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Transaminase mutant and application thereof

A technology of mutants and transaminases, applied in the field of transaminase mutants and their applications, can solve the problems that transaminases are not suitable for industrial production, and achieve the effect of improving catalytic activity

Active Publication Date: 2020-06-05
ASYMCHEM LAB TIANJIN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The main purpose of the present invention is to provide a kind of transaminase mutant and application thereof, to solve the problem that existing transaminase is not suitable for industrialized production

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 1

[0063] Add 30 mg of raw material 1, raw material 2, raw material 3 and raw material 4 to a 5 mL reaction bottle, add Tris-Cl (0.1 M) with pH 8.5, 181 μL of isopropylamine hydrochloride (6 M), 0.9 mg of To PLP, add 300 mg of transaminase (see Table 1 for details), mix well, make a total volume of 1500 μL, and react at 30°C and 200 rpm on a shaker for 16 hours. After the reaction, add 2 times the volume of methanol to the reaction system, mix well, centrifuge at 12000 rpm for 3 min, take the supernatant, and detect it by HPLC with a wavelength of 210 nm. The response characteristics of some mutants are as follows:

[0064] Table 1:

[0065]

[0066] In the above table, the female parent is the mutant SEQ ID NO:1; the conversion rate of the enzyme to the substrate is represented by activity, - represents the initial activity of the female parent, and the multiple of activity increase is represented by +, + represents an increase of 1-5 times, ++ means 5-10 times improvement....

Embodiment 2

[0069] Add 30 mg of raw material 1, raw material 2, raw material 3 and raw material 4 to a 5 mL reaction bottle, add Tris-Cl (0.1 M) with pH 8.5, 181 μL of isopropylamine hydrochloride (6 M), 0.9 mg of To PLP, add 210 mg of transaminase (see Table 2 for details), mix well, make a total volume of 1500 μL, and react at 30°C for 16 hours on a shaker at 200 rpm. After the reaction, add 2 times the volume of methanol to the reaction system, mix well, centrifuge at 12000 rpm for 3 min, take the supernatant, and detect it by HPLC with a wavelength of 210 nm. The response characteristics of some mutants are as follows:

[0070] Table 2:

[0071]

[0072] The above parent is the mutant SEQ ID NO:1; the conversion rate of the enzyme to the substrate is represented by activity, - represents the initial activity of the parent, and the multiple of activity increase is represented by +, + represents an increase of 1-5 times, + + means 5-10 times improvement.

[0073] As can be seen fr...

Embodiment 3

[0075] Add 30 mg of raw material 1, raw material 2, raw material 3 and raw material 4 to a 5 mL reaction bottle, add Tris-Cl (0.1 M) with pH 8.5, 181 μL of isopropylamine hydrochloride (6 M), 0.9 mg of To PLP, add 60 mg of transaminase (see Table 3 for details), mix well, make a total volume of 900 μL, and react at 30°C and 200 rpm on a shaker for 16 hours. After the reaction, add 2 times the volume of methanol to the reaction system, mix well, centrifuge at 12000 rpm for 3 min, take the supernatant, and detect it by HPLC with a wavelength of 210 nm. The response characteristics of some mutants are as follows:

[0076] table 3:

[0077]

[0078] The above parent is the mutant SEQ ID NO:1; the conversion rate of the enzyme to the substrate is represented by activity, - represents the initial activity of the parent, and the multiple of activity increase is represented by +, + represents an increase of 1-5 times, + + indicates an increase of 5-10 times, +++ indicates an incr...

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Abstract

The invention provides a transaminase mutant and application thereof. Compared with an amino acid sequence shown in SEQ ID NO:1, an amino acid sequence of the transaminase mutant includes at least oneof the following mutation sites: L166, K149, K146, A168, H73, F133, H82, E24, V194, T294, A295, G235 and F236. The transaminase mutant has improved catalytic activity for transamination reaction of ketone substrates and is suitable for industrial production of chiral amines.

Description

technical field [0001] The invention relates to the field of enzyme production, in particular to a transaminase mutant and its application. Background technique [0002] Chiral amines are the structural units of many important biologically active molecules and the key intermediates for the synthesis of many chiral drugs. Many important neurological drugs, cardiovascular drugs, antihypertensive drugs, anti-infective drugs, etc. are synthesized using chiral amines as intermediates. The preparation of chiral amines is mainly realized by chemical reduction, which has disadvantages such as harsh reaction conditions, use of toxic transition metal catalysts, and low stereoselectivity of products. At present, the preparation of chiral compounds by biocatalysis is widely used in the pharmaceutical industry and has broad prospects. [0003] ω-aminotransferase can catalyze ketone substrates to efficiently prepare chiral amines through stereoselective transamination. Because of its a...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12P13/00C12P17/12
CPCC12N9/1096C12P13/001C12P13/008C12P17/12C12Y206/01C12N15/70C12R2001/01C12N15/52
Inventor 洪浩詹姆斯·盖吉肖毅张娜焦学成马玉磊牟慧艳曹珊
Owner ASYMCHEM LAB TIANJIN
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