Biosynthesis method for beta-alanine

A technology of alanine and acrylic acid, applied in the field of β-alanine biosynthesis, can solve problems such as low yield, complicated process, incomplete conversion, etc., shorten the reaction process, improve the preparation efficiency, and have little environmental impact Effect

Active Publication Date: 2020-03-27
ZHEJIANG NHU CO LTD +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When the raw material fumaric acid of this method is converted into β-alanine, there is a molecular weight loss, and the atom economy is poor; the reaction needs to be completed in two steps, and the process is complicated, which affects the yield; and the enzyme catalyzes the ammoniation of fumaric acid to synthesize L-aspartic acid The acid reaction is a reversible reaction, cannot be completely converted, and the yield is low
[0010] Further improving the synthesis process of alanine, not only avoiding a series of hazards of chemical methods, but also overcoming the defects of long steps and poor economy of existing biological methods, is still a hot spot of current research

Method used

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  • Biosynthesis method for beta-alanine
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  • Biosynthesis method for beta-alanine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1 Mutation Generation and Screening

[0080] Mutants of aspartase were prepared: T187, M321, K324, N326 and AFIL (quad mutant).

[0081] The wild-type aspartase (Gen Bank No. AB028242.1) is derived from Bacillus sp YM55-1, the nucleotide sequence is shown in SEQ ID NO: 1, the amino acid sequence is shown in SEQ ID NO: 3, Using the recombinant plasmid pET-24a (nucleotide sequence shown in SEQ ID NO: 2) containing the wild aspartase gene as a template, site-directed saturation mutations were performed at T187, M321, K324 and N326.

[0082] Use the software Oligo7 to design blunt-end primers, and the degenerate primers are as follows (wherein N=A, G, C or T; K=G or T):

[0083] Mutation of amino acid T187:

[0084] Forward primer: 5'- NNK GGTATCGGCGGTTTTG-3'

[0085] Reverse primer: 5'-ACCACCCAGACCCAGTGCCGGA-3'

[0086] Mutation of amino acid at position M321:

[0087] Forward primer: 5'- NNK CAATATATCGTAAAAGCTGCTG-3'

[0088] Reverse primer: 5'-ATGTTCTTT...

Embodiment 2

[0098] Expression of embodiment 2 aspartase mutants

[0099] Each transformant prepared in Example 1 (Escherichia coli containing mutants, and Escherichia coli expressing wild-type aspartase as a control) was respectively inoculated in 5 ml of kanamycin-containing (50 μg / ml) In LB medium, culture in a shaker at 37°C and 200rpm until OD 600 When it reaches about 0.4 to 0.5, a seed liquid is obtained.

[0100] Each seed solution was inoculated in autoinduction medium (Qingdao Haibo) at an inoculum amount of 1%, and cultured at 30° C. for 24 hours. The bacterial solution was ultrasonically disrupted, and the supernatant was obtained after centrifugation, which was the corresponding enzyme solution.

Embodiment 3

[0101] The catalysis of embodiment 3 aspartase mutants to acrylic acid

[0102]

[0103] β-Alanine Synthetic Route

[0104] Add 23ml of 25% ammonia water to a 100ml three-necked flask, start stirring at 25rpm, slowly add 5ml of glacial acetic acid (AR), add 6.0g of acrylic acid, stir and heat in a water bath to 48-51°C, the system pH is 9.0-9.2, add MgSO 4 ·7H 2 0.13 mg, add 9 ml of the enzyme solution prepared in Example 2, retest pH 8.9-9.1, and after 24 hours of incubation at 48 to 51° C., get 100 μl of the reaction solution and dilute 1000 times with the mobile phase for HPLC liquid phase analysis.

[0105] Liquid phase conditions: C18 column (25cm), [3% NaH 2 PO 4 2H 2 O (pH3.3-3.5): water = 1: 3]: acetonitrile = 85: 15; flow rate 1ml / min, detection wavelength: 205nm.

[0106] Retention time: β-alanine: 2.66min; Acrylic acid: 5.17min

[0107] Conversion rate=[1-(residual acrylic acid content / initial acrylic acid content)]×100%

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Abstract

The invention relates to the technical field of biology, in particular to a biosynthesis method for beta-alanine, and provides an enzyme which can convert acrylic acid into the beta-alanine. Biosynthesis of the beta-alanine is performed with the enzyme, so that high-temperature and high-pressure reaction related to a chemical method can be avoided, and a side reaction is controlled well; the use of acrylonitrile is avoided; the acrylonitrile has high toxicity and does great harm to the environment; while the crylic acid is taken as a raw material in the biosynthesis method, and the crylic acidhas low toxicity, and has a slight influence on the environment; and compared with an existing biological method, the biosynthesis method only needs one-step reaction, so that the reaction course isshortened, the beta-alanine can be prepared within 24 hours, and the preparation efficiency is increased.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a biosynthetic method of β-alanine. Background technique [0002] β-alanine (β-Alanine) is the only β-type amino acid that exists in nature. It is a non-protein amino acid and is mainly used for the synthesis of substances such as pantothenic acid, coenzyme A, carnosine and anserine. Development plays an essential role. As the research on β-alanine and its derivatives matures, such as camptothecin β-alanine ester, poly-β-alanine, etc., play an increasingly important role in the fields of medicine, fine chemicals, and the environment. role. [0003] There are chemical synthesis method and biosynthesis method for producing β-alanine at home and abroad: [0004] [0005] Synthesis of β-alanine by chemical method [0006] [0007] Biological synthesis of β-alanine route [0008] Among them, the chemical synthesis method is mainly the acrylonitrile method, which uses acrylonitr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/06
CPCC12P13/06
Inventor 周硕赖敦岳何龙丹何梦燕汪钱
Owner ZHEJIANG NHU CO LTD
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