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(+)-gamma-lactamase from microbacterium as well as coding gene and application of (+)-gamma-lactamase

A technology of lactamase encoding and lactamase, which is applied in the field of enzyme engineering, can solve the problems of poor selectivity, restriction, and enzyme instability, and achieve the effects of simple method, low environmental pollution, and good operability

Inactive Publication Date: 2015-05-20
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

(+)-γ-Lactamase is a kind of amidase, which can be used for the synthesis of chiral intermediate (-)-γ-lactam, and a few (-)-γ-lactams have been reported so far , but these (+)-γ-lactamases still have some shortcomings in the resolution process of the prime minister, such as poor enantioselectivity and unstable enzymes, etc., these factors have limited the (+)-γ-lactamase Application in industry

Method used

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  • (+)-gamma-lactamase from microbacterium as well as coding gene and application of (+)-gamma-lactamase
  • (+)-gamma-lactamase from microbacterium as well as coding gene and application of (+)-gamma-lactamase
  • (+)-gamma-lactamase from microbacterium as well as coding gene and application of (+)-gamma-lactamase

Examples

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Effect test

Embodiment 1

[0044] (1) Acquisition of (+)-γ-lactamase 33H4-5540 gene of the present invention

[0045](1) Primer design

[0046] Primers were designed according to the high-throughput screening 33H4-5540 gene sequence, and the primers were synthesized by Shanghai Sangong.

[0047] 33H4-5540 upstream primer:

[0048] 5'-GGAATTC CATATG GCAGCACCCCGCCGCACCGTCGTCCT-3'

[0049] The underline indicates the Nde I restriction site

[0050] 33H4-5540 downstream primer:

[0051] 5'-CCG CTCGAG TCAGAGAGCGACGTGGTCGTGCGTGGCGAT-3'

[0052] The underline indicates the Xho I restriction site

[0053] (2) PCR amplification of the target gene

[0054] Use the whole genome of Microbacteria as a template for PCR amplification. The PCR reaction system is: 2ul genome, 10ul reaction buffer, 10ul high GC enhancer buffer, 2ul upstream and downstream primers, 2ul DNTP (2.5mM), 0.5ul high-fidelity NEBQ5 polymerase , make up to 50ul with double distilled water. The PCR conditions are: the first step is 98...

Embodiment 2

[0066] The quality of the raw materials used in this example is the same as in Example 1, and the preparation method is similar to that of Example 1. The improvement is that in the application of (+)-γ-lactamase to resolve the racemate γ-lactam, an appropriate amount of The recombinant Escherichia coli cells were added to 1 ml of 110 g / l racemic γ-lactam (prepared in phosphate buffer), and reacted at 20° C.-30° C. for 20-30 minutes. After the reaction, the mixed solution was extracted with 1 ml of ethyl acetate to obtain (-)-γ-lactam.

[0067] The (-)-γ-lactam extracted above is subjected to HPLC detection, the optical purity is 99.8%-99.9%, and the yield is greater than 48%.

[0068] The reagents and instruments used in Example 1 and Example 2 are conventional products on the market unless otherwise specified.

[0069] The basic operation techniques of genetic engineering such as PCR, enzyme digestion, connection and transformation adopted in Example 1 and Example 2 are reco...

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Abstract

The invention discloses (+)-gamma-lactamase from microbacterium as well as a coding gene and an application of the (+)-gamma-lactamase, and belongs to the field of enzyme engineering. The (+)-gamma-lactamase is the following protein (a) or (b): (a): a protein containing an amino acid residue sequence as shown in SEQ ID:1; (b) a protein which has racemate gamma-lactamase splitting activity, contains the amino acid residue sequence as shown in SEQ ID:1 and is obtained through substituting and / or deleting and / or adding at least one or more amino acid residues. The (+)-gamma-lactamase further comprises a coding gene of the (+)-gamma-lactamase. The invention provides (+)-gamma-lactamase with activity of efficiently splitting racemate gamma-lactamase and a coding gene of the (+)-gamma-lactamase, which are expected to be applied to the industry. The enzyme has low requirements on temperature, can reach maximal activity at 20-30 DEG C, is very good in stability tolerance, and low in energy consumption when applied to the industry. The method for splitting racemate gamma-lactamase by utilizing the (+)-gamma-lactamase is simple and convenient, good in operability and small in pollution.

Description

technical field [0001] The invention belongs to the field of enzyme engineering, and specifically relates to a (+)-γ-lactamase capable of splitting racemate γ-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-A An important chiral intermediate of influenza virus and avian influenza virus drug Peramivir. The traditional method is to use chemical synthesis to prepare the chiral intermediate, but the cost of the chemical method is high, the steps are cumbersome, and the heavy metal catalyst used in the reaction process will cause serious pollution to the environment. The bioenzyme resolution method has the characteristics of high efficiency, energy saving and environmental friendliness in the process of synthesizing the chiral intermediate (-)-γ-lactam. [0003] Enzymes that can specifically hydrolyze (+)-γ-lactam in racemate γ-lactam ...

Claims

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Application Information

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IPC IPC(8): C12N9/86C12N15/55C12N15/70C12N1/21C12P41/00C12P17/10
CPCC12N9/86C12P17/12C12P41/001C12Y305/02
Inventor 郑国钧高帅华黄蓉朱绍洲李红霞
Owner BEIJING UNIV OF CHEM TECH
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