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Disulfide-bond-introduced omega-aminotransferase mutant and application thereof

A technology of mutants and transaminases, which is applied in the field of ω-transaminase mutants, can solve problems such as further improvement of thermal stability, and achieve the effect of improving thermal stability

Active Publication Date: 2016-09-21
杭州快格科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Experiments show that the half-life of the wild-type enzyme at 40°C is only 6.9 minutes, and its thermal stability needs to be further improved

Method used

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  • Disulfide-bond-introduced omega-aminotransferase mutant and application thereof
  • Disulfide-bond-introduced omega-aminotransferase mutant and application thereof
  • Disulfide-bond-introduced omega-aminotransferase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] First upload the PDB file (PDB ID: 4CE5) of Aspergillus terreus ω-transaminase to Disulfideby Design (DbD, http: / / cptweb.cpt.wayne.edu / DbD2) and Disulfide Bonds in Proteins (MODIP, http : / / caps.ncbs.res.in / dsdbase / modip.html) website, the above software fully considered the C-S bond rotation angle of two cysteines, C α distance between and C β Using bioinformatics to calculate and analyze the bond length, bond angle and energy to predict the feasible introduction of 8 disulfide bond sites, they are R131C and D134C, N25C and A28C, D113C and Y146C, E213C and V234C , A44C and L48C, M150C and M280C, A32C and F142C, R161C and Y201C. According to the crystal structure of ω-transaminase, the temperature factor (B-factor) value of amino acid residues in the G129-D134 region is higher. For proteins, if the B-factor value of a certain amino acid residue is higher, it indicates that the The structure where the amino acid residues are located is more unstable. Therefore, in the ...

Embodiment 2

[0031] Site-directed mutagenesis was performed on R131C and D134C of the ω-transaminase gene locus in turn by site-directed mutagenesis PCR. The primers for site-directed mutagenesis are shown in Table 1, thereby obtaining a mutant containing two cysteines at the same time. The mutant was sequenced to verify that its nucleotide sequence was mutated at codons 497 and 499, and the codes of arginine (R, Arg) and aspartic acid (D, Asp) were encoded by positions 131 and 134. The codons CGT and GAT are all mutated to the codon TGC encoding cysteine ​​(C, Cys).

[0032] Table 1 Primers for site-directed mutagenesis.

[0033] Primer name

Sequence 5'→3'

R131C-F

GGTGCGAGGAACTTGCCCGGAAGATATAGTG

R131C-R

CTATATCTTCCGGGCAAGTTCCTCGCACCCC

D134C-F

GGAACTTGCCCGGAATGCATAGTGAACAACCTGTAC

D134C-R

ACAGGTTGTTCACTATGCATTCCGGGCAAGTTCCTC

[0034]Using the pET28a-ω-opt-TA plasmid containing the wild-type ω-transaminase gene as a template, t...

Embodiment 3

[0038] Transform the plasmid with the double mutant ω-transaminase mutant gene sequenced correctly in Example 2 into E.coli BL21(DE3), pick a single colony and inoculate it into a test tube with 5 mL of LB liquid medium, at 37°C , 200r / min under the condition of cultivating overnight. Inoculate the 100mL LB medium (10g of tryptone, 5g of yeast powder, 10g of sodium chloride, and adjust the pH of ), cultivated to OD at 37℃, 180r / min 600 When the value is 0.4-0.6, add an appropriate volume of IPTG (final concentration: 0.5mmol / L), and then induce culture at 25°C and 150r / min for 18h, then collect the bacteria.

[0039] The collected bacteria were washed twice with phosphate buffer, resuspended in cell-breaking buffer, and ultrasonically disrupted. The working conditions of ultrasonic cell disruption were: power 300W, working for 3s, intermittent for 6s, and ultrasonicating for 8min. The broken cells were centrifuged at 12000r / min and 4°C for 30min, and the supernatant was coll...

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Abstract

The invention provides a disulfide-bond-introduced omega-aminotransferase mutant and application thereof. The amino acid sequence of the omega-aminotransferase mutant is disclosed as SEQ ID NO.1. The omega-aminotransferase mutant is obtained by respectively mutating 131st-site arginine and 134th-site aspartic acid of Aspergillus terreus omega-aminotransferase into cysteines. Compared with the wild type, the half-inactivation temperature of the omega-aminotransferase mutant is enhanced by 1.6 DEG C, the half life at 40 DEG C is 10.4 minutes which is prolonged by 3.5 minutes (50.7%), and the heat stability is greatly enhanced.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a disulfide bond-introduced omega-transaminase mutant and application thereof. Background technique [0002] Transaminases can catalyze the transfer reaction of amino groups from amino donors to amino acceptors, with high regioselectivity and stereoselectivity. Transaminase can not only split racemic amines through kinetics, but also generate chiral amines through asymmetric synthesis of ketones. Compared with traditional chemical catalytic methods, transaminases are more attractive and competitive, and have become industrially used to produce amino acids and chiral amines. One of the commonly used enzymes for important pesticides or pharmaceutical intermediates such as amino alcohols and amino sugars. According to the multiple sequence alignment in the PFAM database, transaminases can be divided into 5 categories: aspartate aminotransferase, aromatic transaminase, ω-transaminase, b...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12P7/26
CPCC12N9/1096C12P7/26C12Y206/01
Inventor 黄俊谢东芳梅乐和王宏鹏胡升方卉赵伟睿吕常江李伊莹
Owner 杭州快格科技有限公司
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