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Transaminase mutant and application thereof

A kind of mutant, transaminase technology, applied in transaminase mutant and its application field, can solve problems such as unfavorable reaction balance, enzyme instability and the like

Active Publication Date: 2022-05-24
ASYMCHEM LAB TIANJIN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, few of the reported wild ω-TAs can be directly used in the pharmaceutical industry, which is mainly limited by the instability of the enzyme and the unfavorable reaction balance.

Method used

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  • Transaminase mutant and application thereof
  • Transaminase mutant and application thereof
  • Transaminase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0078] According to an exemplary embodiment of the present invention, a recombinant plasmid is provided. The recombinant plasmid contains any of the above DNA molecules. The DNA molecules in the above-mentioned recombinant plasmids are placed in appropriate positions of the recombinant plasmids, so that the above-mentioned DNA molecules can be replicated, transcribed or expressed correctly and smoothly.

[0079] Although the term "containing" is used in the present invention to define the above-mentioned DNA molecule, it does not mean that other sequences irrelevant to its function can be arbitrarily added to both ends of the DNA sequence. Those skilled in the art know that in order to meet the requirements of the recombination operation, it is necessary to add suitable restriction endonuclease enzyme cleavage sites at both ends of the DNA sequence, or additionally add a start codon, a stop codon, etc. Therefore, if using Closed formulations to qualify will not truly cover th...

Embodiment 1

[0090] Template and its mutants catalyze the formation of chiral amines from ketone compounds. The activity was tested under reaction condition M. The results showed that Template had poor catalytic activity to the substrate, while the Template mutant had better catalytic activity, and the mutant T291A+ G292A+ The ee value of I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R +Y69H+T70G+G160T+T106S-12aa transaminase mutant increased to more than 90%. After the transformation of the present invention, the template mutants have obtained the catalytic activity for the substrate with larger steric hindrance, the activity of some mutants is greatly improved, and the stereoselectivity of the mutants is also greatly improved, expanding the substrate spectrum . The reaction formula is as follows, and the results are shown in Table 4.

[0091]

[0092] Table 4

[0093]

[0094]

[0095] Remark:

[0096] 1) Reaction conditions M: 0.3 wt enzyme, PLP 1 g / L, DMSO 40%, I...

Embodiment 2

[0100] Template and its mutants catalyze the formation of chiral amines from ketone compounds. The activity was tested under the reaction condition N. The results showed that Template had no catalytic activity to the substrate, while the Template mutant had better catalytic activity. After the modification of the present invention, Template mutants expand the spectrum of catalytic substrates. The reaction formula is as follows, and the results are shown in Table 5.

[0101]

[0102] table 5

[0103]

[0104]

[0105] Remark:

[0106] Reaction conditions M: 1 wt enzyme, PLP 1 g / L, DMSO 20%, IPN 10 eq, 100 V, 0.2 M boric acid pH=10.5, 50 °C, 16 h;

[0107] N.D. means no product generation detected, - means 0~10%, + means 10~20%, ++ means 20~50%, +++ means 50~80%, ++++ means 80~90%, + ++++ means 90%~95%, ++++++ means >95%.

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Abstract

The invention discloses a transaminase mutant and application thereof. The transaminase mutant is obtained by mutation of an amino acid sequence as shown in SEQ ID NO: 1, and the mutation at least comprises: T291A; or the amino acid sequence of the transaminase mutant has a mutation site in the mutated amino acid sequence, and the amino acid sequence has more than 80% of homology with the mutated amino acid sequence, preferably has 85% of homology, and more preferably has 90% of homology. According to the transaminase mutant disclosed by the invention, the substrate spectrum of enzyme is expanded, and the catalytic ability of the enzyme to a large steric hindrance substrate is improved; meanwhile, the stability is enhanced, and catalytic reaction can be carried out under the conditions of high-concentration solvent and high temperature; in addition, the stereoselectivity of the transaminase mutant is also greatly improved, chiral amine can be produced more efficiently, the production cost and post-treatment difficulty are reduced, and the method is suitable for being popularized and used for industrial production of chiral amine.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular, to a transaminase mutant and its application. Background technique [0002] Chiral amines refer to a class of small-molecule compounds whose chiral centers contain amino groups, and are key intermediates for many medicines and pesticides. Among them, chiral amines are important intermediates in the synthesis of neurological drugs, cardiovascular drugs, antihypertensive drugs, anti-infective drugs and vaccines. At present, 40% to 50% of chiral drugs are chiral amine compounds. Efficient and convenient synthesis of chiral amine compounds is an important research direction in organic synthetic chemistry. For example, the diabetes drug sitagliptin (Merck and Codexis) and the broad-spectrum contact herbicide glufosinate-ammonium (Bayer) both have chiral amine chemistry modules. [0003] At present, the methods for preparing chiral amines mainly include chemical method, biological ...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N15/81C12N1/19C12N1/21C12P13/00C12R1/19C12R1/645
CPCC12N9/1096C12N15/70C12N15/81C12P13/001
Inventor 洪浩詹姆斯·盖吉肖毅张娜焦学成张克俭王磊赵军旗凌君徐新生李瑞王祖建
Owner ASYMCHEM LAB TIANJIN
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