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Amino lyase mutant protein and coding gene and application thereof

An amino acid and protein technology, applied in the directions of lyase, application, genetic engineering, etc., can solve the problems of complex chemical synthesis process, low overall yield and high production cost

Active Publication Date: 2018-11-23
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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  • Amino lyase mutant protein and coding gene and application thereof
  • Amino lyase mutant protein and coding gene and application thereof
  • Amino lyase mutant protein and coding gene and application thereof

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 L-aspartate deaminase protein (aspB protein) derived from Bacillus subtilis found 37 amino acid sites. Four important amino acid sites were screened. These 4 amino acid sites are mutated in different forms, and the obtained mutant proteins all have higher aminolyase activity, can catalyze the ammonia addition reaction with trans-2-butenoic acid as a substrate simultaneously, and can be used for the production of ( R) 1-propyl(2-amino)carboxylic acid in high yield and meeting the requirement of 100% stereoselectivity.

[0135] The aspB protein is shown in SEQ ID NO: 2 in the sequence listing, and the encoding gene (aspB gene) of the aspB protein is shown in SEQ ID NO: 1 in 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 mutated forms

[0138]

Embodiment 2

[0139] Embodiment 2, the preparation of recombinant bacteria

[0140] The wild-type aspB protein is shown in SEQ ID NO: 2 of the Sequence Listing, and the encoding gene (aspB gene) of the wild-type aspB protein is shown in SEQ ID NO: 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, and the recombinant expression vector pET21a-aspB was obtained (sequencing verification was correct). The DNA molecule shown in SEQ ID NO: 1 encodes the protein shown in SEQ ID NO: 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 (sequencing verification is correct). Double-stranded DNA molecule 1 is obtained by point m...

Embodiment 3

[0181] Example 3. Determination of enzyme activity of aminotransferase mutant protein

[0182] The wild-type recombinant bacteria and mutant recombinant bacteria 1-34 obtained in Example 2 were used to carry out the following experiments respectively:

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

[0184] 2. After completing step 1, centrifuge at 12,000 rpm to collect the bacteria.

[0185] 3. Use 50 mM Tris (pH 7.5), 2 mM MgCl for the cells collected in step 2 2 After 1ml of resuspended, ultrasonic (power of 25W) was broken for 30s to obtain the broken liquid of bacterial cells.

[0186] 4. The bacterial cell fragmentation solution obtained in step 3 was heated at 60° C. for 30 min, and then centrifuged at 12,000 rpm to collect the supernatant, which was the protein solution....

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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 present invention relates to an amino cleavage enzyme mutant protein and its encoding gene and application. Background technique [0002] Optically pure (R) 1-propyl (2-amino) carboxylic acid and its derivatives are important chemical raw materials widely used in the synthesis of chemical products, food and APIs. At present, the methods for producing (R) 1-propyl(2-amino)carboxylic acid and its derivatives mainly include chemical synthesis method and method combining chemical synthesis with biological enzyme catalysis. [0003] The biological enzymatic conversion method is mainly prepared by decarboxylase, and the production cost is relatively high due to its high raw material price and low utilization rate of raw materials. The chemical synthesis method is complicated in process, uses a large amount of toxic organic solvents in the reaction process, causes large environmental pollution, severe reaction conditions, low overall yield, low stereosel...

Claims

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Application Information

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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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