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A kind of amino lyase mutant protein and its coding gene and application

An amino and gene technology, applied in amino lyase mutant protein and its coding gene and application field, can solve the problems of high raw material price, low stereoselectivity, poor stereoselectivity, etc.

Active Publication Date: 2020-04-10
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The bioenzyme conversion method is mainly prepared by decarboxylase, and the production cost is relatively high due to the high price of raw materials and low utilization rate of raw materials
The chemical synthesis method is complex in process, a large amount of toxic organic solvents are used in the reaction process, the environment is polluted, the reaction conditions are severe, the overall yield is low, the stereoselectivity is low, and the production cost is also at a relatively high level
[0004] Optically pure aminoalcohol compounds are an important raw material of the raw material drug dulutevir. It is difficult to synthesize optically pure (R) 1-propyl (2-amino)methanol by chemical synthesis, and the reaction steps are many, the yield is low, and the three-dimensional Poor selectivity and other reasons lead to high cost and difficult to realize industrialization

Method used

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  • A kind of amino lyase mutant protein and its coding gene and application
  • A kind of amino lyase mutant protein and its coding gene and application
  • A kind of amino lyase mutant protein and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0133] Example 1, Screening and Determination of Mutation Sites

[0134] Sequence analysis, mutation and functional verification of the L-aspartate deaminase protein (aspB protein) derived from Bacillus subtilis revealed 37 amino acid sites. Four important amino acid sites were screened out. These 4 amino acid sites are mutated in different forms, and the obtained mutant proteins all have higher amino lyase activity, and can catalyze the hydrogenation reaction with trans-2-butenoic acid as the substrate at the same time, which can be used in the production of ( R) 1-propyl (2-amino) formic acid, the yield is high and meets the requirement of 100% stereoselectivity.

[0135] The aspB protein is shown in sequence 2 of the sequence listing, and the coding gene of the aspB protein (aspB gene) is shown in sequence 1 of the sequence listing.

[0136] The four amino acid sites and their mutant forms are shown in Table 1.

[0137] Table 14 amino acid sites and their mutant forms

...

Embodiment 2

[0139] Embodiment 2, the preparation of recombinant bacteria

[0140] The wild-type aspB protein is shown in sequence 2 of the sequence listing, and the coding gene (aspB gene) of the wild-type aspB protein is shown in sequence 1 of the sequence listing.

[0141] 1. Construction of wild-type recombinant expression vector

[0142] The double-stranded DNA molecule shown in Sequence 1 was inserted between the EcoR I and Not I restriction sites of the pET21a vector to obtain the recombinant expression vector pET21a-aspB (sequencing verification was correct). The DNA molecule shown in sequence 1 encodes the protein shown in sequence 2.

[0143] 2. Construction of mutant recombinant expression vector

[0144] 1. Insert the double-stranded DNA molecule 1 between the EcoR I and Not I restriction sites of the pET21a vector to obtain the recombinant expression vector pET21a-1 (sequence verification is correct). Double-stranded DNA molecule 1 is obtained by point-mutating the wild-typ...

Embodiment 3

[0181] Embodiment 3, the enzyme activity assay of aminoacidase mutant protein

[0182] Adopt the wild-type recombinant bacterium that embodiment 2 obtains and mutant recombinant bacterium 1-34 to carry out following experiment respectively:

[0183] 1. Inoculate the bacteria to be tested into 5ml LB liquid medium (amphicillin resistance: 100μg / ml), shake at 37°C and 200rpm until the OD of the bacteria solution 600nm =1-2, add 30ppm IPTG, continue to culture at 30°C and 200rpm shaking until 24h.

[0184] 2. After completing step 1, collect the bacterial cells by centrifugation at 12000 rpm.

[0185] 3. Use 50mM Tris (pH 7.5), 2mM MgCl to the bacteria collected in step 2 2 After resuspending 1ml, ultrasonically (power 25W) disrupted for 30s to obtain the cell disrupted liquid.

[0186] 4. Heat the broken cell solution obtained in step 3 at 60°C for 30 minutes, then centrifuge at 12,000 rpm, and collect the supernatant, which is the protein solution.

[0187] The protein sol...

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PUM

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Abstract

The invention discloses amino lyase mutant protein and a coding gene and application thereof. The protein is obtained by conducting mutation on at least one of 187-326 site amino acid residue shown inthe sequence 2 of the sequence table. The invention further discloses a method for utilizing the protein to prepare (R) 1-propyl(2-amino)formic acid. The protein provided by the invention has the relatively high amino lyase activity, and meanwhile can catalyze an ammonification reaction which taking trans-2-butenoic acid as a substrate to produce (R) 1-prypyl(2-amino)formic acid, the yield is high, the requirements of 100% stereoscopic selectivity is satisfied, and the protein has the very wide application prospects.

Description

technical field [0001] The invention relates to an amino lyase mutant protein, its coding gene and application. Background technique [0002] Optically pure (R)1-propyl(2-amino)formic acid and its derivatives are important chemical raw materials widely used in the synthesis of chemical products, food and APIs. Currently, the methods for producing (R)1-propyl(2-amino)formic acid and its derivatives mainly include chemical synthesis and the combination of chemical synthesis and biological enzyme catalysis. [0003] The bio-enzyme conversion method is mainly prepared by decarboxylase, and the production cost is relatively high due to the high price of raw materials and the low utilization rate of raw materials. The chemical synthesis process is complex, the reaction process uses a large amount of toxic organic solvents, the environment is polluted, the reaction conditions are severe, the overall yield is low, the stereoselectivity is low, and the production cost is also at a r...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C12N15/60C12P13/04
CPCC12N9/88C12P13/04
Inventor 李瑞峰吴边宋璐田玉娥丰婧
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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