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Transaminase mutant and application thereof

A technology of transaminase and mutant, which is applied to the transaminase mutant and its application field, can solve the problem that the transaminase is not suitable for industrial production, and achieve the effects of improving enzyme specificity, reducing cost and improving enzyme activity

Active Publication Date: 2018-03-23
天津凯诺医药科技发展有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention aims to provide a transaminase mutant and its application to solve the technical problem that the wild-type transaminase in the prior art is not suitable for industrial production

Method used

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

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Experimental program
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Embodiment approach

[0032] According to a typical embodiment of the present invention, a recombinant plasmid is provided. The recombinant plasmid contains any one of the above DNA molecules. The DNA molecules in the above recombinant plasmids are placed in appropriate positions of the recombinant plasmids, so that the above DNA molecules can be replicated, transcribed or expressed correctly and smoothly.

[0033] Although the qualifier used in the present invention is "contains" when limiting 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 recombination operations, it is necessary to add suitable restriction endonuclease cutting sites at both ends of the DNA sequence, or additionally add start codons, stop codons, etc., therefore, if using A closed-ended statement will not truly cover these situations.

[0034] The ...

Embodiment 1

[0045] In a 10mL reaction bottle, add 40mg of isopropylamine into 1mL of 0.2M phosphate buffer solution, adjust the pH to 7.0~7.5, add transaminase SEQ ID NO: 1 with a mass of 120mg, add 0.4mg of pyridoxal phosphate, mix well and drop into 40 mg dissolved in 0.2 mL DMSO The pH of the system is 7.0-7.5, and the mixture is stirred at a constant temperature of 35°C±3°C for 16h. The conversion rate of the system was detected by GC. According to the above steps, the reaction data of the mutant transaminase whose sequence number is SEQ ID NO: 13-26 is as follows in Table 1:

[0046] Table 1

[0047] serial number

mutation site

16h

Enzyme dosage

SEQ ID NO: 1

none

21.3％

3wt

SEQ ID NO: 13

G72L

36.8％

2wt

SEQ ID NO: 14

L76A

47.1％

2wt

SEQ ID NO: 15

L107I

47.7％

2wt

SEQ ID NO: 16

S125A

38.9％

2wt

SEQ ID NO: 17

S132A

52.8％

2wt

SEQ ID NO: 18

K92G

39....

Embodiment 2

[0051] Generally speaking, the performance of mutants with single-point mutations is difficult to be significantly different from that of the mother parent, and the combination of mutation points can obtain better mutants. Therefore, the mutation sites are randomly recombined by DNA shuffling, a mutation library is established, and then screened to try to obtain better mutants.

[0052]DNA shuffling is the sexual recombination of genes at the molecular level. A group of homologous genes were digested into random fragments with nuclease I, and these random fragments were used to form a library, which were used as primers and templates for PCR amplification. Template exchange and genetic recombination occur when fragments of one gene copy serve as primers for another gene copy.

[0053] Enzyme solution preparation method: centrifuge the 96-well plate to remove the supernatant medium, add 200 μl of enzymatic hydrolysis solution (lysozyme 2 mg / mL, polymyxin 0.5 mg / mL, pH=7.0) int...

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Abstract

The invention discloses a transaminase mutant and application thereof. The amino acid sequence of the transaminase mutant is obtained by mutation of an amino acid sequence shown in SEQ ID NO:1, and the mutation at least comprises one of the following mutation sites: the 19th site, the 41st site, the 43rd site, the 72nd site, the 76th site, the 92nd site, the 107th site, the 125th site, the 132nd site, the 226th site, the 292nd site, the 295th site, the 308th site and the 332nd site; the 19th mutation is serine, the 41st mutation is serine, the 43rd mutation is asparagine, and the 72nd glycinemutation is leucine; or the amino acid sequence of the transaminase mutant has mutation sites in a mutated amino acid sequence, and has 80% or more homology with the mutated amino acid sequence. The transaminase mutant disclosed by the invention greatly reduces the cost in industrial production of chiral amine.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a transaminase mutant and its application. Background technique [0002] Chiral amines widely exist in nature and are the structural units of many important biologically active molecules, as well as important intermediates in the synthesis of natural products and chiral drugs. Many chiral amines contain one or more chiral centers. The pharmacological activity, metabolic process, metabolic rate and toxicity of different chiral drugs are significantly different. Usually one enantiomer is effective, while the other enantiomer are inefficient or ineffective, or even toxic. Therefore, how to efficiently and stereoselectively construct compounds containing chiral centers is of great significance in pharmaceutical research and development. [0003] ω-transaminase (ω-TA) belongs to the transferase class, and like other transaminases, it catalyzes a process of amino and ketone exchange. In...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12P17/12
CPCC12N9/1096C12P17/12C12Y206/01
Inventor 洪浩詹姆斯·盖吉卢江平焦学成张娜李瑞张克俭
Owner 天津凯诺医药科技发展有限公司
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