Aminopherase for producing L-2-aminobutyric acid

A technology of aminobutyric acid and threonine deaminase, which is applied in the field of biocatalysis, can solve the problems of low conversion concentration and low enzyme activity, and achieve the effect of overcoming low conversion rate, improving enzyme activity and realizing industrialization

Active Publication Date: 2016-03-30
湖州颐盛生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current conversion concentration is low, only 0.1M, and the activity of the enzyme is also relatively low [ParkES, DongJY, ShinJS., Organic & biomolecula

Method used

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  • Aminopherase for producing L-2-aminobutyric acid
  • Aminopherase for producing L-2-aminobutyric acid
  • Aminopherase for producing L-2-aminobutyric acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] 1.1 Construction of recombinant Escherichia coli expressing various sources of ω-transaminase (ω-TA)

[0061] Select Ochrobactrumanthropi, Aquamicrobiumdefluvii, Bradyrhizobiumsp.Ec3.3, Brucellaneotomae, Novosphingobiumacidiphilum, Ochrobactrumintermedium, Pseudaminobacteralicylatoxidans, Brucellaabortus, ParacoccusdenitrificansPD1222 sources of ω-transaminase, and synthesize the basic transaminase gene sequence as Restriction endonuclease sites NdeI and BamHI were designed at the end, subcloned into the corresponding sites of the vector pET24a (Novagen) to obtain recombinant plasmids, transformed into E. coli expression host BL21 (DE3), and obtained recombinant E. coli expressing ω-transaminase.

[0062] 1.2 Comparison of the ability of ω-transaminases from different sources to catalyze the production of L-2-aminobutyric acid

[0063] Shake flask fermentation of ω-transaminase expression strains: prepare liquid medium TB (pH7.0-7.5), containing 12g / L peptone, 24g / L imp...

Embodiment 2

[0069] Example 2 Site-directed mutagenesis

[0070] 2.1 Site-directed mutagenesis of ω-transaminase from multiple sources

[0071] Park et al. (2014) carried out molecular modeling of PDTA by X-ray diffraction technology, and obtained the active site and substrate-related information in the enzyme structure (PBDID: 4GRX). They found that the type of substrate recognized by Lpocket of PDTA is determined by the spatial structure of Spocket regulated by the V153 site. Subsequent site-directed mutation at this site resulted in the mutant V153A with improved enzymatic activity for specific substrates, further confirming the key role of the V153 site as the active site of the enzyme. [Park ES, et al., Advanced Synthesis & Catalysis, 2014, 356(1): 212–220]

[0072] Park et al. (2012) have reported that the V154 site of OATA corresponds to the V153 site of PDTA [ParkES, KimM, ShinJS., ApplMicrobiolBiotechnol, 2012, 93(6):2425-2435]. The inventors compared the amino acid sequences o...

Embodiment 3

[0082] Example 3 Site-directed saturation mutation

[0083] 3.1 Construction of site-specific saturation mutation library

[0084] After site-directed mutation, compared with the corresponding wild type, the catalytic ability of ADTA / V154A, BNTA / V154A, BSTA / V154A, OITA / V154A, PSTA / V154A was significantly improved, proving that the V154 site determines the activity of ω-transaminase key active sites. Use degenerate primers to make site-directed saturation mutations at these sites, and construct a mutation library.

[0085] Table 4 Site-directed saturation mutation sites and primer design of ω-transaminase from various sources

[0086] name

mutation site

Mutation Primer

Primer sequence (5'-3')

OATA

V154

OATA-V154-F

cgcggctatcacggtNNKacgattgcctctgcc

ADTA

V154

ADTA-V154-F

cgcggttatcatggcNNKaccattgcatcggct

BATA

V154

BATA-V154-F

cgcggttatcatggcNNKaccattgccagcgca

BSTA

V154

BSTA-V154-F

c...

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Abstract

The invention establishes aminopherase through genetic engineering. Compared with ochrobactrum anthropi based wild w-aminopherase coming from ochrobactrum anthropi, the enzyme activity of aminopherase is remarkably improved, and aminopherase can be used for industrially producing L-2-aminobutyric acid.

Description

technical field [0001] The invention belongs to the technical field of biocatalysis, and in particular relates to a transaminase constructed by genetic engineering for producing L-2-aminobutyric acid. Background technique [0002] L-2-aminobutyric acid, the English name is L(+)-2-Aminobutyric acid, the molecular formula is C4H9NO2, and the CAS No. is 1492-24-6. It is a non-natural chiral α-amino acid. L-2-Aminobutyric acid is the main raw material for the production of a new type of antiepileptic drug, levetiracetam, and is also a key chiral precursor for the synthesis of the antibacterial and anti-tuberculosis drug ethambutol, as well as many chiral drugs. Market demand volume is increasing substantially. [0003] The production of L-2-aminobutyric acid includes chemical and biological methods. The production process of chemically producing L-2-aminobutyric acid can be found in Chinese invention patents ZL200610030420.X and ZL201210034250.8. This process uses 2-bromobutyr...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P13/00
CPCC12N9/1096C12P13/00C12Y206/01
Inventor 陶荣盛朱傅赟沈青孙梁栋沈正权蒋宇杨晟
Owner 湖州颐盛生物科技有限公司
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