamase with racemate gamma-lactam resolution activity as well as encoding gene and applications thereof

A technology of lactamase and lactam, which is applied in the field of enzyme engineering, can solve the problems of enzyme instability and low selectivity in the resolution of racemic γ-lactam substrates

Inactive Publication Date: 2014-08-06
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few studies on (-) γ-lactamase, but there are disadvantages such as low resolution selectivity for racemic γ-lactam substrates and unstable enzymes.

Method used

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  • Gamma-lactamase with racemate gamma-lactam resolution activity as well as encoding gene and applications thereof
  • Gamma-lactamase with racemate gamma-lactam resolution activity as well as encoding gene and applications thereof
  • Gamma-lactamase with racemate gamma-lactam resolution activity as well as encoding gene and applications thereof

Examples

Experimental program
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Effect test

Embodiment Construction

[0029] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0030] (1) Obtaining of (-)γ-lactamase B-J gene of the present invention

[0031] (1) (-) γ-lactamase B-J genome was extracted from Bradyrhizobium soybean.

[0032] (2) Primer design

[0033] Primers were designed according to known gene sequences, and the primer sequences were as follows (synthesized by Shanghai Sangong);

[0034] B-J upstream primers:

[0035] 5'-ggaatt ccatatg atgccccaccatcaccaccaa-3'

[0036] The underline indicates the NdeI restriction site

[0037] B-J downstream primers:

[0038] 5'-ccc aagctt cgccttgaagaacgccagcagg-3'

[0039] (3) PCR amplification and gene cloning

[0040] PCR amplification was performed using genomic DNA as a template. The PCR reaction system was: 1 μl genomic DNA, 2.5 μl 10×PCR Buffer, 1 μl each of upstream and downstream primers, 1 μl dNTP (2.5 mM), 1 μl Taq enzyme (5 U / μl), and ddH 2 O to make up to 25 μl. ...

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Abstract

The invention discloses gamma-lactamase with racemate gamma-lactam resolution activity as well as an encoding gene and applications thereof, namely, bradyrhizobium japonicum (Bradyrhizobium japonicum USDA6) derived gamma-lactamase as well as an encoding gene and applications thereof, and provides an application of the gamma-lactamase in the hydrolytic resolution of racemate gamma-lactam. The gamma-lactamase disclosed by the invention is a protein as the following (a) or (b): (a) a protein consisting of amino acid residue sequences shown in SEQ ID No:1 in a sequence table; and (b) a protein formed by that amino acid residue sequences shown in SEQ ID No:1 in a sequence table are replaced and / or deleted and / or added by one or more amino acid residues, with racemate gamma-lactam resolution activity, and derived from SEQ ID No:1. Racemate gamma-lactam is subjected to hydrolytic resolution by using the gamma-lactamase, the resolution activity of the gamma-lactamase is close to 100%, and then gamma-lactam is prepared by using a catalytic product amino acid.

Description

technical field [0001] The invention belongs to the field of enzyme engineering, and in particular relates to a (-)γ-lactamase with the activity of splitting racemic γ-lactam and its coding gene, and the application of the (-)γ-lactamase. Background technique [0002] (-)2-Azabicyclo[2,2,1]hept-5-en-3-one (abbreviated as (-)γ-lactam) is the preparation of antiviral drug abacavir and anti-influenza drug paramil Wei and other important intermediates. Compared with chemical synthesis methods, which are cumbersome and easy to cause environmental pollution, biological enzymatic methods have many advantages such as high efficiency, simplicity, and environmental friendliness in the process of synthesizing chiral (-)γ-lactams. [0003] [0004] (-)γ-lactamase Enzyme capable of hydrolytic resolution of racemic γ-lactams. (-)γ-amidase is a kind of amidase, and amidase is an enzyme in the nitrilase family, which can effectively break the carbon-nitrogen bond in the acyl group to f...

Claims

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Application Information

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IPC IPC(8): C12N9/86C12N15/55C12N15/70C12N1/21C12P41/00C12P17/10C12R1/19
CPCC12N9/86C12P17/10C12P41/001
Inventor 郑国钧任璐
Owner BEIJING UNIV OF CHEM TECH
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