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Transaminase mutants and application thereof

A transaminase and mutant technology, applied in the field of enzyme engineering, can solve the problems of large column pressure, weak strength, continuous flow reaction, etc., and achieve the effect of increasing possibility, high space-time yield, and reducing emulsification

Pending Publication Date: 2021-12-21
ASYMCHEM LAB TIANJIN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the strength of the cross-linked enzyme is weak because there is no solid support. If it is filled in the pipeline, a huge column pressure will be generated when the reaction solution flows through, which will make the continuous flow reaction impossible.

Method used

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  • Transaminase mutants and application thereof
  • Transaminase mutants and application thereof
  • Transaminase mutants and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1: Mutant 50°C, pH 10, 50% DMSO tolerance test

[0080] The crude enzyme was treated at 50°C, pH 10, and DMSO concentration was 50% for 1h, then in a 10mL reaction flask, 0.1g of substrate 2 was added, and 4eq of isopropylamine hydrochloride and 0.6-1mg of PLP (5'- pyridoxal phosphate), and then add 4 mg of the enzyme after the above treatment, and stir for 16 hours at 50° C., pH 10, and DMSO concentration of 50% in an environment with constant temperature. The conversion rate of the system was detected by HPLC, and the mutant reaction data are shown in Table 5.

[0081] table 5:

[0082]

[0083]

Embodiment 2

[0084] Example 2: Mutant 60°C, pH 10, 60% DMSO tolerance test

[0085] The crude enzyme was treated for 1 h at 60°C, pH 10, 60% DMSO concentration environment, then in a 10mL reaction flask, 0.1g substrate 2 was added, and 4eq isopropylamine hydrochloride and 0.6mg PLP (5' - pyridoxal phosphate), and then add 5 mg of the above-mentioned enzyme, and continue to stir at 60° C., pH 10, and 60% MeOH concentration environment for 16 hours. The conversion rate of the system was detected by HPLC, and the reaction data of the mutants are shown in Table 6 below.

[0086] Table 6:

[0087] mutation site Conversion rate(%) Female parent (TA-Cv-1) 34.4 W60Y 84.3 Y168A 44.6 V379W 85.8 V379L 77.9 V379M 83.1 C418Q 69.0 C418W 75.2

Embodiment 3

[0088] Example 3: Mutant 60°C, pH 10, 70% DMSO tolerance detection

[0089] Treat the crude enzyme at 60°C, pH 10, 70% DMSO concentration for 1h, then add 0.1g of substrate 2, 4eq of isopropylamine hydrochloride and 0.6-1mg of PLP (5'- pyridoxal phosphate), and then add 10 mg of the enzyme after the above treatment, and continue stirring at 60° C., pH 10, and 70% DMSO concentration environment for 16 hours. The conversion rate of the system was detected by HPLC, and the reaction data of the mutants are shown in Table 7.

[0090] Table 7:

[0091]

[0092]

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PUM

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Abstract

The invention provides transaminase mutants and application thereof. The transaminase mutants are subjected to amino acid mutation on the basis of a sequence shown in SEQ ID NO: 1, and the amino acid mutation is single-point mutation or combined mutation of W60Y, Y168A, V379W, V379L, V379M, C418Q and C418W. The activity, stability, temperature tolerance, pH tolerance and organic solvent tolerance of the transaminase mutants are improved, the problem that transaminase is poor in tolerance in an extreme environment in the prior art is solved, and the transaminase mutants are suitable for the field of enzyme engineering.

Description

technical field [0001] The invention relates to the field of enzyme engineering, in particular to a transaminase mutant and its application. Background technique [0002] Chiral amines are important intermediates for the synthesis of many chiral drugs, and are also the main components of optically pure drugs containing amino groups. Neurological drugs, vascular drugs, antihypertensive drugs, anti-infective drugs and vaccines all use chiral amines as intermediates. Transaminases use ketones as raw materials to efficiently produce chiral amines through stereoselective transamination. However, the free enzyme enzymatic method has many problems in the scale-up production application, such as low enzyme activity and large enzyme dosage, which lead to increased fermentation costs; it is easily denatured and inactivated by the influence of organic solvents in the reaction system, and the recovered product after post-treatment is emulsified. , a large amount of three wastes are pr...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N15/81C12N1/21C12N1/19C12N11/082C12N11/089C12N11/087C12N11/14C12P17/10C12P17/12C12P13/00C12R1/19
CPCC12N9/1096C12N15/70C12N15/81C12N11/082C12N11/089C12N11/087C12N11/14C12P17/10C12P17/12C12P13/001C12P13/008C12Y206/01Y02P20/50
Inventor 洪浩詹姆斯·盖吉肖毅张娜罗杰斯卡·维亚撒·威廉姆斯崔瑜霞赵佳东高妍妍傅涵
Owner ASYMCHEM LAB TIANJIN
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