Biosynthesis method of beta-alanine

A biosynthesis and alanine technology, applied in the biological field, can solve the problems of unsatisfactory stability of L-aspartate-α-decarboxylase, increase the cost of separation and purification, unfavorable industrial production, etc., so as to eliminate the need for separation and purification , Avoid the use of antibiotics, the effect of fast production rate

Inactive Publication Date: 2018-05-18
INNOBIO CORP LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

According to the current reports, most of the host strains of genetically engineered bacteria are mainly Escherichia coli, but Escherichia coli is not a recognized biosafety strain, and there are biosafety risks such as endotoxin contamination during use, and the cells are broken and broken. Will increase the cost of separation and purification
In 2015, Deng Siying and others studied the enzymatic properties of L-aspartate-α-decarboxylase. The stability of L-aspartate-α-decarboxylase from different sources is not ideal. The higher the reaction temperature, the lower the reaction rate. The faster, the greater the loss of enzyme activity, which is unfavorable for the industrial production of the enzyme

Method used

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  • Biosynthesis method of beta-alanine
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  • Biosynthesis method of beta-alanine

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preparation example Construction

[0027] The amino acid mixture mentioned in the preparation of the above seeds and induction medium contains the following components: Arginine 0.2g / L, Aspartic acid 1.0g / L, Glutamic acid 1.0g / L, Isoleucine Acid 0.3g / L, Lysine 0.3g / L, Valine 1.5g / L, Methionine 0.2g / L, Phenylalanine 0.5g / L, Serine 3.75g / L, Tyrosine 0.3g / L And adenine 0.4g / L.

[0028] On this basis, the present invention further provides a biosynthetic method of β-alanine, the method is to use the whole cell of the above-mentioned recombinant Saccharomyces cerevisiae of the present invention to catalyze the conversion of L-aspartic acid into β-alanine ; Wherein the recombinant Saccharomyces cerevisiae contains exogenous L-aspartic acid-α-decarboxylase coding gene. The exogenous L-aspartic acid-α-decarboxylase coding gene mentioned therein has the nucleotide sequence described in SEQ ID NO.1, and the Saccharomyces cerevisiae EBY100 as the exogenous gene carrier is preferably Saccharomyces cerevisiae EBY100.

[0...

Embodiment 1

[0043] Construction and cultivation of surface-displayed recombinant Saccharomyces cerevisiae:

[0044] First, the panD gene was amplified using the Bacillus subtilis 168 genome (GenBank: CP019663.1) as a template, and the PCR product and the expression vector pYD1 were digested and purified by using restriction endonucleases BamH I and Xho I, and ligated overnight, and then It was transformed into Escherichia coli DH5α, the ampicillin resistance gene was used to screen the transformant, and it was verified by PCR and enzyme digestion and DNA sequencing, and then stored in the plate medium after verification. The result of the recombinant vector is as figure 1 shown. Secondly, extract the recombinant expression vector, and transform it into Saccharomyces cerevisiae EBY100 competent cells, use the defect-type screening to obtain positive transformants, activate the positive transformants and inoculate them into the seed medium, and wait for the cells to grow to OD 620 =1.0-5...

Embodiment 2

[0047] Effects of different reaction pH on the catalytic synthesis of β-alanine by recombinant Saccharomyces cerevisiae displayed on the surface:

[0048] The cultivation of recombinant Saccharomyces cerevisiae displayed on the surface was the same as in Example 1, and the cells were collected by centrifugation for catalytic reaction. The effects of different reaction pH 5 (acetate buffer) and 6 / 7 / 8 / 9 (phosphate buffer) on the catalytic synthesis of β-alanine by recombinant Saccharomyces cerevisiae displayed on the surface were studied. Dissolve 2.66g L-aspartic acid in 90mL buffer solution, maintain the above pH with 6M NaOH solution and 10% hydrochloric acid, add recombinant Saccharomyces cerevisiae into the reaction system, and make the biomass OD in the reaction solution 620 =1.0~5.0, control the temperature at 37°C, take samples every hour to measure β-alanine, until L-aspartic acid is completely converted, centrifuge to get the supernatant, and use high performance liqui...

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Abstract

The invention discloses a biosynthesis method of beta-alanine, which includes reaction of transforming and compounding beta-alanine by recombined brewing yeast full cell catalyzed L- asparaginic acid,wherein the recombined brewing yeast contains exogenous L- aspartic acid-alpha- decarboxylase encoding gene. The method and application have advantages of low-priced and easy-acquired raw materials,simple and easy-controlled operation, green and high-efficient synthetic route, good in biological safety, quick in reaction rate, high in converting rate, and others; the method and application alsolay a foundation of the industrialization of beta-alanine.

Description

technical field [0001] The invention relates to a simple and efficient microbial synthesis method for beta-alanine, in particular to a method for biotransforming and synthesizing beta-alanine using recombinant genetically engineered yeast, and belongs to the field of biotechnology. Background technique [0002] β-alanine, also known as 3-alanine, is the only β-type amino acid that exists in nature. β-alanine is widely used in the fields of medicine, food, beauty, feed and chemical industry, such as the synthesis of calcium pantothenate, carnosine, disodium pamidronate, balsalazide and other medicines or feed and food additives. At present, the production of β-alanine mainly includes chemical synthesis and biological methods. The chemical method was used earlier and more widely, but it often has the disadvantages of serious pollution, many side reactions (products), and low crystal purity; while the biological enzyme method It has the advantages of high substrate selectivity...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/06C12N15/81C12N15/60C12R1/865
CPCC12N9/88C12N15/81C12P13/06C12Y401/01011
Inventor 洪皓范超刘军吴文忠
Owner INNOBIO CORP LTD
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