Enzymatic preparation of beta alanine

An alanine and amino acid technology, applied in the directions of enzymes, lyases, carbon-carbon lyases, etc., can solve the problems of complicated product purification steps and environmental pollution of nitrile substances.

Active Publication Date: 2020-07-24
ZHEJIANG HUARUI BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of these methods require strong acid and strong alkali, high temperature and high pressure conditions, and the product purification steps are cumbersome, and there are problems of environmental pollution caused by nitrile substances

Method used

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  • Enzymatic preparation of beta alanine
  • Enzymatic preparation of beta alanine

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1 Aspartic Acid Decarboxylase Genetic Engineering Bacteria Construction

[0042] Synthesize the sequence SEQ ID NO:3 through the whole gene sequence, design restriction sites NdeI and BamHI at both ends, clone into the corresponding sites on the pSH plasmid, obtain the recombinant plasmid pSH-Pandcg, and then transform it into E. coli for expression by calcium chloride method In the host BL21 (DE3) competent cells, coat the LB medium plate containing kanamycin, cultivate overnight at 37°C, pick a single colony, inoculate it into a test tube containing LB medium, cultivate overnight, collect the bacteria by centrifugation, and extract The plasmid was extracted, the gene sequence was confirmed to be correct, and the recombinant genetically engineered strain expressing the wild-type Pandcg enzyme was obtained.

Embodiment 2

[0043] Example 2 Error-prone PCR method constructs aspartate decarboxylase (Pandcg) random mutation point library

[0044] Using SEQ ID NO:3 as a template, an error-prone PCR technique was used to construct a random mutant library.

[0045] Forward primer Pandcg-Nde-F: 5'-ATGTACCTGCGCACCATCCTCGGAAG-3',

[0046] Reverse primer Pandcg-Xho-R: 5'-CTCGAGCTAAATGCTTCTCGACGTCAAAAGC-3'.

[0047] 50μL error-prone PCR reaction system includes: 50ng plasmid template pSH-Pandcg, 30pmol pair of primers Pandcg-Nde-F and Pandcg-Xho-R, 1×Taq buffer, 0.2mM dGTP, 0.2mM dATP, 1mM dCTP, 1mM dTTP, 7mMMgCl 2 , (0mM, 0.05mM, 0.1mM, 0.15mM, 0.2mM) MnCl 2 , 2.5 units of Taq enzyme (fermentas). The PCR reaction conditions were: 95°C for 5 minutes; 94°C for 30s, 55°C for 30s, 72°C for 2min / kbp; 30 cycles; 72°C for 10min. The 2.0kb random mutation fragment was recovered from the gel as a large primer, and MegaPrimer PCR was performed with KOD-plus DNA polymerase: 94°C for 5min; 98°C for 10s, 60°C for...

Embodiment 3

[0048] The high-throughput screening of embodiment 3Pandcg mutant library

[0049] 3.1 Select the transformant in the mutant library, inoculate it into a 96-well deep-well culture plate containing 700 μg / mL of LB medium, the medium contains 100 μg / mL kanamycin, culture at 37°C for 6 hours, and add a final concentration of 0.1 mM IPTG , cooled to 25°C, and incubated overnight. Centrifuge at 5000rpm for 10min, discard the supernatant, freeze at -70°C for 1h, and thaw at room temperature for 30min. Add 200 μL of 0.1M potassium phosphate buffer (pH 8.0) to resuspend the bacteria for the determination of Pandcg enzyme activity.

[0050] 3.2 Determination and screening of mutants with high enzyme activity

[0051] Substrate reaction solution: 200 g / L L-aspartic acid, adjusted pH with NaOH to dissolve.

[0052] Termination reaction solution: 0.5ml of 1M NaOH solution.

[0053] Homogenize 80 μL of the cell suspension in step 3.1 above to break the wall, add 80 μL of the substrate ...

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Abstract

A high enzyme activity aspartate decarboxylase mutant can be obtained by a directed evolution method, the high enzyme activity aspartate decarboxylase mutant can efficiently catalyze decarboxylation of aspartic acid to produce beta-alanine, and by optimization of the reaction process, the aspartic acid substrate concentration can reach 250g / L. After 24 hours of conversion, the conversion rate canreach above 98.3%, and the high enzyme activity aspartate decarboxylase mutant has industrial application prospects.

Description

technical field [0001] The invention belongs to the field of biocatalysis, in particular, relates to an aspartate decarboxylase mutant, and relates to the use of the aspartate decarboxylase mutant or its expressing microorganism in the production of beta-alanine. Background technique [0002] Beta-alanine, namely β-alanine (β-alanine), is the only naturally occurring β-type amino acid in nature. In recent years, its role in medicine, chemical industry, food, environment and other fields has become increasingly prominent (Biotechnology and Bioengineering, 2012, 109(10):2437-2459.). In the field of medicine, it is mainly used for the synthesis of pantothenic acid and calcium pantothenate, and it is also the raw material for the synthesis of carnosine, sodium pamidionate, balsalazide and other drugs. In the field of food, it can be used as seasoning. In the environmental field, it can be used to prepare water purification flocculants, etc. (Nature Chemical Biology, 2012, 8(6)...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C12N15/60C12P13/06
CPCC12N9/88C12P13/06C12Y401/01
Inventor 范文超王金刚梁岩袁圣伦任亮
Owner ZHEJIANG HUARUI BIOTECHNOLOGY CO LTD
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