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Aspartase mutant and applications thereof

An aspartase and mutant technology, applied in application, enzyme, lyase and other directions, can solve the problem of low catalytic activity, and achieve the effects of high stereoselectivity, high conversion rate and environmental friendliness

Active Publication Date: 2020-04-21
ABIOCHEM BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The technical problem to be solved by the present invention is to solve the defect that the enzymatic activity of catalyzing (E)-4-(2,4,5-trifluorophenyl)but-2-enoic acid is not high in the prior art, therefore, The invention provides an aspartase mutant and its application

Method used

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  • Aspartase mutant and applications thereof
  • Aspartase mutant and applications thereof
  • Aspartase mutant and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Construction of AspB (aspartase, aspartase) mutant library

[0059] The AspB enzyme derived from Bacillus sp.YM55-1 was retrieved from NCBI, PDB No. 1J3U_A, the amino acid sequence is SEQ ID NO.2, the gene sequence is SEQ ID NO.1, targeting the 187th and 321st positions of SEQ ID NO.2 Position, 324, 326, 358 mutation library construction designed primer sequences are shown in Table 3:

[0060] Table 3 Primer sequence list

[0061]

[0062] Wherein, N represents any nucleotide in A, G, C, T, M represents A or C, and K represents G or T; it is selected according to the coding nucleotide of the amino acid to be mutated into at the site , such as NNK in the C187NNK forward primer can represent AAG (lysine), AAT (aspartic acid), AGG (arginine) or AGT (serine), etc., and the nucleotides corresponding to specific amino acids can be found in the table 2.

[0063] The gene AspB was synthesized according to the sequence of SEQ ID NO.1 in the sequence listing. The ...

Embodiment 2

[0071] Embodiment 2 High-throughput screening mutant library

[0072] Screen according to the following experimental steps

[0073] The first step of primary screening is carried out by using the screening method of nylon membrane transfer. When the transformant grows on the plate in Example 1, use the sterilized nylon membrane to blot the colony on the plate, and then use 500 μL of the colony on the membrane to contain 80 μg / mL ampicillin, 0.1mM IPTG, and 0.01mM MgCl 2 Wash the colonies with the TB medium, transfer the washed bacterial solution to a 96-well plate for induction, and induce overnight at 200 rpm at 25°C, then centrifuge at 4000 rpm for 20 minutes to collect the bacteria, discard the culture medium, and finally add 60 μL bugbuster to lyse, and lyse at 30°C for 2 hours. The obtained enzyme lysate was reacted according to the reaction system in Table 6, and reacted at 45° C. and 220 rpm for 3 days, and the reaction effect was detected by HPLC.

[0074] Table 6 Pr...

Embodiment 3

[0089] Example 3 Application of AspB mutant in the preparation of (R)-3-amino-4-(2,4,5-trifluorophenyl)-butyric acid

[0090]

[0091] Accurately weigh 60.6 mg of Tris base, 216 mg of (E)-4-(2,4,5-trifluorophenyl)but-2-enoic acid, 0.5 g of NH 4Cl, add to 7mL water containing 5% DMSO (dimethyl sulfoxide), adjust the pH to 8.5 with ammonia water, finally add 1g of the fungus slime prepared according to the method in Example 2 to the reaction system, and finally make up 2-3mL of water to the final volume 10mL. At 45°C, 220rpm, overnight 12h reaction, HPLC analysis conversion rate (after this embodiment does not directly detect the ee of product (R)-3-amino-4-(2,4,5-trifluorophenyl)-butyric acid value, but the product is continued to react to obtain (3R)-N-tert-butoxycarbonyl-3-amino-4-(2,4,5-trifluorophenyl)-butyric acid after detection ee value), after Add 218 mg of di-tert-butyl dicarbonate (Boc anhydride) and react for 48 hours, take the reaction solution to measure the e...

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Abstract

The invention discloses an aspartase mutant, which comprises the following mutations M321V, K324T and N326S on an amino acid sequence represented by SEQ ID NO.2, and has high enzymatic activity compared with wild-type aspartase in catalysis of (E)-4-(2,4,5-trifluorophenyl)2-butenoic acid. The invention also discloses applications of the aspartase mutant in preparation of (R)-3-amino-4-(2,4,5-trifluorophenyl)-butyric acid. According to the invention, with the application of the aspartase mutant in an enzyme catalytic reaction to prepare a sitagliptin chiral intermediate (R)-3-amino-4-(2,4,5-trifluorophenyl)-butyric acid, the conversion rate is high, the stereoselectivity is high, the yield is high, the production cost is low, the environment is friendly, and industrial large-scale production is facilitated.

Description

technical field [0001] The invention relates to an aspartase mutant and its application. Background technique [0002] Diabetes mellitus is a metabolic disease that occurs due to changes in insulin secretion, resulting in insulin deficiency, or decreased insulin activity, or under the combined influence of the two. The disease is characterized by hyperglycemia, accompanied by metabolic disorders of protein, sugar and fat. Diabetes and its complications are the third most harmful to human health after cardiovascular diseases and tumors, becoming an important disease that endangers human health. The International Diabetes Federation predicts that by 2030, the total number of people suffering from diabetes will exceed 435 million. And my country has become one of the countries with the fastest growing rate of diabetes prevalence in the world. At present, there are about 40 million diabetic patients, second only to India, ranking second in the world. Among the four types of d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12P13/04
CPCC12N9/88C12N15/70C12P13/04C12Y403/01001
Inventor 田振华程占冰秦丽军徐艳冰刘巧
Owner ABIOCHEM BIOTECH CO LTD
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