Genetic engineering strain of L-aspartic acid-alpha-decarboxylase and application

A genetically engineered strain, aspartic acid technology, applied in the field of genetic engineering, can solve the problems of low enzyme activity, difficult product separation and purification, and low yield of β-alanine biosynthesis, and achieve the effect of increasing product concentration

Inactive Publication Date: 2019-03-01
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, there are two main biosynthesis methods. One is to optimize the metabolic pathway and synthesize β-alanine in the process of cultivating microorganisms. Purification brings great difficulties and increases the cost of later purification; another method is to synthesize β-alanine by enzymatic meth

Method used

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  • Genetic engineering strain of L-aspartic acid-alpha-decarboxylase and application
  • Genetic engineering strain of L-aspartic acid-alpha-decarboxylase and application
  • Genetic engineering strain of L-aspartic acid-alpha-decarboxylase and application

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Embodiment 1

[0033] The construction of embodiment 1 L-aspartic acid α-decarboxylase genetically engineered bacteria

[0034] Using the L-aspartic acid-α-decarboxylase gene derived from the wild-type Tribulus chinensis as shown in SEQ ID NO.4 as a template, primers were designed:

[0035] For F49, the sequence information is shown in SEQ ID NO 7; for R49, the sequence information is shown in SEQ ID NO 8.

[0036] For F369, the sequence information is shown in SEQ ID NO 9; for R369, the sequence information is shown in SEQ ID NO 10.

[0037] For F221, the sequence information is shown in SEQ ID NO 11; for R221, the sequence information is shown in SEQ ID NO 12.

[0038] Use the whole plasmid PCR method to obtain the plasmid in which the mutated gene is connected to the expression vector pET 28a(+), digest it with DpnI for about 3 hours, and transfer it into Escherichia coli JM109 by competent heat stimulation. The plasmids were extracted overnight and sent to the sequencing company for se...

Embodiment 2

[0039] The cultivation, induction and expression of embodiment 2 L-aspartic acid α-decarboxylase genetically engineered bacteria

[0040]The obtained wild-type and mutant aspartate-a decarboxylase engineered bacteria were inoculated into 5 mL of LB medium with a kanamycin concentration of 50 μg / mL, and cultured overnight at 37° C. with shaking at 200 r / min. The above culture was inoculated into 2xYT medium with a kanamycin concentration of 50 μg / mL at an inoculum size of 1%, and cultured with shaking at 37°C and 200 r / min until OD 600 0.6–0.8, add a final concentration of 0.2mmol / L IPTG, culture at 20°C for 16–20h, and collect the bacteria by centrifugation at 4000rpm.

Embodiment 3

[0041] The catalytic process of embodiment 3 one-time addition of high-concentration solid substrate

[0042] After the induced strain was collected by centrifugation, the whole cell catalytic reaction was carried out. Cell OD 600 200, pH 6.5, PLP concentration 1mmol / L, reaction temperature 37°C, add solid substrate L-sodium aspartate at one time to a final concentration of 1mol / L. Samples were taken at regular intervals to detect the amount of β-alanine produced. The result is as figure 1 shown. After 23 hours of conversion, 836 mmol / L of β-alanine was produced, and the molar conversion rate was 83.6%.

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Abstract

The invention discloses a genetic engineering strain of L-aspartic acid-alpha-decarboxylase and an application, particularly relates to a method for catalytically synthesizing beta-alanine by the genetic engineering strain of L-aspartic acid-alpha-decarboxylase and application and belongs to the technical field of genetic engineering. According to the method, through establishing and optimizing awhole-cell catalysis process and adopting a substrate batched-adding mode, after a substrate is added in four times, about 123.81g/L of beta-alanine is produced through 23h of conversion of wild typerecombinant strains; the concentration of the product, i.e., beta-alanine can reach 134.72g/L through 23h of conversion of mutant strains K221R; the concentration of the product, i.e., beta-alanine can reach 136.33 g/L through 23h of conversion of mutant strains G369A. By the method, the yield of beta-alanine is increased relatively greatly, and this discovery has an important application value inindustrialized preparation of beta-alanine.

Description

technical field [0001] The present invention relates to a genetically engineered bacterial strain of L-aspartic acid α-decarboxylase and its application, in particular to a method for catalyzing the synthesis of β-alanine with the genetically engineered strain of L-aspartic acid α-decarboxylase and applications, belonging to the technical field of genetic engineering. Background technique [0002] β-alanine is the only β-type amino acid that exists in nature, and it has a wide range of uses: in industry, it is an important raw material for the synthesis of calcium pantothenate, and is also one of the two amino acids for the synthesis of carnosine; in medicine, it is used as a raw material to synthesize and inhibit malignant Sodium pamidronate for tumor bone metastasis and anti-colitis drug balsalazide; it can also be used as an antidote for lead poisoning and for synthetic sweeteners, etc. [0003] In industrial production, the main synthesis method of β-alanine is acrylic ...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12P13/06C12R1/19
CPCC12N9/88C12N15/70C12P13/06C12Y401/01
Inventor 周哲敏王超刘中美周丽崔文璟郭军玲叶文琪
Owner JIANGNAN UNIV
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