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L-aspartate-alpha-decarboxylase with improved thermal stability

A technology of aspartic acid and heat stability, applied in the field of genetic engineering, can solve the problem of low enzyme activity

Active Publication Date: 2018-12-21
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The L-aspartic acid-α-decarboxylase found in the current research generally has the problem of low enzymatic activity, and in industrial production, the enzyme needs to have better thermal stability. Therefore, increasing the L-aspartic acid - The activity and thermostability of α-decarboxylase are very important for the industrial synthesis of β-alanine

Method used

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  • L-aspartate-alpha-decarboxylase with improved thermal stability
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  • L-aspartate-alpha-decarboxylase with improved thermal stability

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Construction of recombinant mutant Escherichia coli BL21 / pET28a-TcpanD

[0049]Using the L-aspartic acid-α-decarboxylase gene derived from the wild-type Tribulus chinensis as shown in SEQ ID NO.5 as a template, primers were designed:

[0050] For F49, the sequence information is shown in SEQ ID NO 9; for R49, the sequence information is shown in SEQ ID NO 10.

[0051] For F369, the sequence information is shown in SEQ ID NO 11; for R369, the sequence information is shown in SEQ ID NO 12.

[0052] For F221, the sequence information is shown in SEQ ID NO 13; for R221, the sequence information is shown in SEQ ID NO 14.

[0053] 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 sequencing. The p...

Embodiment 2

[0054] Example 2 Expression and Purification of Recombinant Mutant L-Aspartic Acid-α-Decarboxylase

[0055] Recombinant wild-type and mutant Escherichia coli BL21 / pET28a-TcpanD were inoculated in 5 mL of LB medium with a kanamycin concentration of 100 μg / mL, cultured overnight at 37°C with shaking at 200 r / min. Inoculate the above overnight culture into 2YT medium containing 100 μg / mL of kanamycin at an inoculum size of 1%, culture at 37°C with shaking at 200 r / min until the OD600 of the bacterial solution reaches 0.6-0.8, and add IPTG to the final concentration 0.2mmol / L, induced culture at 20°C for 16-20h, collected the bacteria and sonicated, and analyzed and identified the expression level of L-aspartic acid α-decarboxylase recombinant protein by Tris-tricine SDS-PAGE method. Proteins were purified by sonication, centrifugation, and His Trap FF affinity chromatography column.

Embodiment 3

[0056] Example 3 Recombinant Mutant Enzyme Activity and Thermal Stability Detection

[0057] The L-aspartic acid α-decarboxylase recombinant Escherichia coli was collected and purified with an affinity chromatography column. After the target protein was detected by SDS-PAGE gel electrophoresis, the concentration of the target protein was measured by the Brandford method.

[0058] (1) Comparison of enzyme activity:

[0059] Definition of enzyme activity: at 37°C and pH 6.5, the amount of enzyme required to convert 1 mM product β-alanine per hour is defined as 1U.

[0060] Definition of specific enzyme activity: the number of enzyme activity units per gram of protein.

[0061] Determination of L-aspartic acid-α-decarboxylase activity: A large number of Escherichia coli capable of normal expression were cultured, mature cells were collected by centrifugation, resuspended with 50mM phosphate buffer (pH 6.5), and resuspended twice by repeated centrifugation. After several times,...

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Abstract

The invention discloses L-aspartate-alpha-decarboxylase with improved thermal stability and belongs to the technical field of gene engineering. Three enzyme mutants including K49R, G369A and K221R areobtained through site-directed mutation of L-aspartate-alpha-decarboxylase from tribolium castaneum, recombinant plasmid of the mutants is transformed into escherichia coli BL21, and a substrate L-aspartic acid is catalyzed to produce beta-alanine after expression separation and purification. At the treatment temperature of 50 DEG C, K49R can improve the thermal stability by 14% as compared withwild enzyme, G369A can improve the thermal stability by 20% as compared with wild enzyme, and K221R can improve the thermal stability by 23% as compared with wild enzyme, wherein K221 can make the enzyme activity reach about 320 U / g and has the enzyme activity improved by 23% as compared with the wild enzyme, and the discovery has important study value for industrial preparation of beta-alanine.

Description

technical field [0001] The invention relates to an L-aspartic acid-alpha-decarboxylase with improved thermal stability, which belongs to the technical field of genetic engineering. Background technique [0002] L-aspartate-a-decarboxylase (L-aspartate-a-decarboxylase, EC4.1.1.11, panD) catalyzes L-aspartic acid to generate β-alanine, which is important in the biosynthetic pathway of pantothenic acid Regulatory enzymes. [0003] β-alanine is the only β-type amino acid that exists in nature, and it has a wide range of uses. In industry, β-alanine is an important raw material for the synthesis of calcium pantothenate and one of the two amino acids for the synthesis of carnosine; in medicine, β-alanine can be used as a raw material for the synthesis of pamidronic acid that inhibits bone metastasis of malignant tumors Sodium and the anti-colitis drug balsalazide, but also as an antidote for lead poisoning and as a synthetic sweetener. [0004] In industrial production, the mai...

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
CPCC12N9/88C12N15/70C12P13/06C12Y401/01011
Inventor 周哲敏王超刘中美周丽崔文璟郭军玲薛岚
Owner JIANGNAN UNIV
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