L-aspartate-alpha-decarboxylase and application thereof

An aspartic acid and decarboxylase technology, applied in the field of bioengineering, can solve the problems of large amount of enzyme, reduced amount of enzyme, lower cost, etc., and achieves the effect of less amount of enzyme, rapid reaction and overcoming large amount of enzyme.

Pending Publication Date: 2021-05-11
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to overcome the defects that the L-aspartic acid-α-decarboxylase derived from prokaryotic sources is easily inactivated and cannot be reused in the prior art, resulting in too much enzyme consumption and high cost when preparing β-alanine. Provided is an L-aspartic acid-α-decarboxylase derived from eukaryotes that can be used repeatedly, which can be used repeatedly in the process of catalyzing the preparation of β-alanine, and at the same time, the amount of enzyme is effectively reduced , the cost is greatly reduced

Method used

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  • L-aspartate-alpha-decarboxylase and application thereof
  • L-aspartate-alpha-decarboxylase and application thereof
  • L-aspartate-alpha-decarboxylase and application thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0044]Preparation method of derivatizer: Take 0.0343g o-phthalaldehyde and 0.1472g N-acetyl-L-cysteine, add 5mL absolute ethanol, and then dilute to 25mL with 0.1M sodium borate (pH=9.5).

[0045] Derivatization reaction: take 300 μL of the sample to be tested, add 200 μL of sodium borate buffer (0.1M pH=9.5), then add 200 μL of derivatizing agent, mix by inverting repeatedly for 6-8 times, derivatize in the dark for 2 minutes, and then inject the sample.

[0046] HPLC system: Agilent 1100; Chromatographic column: Welch Ultimate® AQ-C18 (4.6×250mm, 5μm);

[0047] Mobile phase: 50mmol / L sodium acetate aqueous solution:methanol=45:55 (volume ratio).

[0048] Flow rate: 0.8mL / min;

[0049] Column temperature: 35°C;

[0050] Detection wavelength: 334nm.

[0051] Among them, the L-aspartic acid detection picture is as follows figure 1 Shown, L-aspartic acid elution time is 2.392min.

[0052] β-alanine liquid phase detection pictures such as figure 2 Shown, β-alanine peak ti...

Embodiment 1

[0054] Construction of High Expression Engineering Bacteria

[0055] (1) The peach aphid obtained from the NCBI database ( Myzus persica ) The protein sequence XP_022171514 of cysteine ​​sulfinic acid decarboxylase, codon optimized to SEQ ID NO.3, was synthesized by Nanjing GenScript Biotechnology Co., Ltd., using Nde I and Bam The HI restriction site was cloned into the pET28a(+) vector to obtain the pET28a(+)-MpADC recombinant plasmid.

[0056] (2) Transform the resulting recombinant vector into Escherichia coli BL21 (DE3) competent cells.

[0057] (3) In a sterile environment, take 100 μL of the cells and evenly spread the cells on the LB solid medium plate (Kan resistance) with a coating stick. 12-14h).

[0058] (4) Streak the LB plate (Kan resistance) and incubate at 37°C for 12 hours, then connect to a 5mL LB test tube (Kan resistance) and culture overnight.

[0059] (5) Inoculate TB medium containing 45 μg / mL of kanamycin with 2% inoculum, incubate at 37°C for 4...

Embodiment 2

[0061] The rest of the steps in Example 2 are the same as those in Example 1, the difference lies in step (5), which is as follows: Inoculate 1% engineered bacteria into TB medium containing 30 μg / mL of kanamycin, culture at 30°C for 5 hours, and then add The inducer IPTG was used to a final concentration of 0.2mmol / L, and the temperature was lowered to 20°C to induce the expression of the target protein. The culture was continued for 25 hours, and the fermentation was terminated. The bacteria were collected for later use.

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Abstract

The invention relates to the field of bioengineering, in particular to L-aspartate-alpha-decarboxylase. The L-aspartate-alpha-decarboxylase has an amino acid sequence and a gene sequence shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 in SEQUENCE LISTING. The gene is synthesized in vitro after codon optimization based on a gene sequence encoding cysteinesulfinate decarboxylase in genome of Myzus persicae. The gene is cloned into an expression vector and transformed into Escherichia coli to construct a high-expression engineering strain. After the engineering strain is cultured and induced, it is found that the enzyme has activity of L-aspartate-alpha-decarboxylase, and the enzyme can convert L-aspartic acid into beta-alanine under proper conditions, overcome a defect that prokaryotic-derived L-aspartate-alpha-decarboxylase in the prior art is easy to inactivate, and can be used for industrial production of beta-alanine under conditions of small enzyme dosage and low cost.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to an L-aspartic acid-α-decarboxylase and its application. Background technique [0002] L-aspartate-α-decarboxylase (L-aspartate-α-decarboxylase, EC4.1.1.11, ADC), also known as L-aspartate-1-decarboxylase, can catalyze L-aspartate The acid removes the carboxyl group at the α position to generate β-alanine. [0003] There are two main types of ADCs currently reported. The first type of ADC is found in bacteria and archaea, and this type of ADC uses the pyruvyl group as the active center; the second type of ADC is found in some archaea and insects, and requires the coenzyme pyridoxine phosphate Aldehyde (PLP) completes the catalytic activity. This type of enzyme usually has a variety of catalytic capabilities and poor catalytic specificity, but has no substrate-dependent inactivation. [0004] β-Alanine, also known as 3-aminopropionic acid, is an important precursor substance for the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12P13/06C12R1/19
CPCC12N9/88C12Y401/01011C12P13/06
Inventor 郭倩柳鹏福储消和陈艳竹国津韩笑笑
Owner ZHEJIANG UNIV OF TECH
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