Preparation method of transaminase mutant and chiral amine compound

A transaminase and mutant technology, applied in the field of enzyme catalysis, can solve the problem of low transaminase activity, and achieve the effects of high enzyme activity, wide substrate spectrum, and strong ability to withstand extreme environments

Pending Publication Date: 2022-08-09
ASYMCHEM LIFE SCI TIANJIN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The main purpose of the present invention is to provide a method for preparing transaminase mutants and chiral amine compounds to solve the problem of low transaminase activity in the prior art

Method used

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  • Preparation method of transaminase mutant and chiral amine compound
  • Preparation method of transaminase mutant and chiral amine compound
  • Preparation method of transaminase mutant and chiral amine compound

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0233] Using the method described above, site-directed mutagenesis was performed on the basis of the parental SEQ ID NO:1. The specific mutation sites are shown in Table 1, and the catalytic activity of the mutants was detected according to the following reaction conditions:

[0234] 1mL reaction system includes 2mg substrate 1 or substrate 2 or substrate 3 or substrate 4, 1mg PLP, 2mg isopropylamine hydrochloride, 400μL crude enzyme solution (prepared by 200mg wet bacteria mud), pH8.0 100mM phosphoric acid Salt buffer, react at 50°C for 42h. The above-mentioned wet bacteria mud is obtained by centrifuging the corresponding mutant Escherichia coli fermentation broth, and the crude enzyme liquid is obtained by adding pH8.0100mM phosphate buffer to the obtained wet bacteria mud, homogenizing the walls by ultrasonic or homogenizer, and then concentrating.

[0235] The test results are shown in Table 1.

[0236] Table 1:

[0237]

[0238] Note: In the above table, 0 means th...

Embodiment 2

[0240] On the basis of Example 1, the combined mutation was performed, and the activity of the combined mutation was screened according to the same reaction conditions as Example 1. The results are shown in Table 2.

[0241] Table 2:

[0242]

[0243] Note: In the above table, 0 means the conversion rate is less than 1%, + means the conversion rate is greater than or equal to 1% and less than 5%, ++ means the conversion rate is greater than or equal to 5% and less than or equal to 10%, and +++ means the conversion rate is greater than or equal to 10% and less than 15%, ++++ means the conversion rate is greater than or equal to 15% and less than 20%.

Embodiment 3

[0245] On the basis of Example 2, multiple rounds of saturation mutation were carried out, and the catalytic activity of the mutant was detected according to the following reaction conditions:

[0246] 1mL reaction system includes 4mg substrate 1 or substrate 2 or substrate 3 or substrate 4, 1mg PLP, 2mg isopropylamine hydrochloride, 400μL crude enzyme solution (prepared by 200mg wet bacteria mud), pH8.0 100mM phosphoric acid Salt buffer, 50 ℃ reaction 18h. The results are shown in Table 3.

[0247] table 3:

[0248]

[0249]

[0250] Note: In the above table, 0 means the conversion rate is less than 1%, + means the conversion rate is greater than or equal to 1% and less than 5%, ++ means the conversion rate is greater than or equal to 5% and less than 10%, and +++ means the conversion rate is greater than or equal to 10 % and less than 15%, ++++ means the conversion rate is greater than or equal to 15% and less than 20%, +++++ means the conversion rate is greater than...

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Abstract

The invention provides a transaminase mutant and a preparation method of a chiral amine compound. The transaminase mutant comprises (a) a protein with an amino acid sequence as shown in SEQ ID NO: 1; or (b) a protein with an amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 3; or (c) a protein which is subjected to amino acid mutation and has a transaminase function on at least one of the following sites of the amino acid sequence in (b): Y89, L380, N86, Y85, T91, P83, K90, S417, S424, F301, G164, T452 and the like; and (d) protein which has more than 80% of homology with the amino acid sequence limited by any one of (a), (b) or (c) and has a transaminase function. The method can solve the problem of low transaminase activity in the prior art, and is suitable for the field of enzyme catalysis.

Description

technical field [0001] The present invention relates to the field of enzyme catalysis, in particular, to a method for preparing a transaminase mutant and a chiral amine compound. Background technique [0002] Chiral amines refer to a class of small-molecule compounds whose chiral center contains an amino group. They are the structural units of many important biologically active molecules and key intermediates for the synthesis of many chiral drugs. At present, the preparation of chiral amines is mainly achieved by chemical methods, biological resolution methods and biological asymmetric methods. The chemical method has disadvantages such as long reaction route, harsh conditions, use of toxic transition-state metal catalysts, and low product stereoselectivity; the theoretical maximum yield of biological separation method is only 50%, so there are certain limitations in scale-up production. sex. [0003] Asymmetric synthesis catalyzed by transaminase has become the preferred...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P13/00C12P17/12C12R1/19
CPCC12N9/1096C12N15/70C12P13/001C12P17/12C12Y206/01C12N2800/101
Inventor 洪浩詹姆斯·盖吉张娜焦学成马玉磊牟慧艳玄美娟张燕青张仁宽
Owner ASYMCHEM LIFE SCI TIANJIN
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