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(+)Gamma-lactamase, its coding gene and application

A technology of lactamase encoding and lactamase, which is applied in the field of enzyme engineering, can solve the problems of limited industrial application, poor selectivity, and enzyme instability, and achieve the effects of high temperature tolerance, low cost, and stable properties

Inactive Publication Date: 2014-04-16
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main shortcomings of the (+)γ-lactamase resolution process for racemic γ-lactams currently reported are: (1) (+)γ-lactamase resolution of racemic γ-lactams Poor selectivity; (2) The selected enzyme is unstable, which limits its industrial application

Method used

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  • (+)Gamma-lactamase, its coding gene and application
  • (+)Gamma-lactamase, its coding gene and application
  • (+)Gamma-lactamase, its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] (1) Acquisition of (+) γ-lactamase A-P gene of the present invention

[0040] (1) (+) γ-lactamase A-P genome Japan Bioresource Center (NBRC) direct purchase

[0041] (2) Primer design

[0042] Primers were designed according to the known (+) γ-lactamase A-P gene sequence, the primer sequence is as follows (synthesized by Shanghai Sangong);

[0043] A-P upstream primers:

[0044] 5'-GGAATTC CATATG GTAACAAGGATAACT-3'

[0045] The underline indicates the NdeI restriction site

[0046] A-P downstream primers:

[0047] 5'-CCC AAGCTT CTACTCTGCTAGCTTCTTC-3'

[0048] The underline indicates the HindⅢ restriction site

[0049] (3) PCR amplification and gene cloning

[0050] Use the genomic DNA as a template for PCR amplification. The PCR reaction system is: 1 μL of genomic DNA (100 μg / μL), 2.5 μL of 10×PCR Buffer, 1 μL of upstream and downstream primers, 1 μL of dNTP (2.5 mM, GenStar, Cat#A114 -01), 1mL Taq enzyme (5U / μL, TaKaRa Taq TM , DR001A), with ddH 2 O to 25...

Embodiment 2

[0060] The quality of the raw materials used in this example is the same as that of Example 1. For the preparation method, refer to Example 1. The improvement is: (+) γ-lactamase is used to resolve the racemic γ-lactam. Take 5 mg of recombinant large intestine Bacillus cells were suspended in 450μL 150mmol pH6.0~8.5 phosphate buffer, 50μL 5g / L (-)γ-lactam substrate was added to the phosphate buffer, and reacted at 60~120℃ for 10~ 24 hours. The above reaction mixture was centrifuged, the supernatant was taken, and 250 μL of ethyl acetate was extracted from the supernatant to obtain the (-)γ-lactam.

[0061] 100 μL of the (-)γ-lactam was taken for HPLC (Shimadzu, Essentia LC-15C) detection, and the optical purity was 91.4%-99.9%, and the yield was greater than 45%.

[0062] The reagents and instruments used in Example 1 and Example 2 are conventional products in the market unless otherwise specified.

[0063] In Example 1 and Example 2, the basic operation methods of genetic e...

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Abstract

The invention discloses an Aeropyrum pernix derived (+)gamma-lactamase, its coding gene and application. The (+)gamma-lactamase provided in the invention is the protein of following (a) or (b): (a) a protein composed of an amino acid residue sequence in SEQ ID No:1 of a sequence table; and (b) an SEQ ID No:1 derived protein that is composed of an amino acid residue sequence obtained by subjecting the SEQ ID No:1 amino acid residue sequence in the sequence table to substitution and / or deletion and / or adding by one or multiple amino acid residues and has racemate gamma-lactam resolution activity. By applying the (+)gamma-lactamase to hydrolytic resolution of the racemate gamma-lactam, (-)gamma-lactam with optical purity of 91.4%-99.9% can be obtained, and the yield is more than 45%.

Description

technical field [0001] The invention belongs to the field of enzyme engineering, and in particular relates to a (+) gamma-lactamase with the activity of splitting racemate gamma-lactam, its coding gene and application. Background technique [0002] (-)2-Azabicyclo[2,2,1]hept-5-en-3-one (abbreviated as (-)γ-lactam) is a synthetic antiviral drug such as abacavir, anti-influenza A and An important intermediate of the bird flu drug Peramivir. Traditionally, (-)γ-lactams are prepared by chemical synthesis, but this method has high cost and complicated steps, and the heavy metal catalyst used in the catalytic process will cause serious pollution to the environment. In the process of synthesizing chiral (-) γ-lactams, biological enzymatic method has many advantages such as high efficiency, simplicity, energy saving, and environmental friendliness. [0003] [0004] Enzymes capable of hydrolytic resolution of racemic γ-lactams are called (+)γ-lactamases. (+)γ-lactamase belongs...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/86C12N15/55C12N15/63C12N5/10C12N1/21C12P41/00C12P17/10C12R1/01C12R1/19
Inventor 郑国钧朱绍洲任璐
Owner BEIJING UNIV OF CHEM TECH
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