Transaminase mutant and its application

A technology of mutant and transaminase, applied in transaminase mutant and its application field, can solve the problems of loss of activity, poor water solubility, variability of wild transaminase and the like

Active Publication Date: 2019-09-06
ASYMCHEM LIFE SCI TIANJIN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In industrial production of chiral amines, due to the poor water solubility of most of the existing substrates and amino donors, in order to improve the generation of chiral amine products, it is necessary to increase the content of organic solvents in the reaction system, or use basic amino donors ( Such as isopropylamine), created extremely harsh reaction conditions, made the wild transaminase extremely variable and lost its vitality, so it is necessary to adapt to the needs of industrial production by transaminases that are well tolerant to organic solvents and high pH

Method used

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  • Transaminase mutant and its application
  • Transaminase mutant and its application
  • Transaminase mutant and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0095] According to a typical embodiment of the present invention, the mutation further includes at least one of the following mutation sites or combinations of mutation sites: 7th, 32nd, 96th, 164th, 171st, 186th, 252nd, 384th, 389th, 391st, 394th, 404th, 411th, 420th, 423rd, 424th, 442nd, 452nd and 456th Preferably, the mutation further includes at least one of the following mutation sites: K7N, Q32L, K96R, V164L, E171D, S186G, V252I, Y384F, I389M, I389F, D391E, N394D, L404Q, L404Q, G411D, Q420R, Q420K, M423K, E424R, E424K, E424Q, R442H, R442L, G452S and K456R. Mutations are carried out by site-directed mutagenesis to change its amino acid sequence to achieve changes in protein structure and function, and then through directional screening methods to obtain transaminases with the above mutation sites, so these transaminase mutants have good resistance to organic solvents tolerance and high pH tolerance, and has high soluble expression characteristics and high activity chara...

Embodiment 1

[0116] Catalytic activity of ArS-ωTA mutant and wild enzyme on substrate 1 in organic solvent-free system:

[0117]

[0118] In a 10mL reaction bottle, weigh 100mg of the raw material, add 1mg of pyridoxal 5'-phosphate, add 2mM isopropylamine hydrochloride, add 250μL of crude enzyme solution of ArS-ωTA mutant or wild enzyme (0.05g of mutant wet cells are subjected to sonication Broken to obtain 20% crude enzyme solution (pH=8.5), add 0.41mL of 100mM PB8.5 to make the final volume of the system to 1mL, stir at 30°C for 16h, centrifuge the system at 12000rpm for 5min, take a sample of 200μL and add 2mL of acetonitrile to dissolve it. After being centrifuged at 12000rpm for 5min, the product conversion rate was detected by HPLC. The mutant information and results are shown in Table 9.

[0119] Table 9

[0120]

[0121] The results in Table 9 showed that the catalytic activity of the ArS-ωTA mutant to substrate 1 was greatly improved compared with the wild strain. After th...

Embodiment 2

[0123] Catalytic activity of ArS-ωTA wild enzyme and mutants to substrate 1 in organic solvent system (40%) DMSO:

[0124] In a 10mL reaction bottle, weigh 100mg of raw material (same as Example 1), add 1mg 5'-pyridoxal phosphate, add 2mM isopropylamine hydrochloride, add 250μL ArS-ωTA mutant or wild enzyme crude enzyme solution (0.05g The mutant wet cells were sonicated to obtain 20% crude enzyme solution (pH = 8.5), add 100mM PB8.5 0.01mL, add dimethyl sulfoxide 0.4mL to make the final volume of the system 1mL, and stir at 35°C for 16h, After the system was centrifuged at 12000rpm for 5min, 200 μL of the sample was added to 2mL of acetonitrile to dissolve, centrifuged at 12000rpm for 5min, and sent to HPLC to detect the conversion rate of the product. See Table 10 for mutant information and results.

[0125] Table 10

[0126]

[0127] ND: No Product Generation Detected

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Abstract

The invention discloses a transaminase mutant and an application thereof. An amino acid sequence of the transaminase mutant is the amino acid sequence obtained by mutating the amino acid sequence shown as SEQ ID NO: 1, and the mutation includes at least one of the following mutation sites: 3rd, 5th, 8th, 25th, 32nd, 45th, 56th, 59th, 60th, 84th, 86th, 164th, 176th, 178th, 180th, 187th, 197th, 206th, 207th, 242th, 245th, 319th, 324th sites and the like; alternatively, the amino acid sequence of the transaminase mutant has the mutation site in the amino acid sequence in which the mutation is generated. The application of the mutant can increase the reaction rate, improves the stability of the enzyme, reduces the amount of the enzyme, and reduces the difficulty of the post-treatment, so thatthe transaminase mutant can be suitable for industrial production.

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 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] Chiral amines are an important part of the synthesis of a variety of bioactive compounds and active pharmaceutical ingredients. It is estimated that 40% of current drugs are chiral amines and their derivatives, such as neur...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12P17/12C12P17/10C12P13/00
CPCC12N9/1096C12Y206/01C12P17/12C12P17/10C12P13/001C12P13/008C12N15/70C12N15/81
Inventor 洪浩詹姆斯·盖吉张娜刘芳颜俊杰刘冶王祖建
Owner ASYMCHEM LIFE SCI TIANJIN
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