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Alanine-glyoxylate transaminase mutant with improved enzyme activity and application thereof

A technology of glyoxylate transaminase and alanine, which is applied in applications, nucleic acid carriers, enzymes, etc., can solve the problems of production and application that cannot meet the industrial scale and low output

Active Publication Date: 2021-12-28
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above genetic engineering operations can increase the accumulation of 5-ALA in engineering strains, but the final yield is still very low, which cannot meet the production and application of industrial scale

Method used

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  • Alanine-glyoxylate transaminase mutant with improved enzyme activity and application thereof
  • Alanine-glyoxylate transaminase mutant with improved enzyme activity and application thereof
  • Alanine-glyoxylate transaminase mutant with improved enzyme activity and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] pZY48-agxT M Plasmid construction:

[0050] get agxT M Fragment: agxT-F shown in SEQ ID No.5 and agxT-R shown in SEQ ID No.6 as primers, pUC18-agxT shown in SEQ ID No.12 M As a template, agxT shown in SEQ ID No.3 is obtained by PCR method M Fragment;

[0051] Obtain pZY48 fragment: with pZY48-F shown in SEQ ID No.7 and pZY48-R shown in SEQ ID No.8 as primers, with plasmid pZY48 as template (source is Addgene (https: / / www.addgene.org / )), amplified by PCR to obtain the pZY48 fragment shown in SEQ ID No.13;

[0052] Construct pZY48-agxT M Plasmid: agxT will be obtained with the above steps M The pZY48 fragment was ligated with the CPEC method (Quan, J., & Tian, ​​J..(2014). Methods in Molecular Biology, 1116, 103.) to obtain a plasmid named pZY48-agxT M , as shown in SEQ ID No.9, the schematic diagram of the plasmid is figure 1 ;

[0053] Host cell Escherichia coli MG1655-pZY48-agxT carrying mutant gene or mutant enzyme of alanine-glyoxylate aminotransferase M C...

Embodiment 2

[0058] Alanine-glyoxylate aminotransferase activity assay:

[0059] Take control strains MG1655-pZY48-agxT and MG1655-pZY48-agxT M Put the monoclonal strain into fresh LB liquid medium and culture it to OD at 37°C and 220rpm 600 Stop culture when it is 0.6. Take 8 mL of bacterial liquid at 12000 rpm, centrifuge at 4°C for 5 min, resuspend the cell pellet with an equal volume of 50 mM potassium phosphate buffer (pH 7.0), and use an ultrasonic homogenizer (VC130PB, Sonics & Materials, Inc., Newtown, Conn. ) to break up the cells. The crushed mixture was centrifuged at 12000 rpm and 4°C for 10 minutes, and the supernatant was used for enzyme activity test. Transaminase activity was measured by the concentration of pyruvate formed from alanine and glyoxylate. Reaction system: 10mM alanine, 10mM glyoxylic acid, 1mM pyridoxal-5'-phosphate, 50mM potassium phosphate (pH7.0) and an appropriate amount of enzyme, the reaction is carried out at 30°C for 2-6 hours, and then Quantitati...

Embodiment 3

[0061] Applications of alanine-glyoxylate transaminase mutants:

[0062] pZY48-agxT carrying the alanine-glyoxylate transaminase mutant M Its control plasmid pZY48-agxT and the pUC18-hemA-aceA plasmid shown in SEQ ID No.9 were co-transformed into Escherichia coli E.coli MG1655 respectively to obtain bacterial strain MG1655-HAAM and its control bacterial strain MG1655-HAA, and carry out 5- ALA fermentation, the specific steps are as follows:

[0063] (1) Activated bacterial strains: Streak the claim 10 carrying the alanine-glyoxylate transaminase mutant strain MG1655-HAAM and its control strain MG1655-HAA in the LB solid medium, cultivate at 37°C for 10-20h, and make strain rejuvenation;

[0064] (2) Cultivation of seed liquid: Inoculate the activated bacteria obtained in step (1) into LB liquid medium, cultivate at 37°C and 220rpm for 10-20h, and then inoculate the obtained bacterial liquid according to the initial OD 600 =0.02 inoculum amount was inoculated into fresh LB m...

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Abstract

The invention discloses an alanine-glyoxylate transaminase mutant with improved enzyme activity and application thereof. The alanine-glyoxylic acid transaminase mutant is characterized in that the 20th-site isoleucine, the 124-site histidine, the 142-site glycine, the 196-201 aspartic acid, the 231-site tryptophan, the 256-site asparagine, the 341-site asparagine, the 360-site arginine and the 391-site lysine of the alanine-glyoxylic acid transaminase with the starting nucleotide sequence of SEQ ID NO.1 and the amino acid sequence of SEQ ID NO.2 are mutated into threonine respectively, and the amino acid sequence of the alanine-glyoxylic acid transaminase is mutated into threonine, arginine, tryptophan, tyrosine, glycine, cysteine, leucine, glycine, cysteine, glutamic acid; the 84-site cysteine, the 207-site serine, the 217-site threonine and the 293-site lysine are synonymously mutated to obtain the alanine-glyoxylate transaminase with the mutant amino acid sequence of SEQ ID NO.3 and the mutant amino acid sequence of SEQ ID NO. 4. The alanine-glyoxylate transaminase mutant is used for improving the yield of 5-ALA.

Description

technical field [0001] The invention relates to an alanine-glyoxylate transaminase mutant with improved enzyme activity and its application, belonging to the fields of genetic engineering and application and microorganism technology. Background technique [0002] Aminolevulinic acid (5-aminolevulinic acid, 5-ALA), as an important high value-added bio-based chemical product, is widely used in medicine, agriculture, feed and other fields. At present, the synthesis of 5-ALA is mainly produced by chemical synthesis, but the high cost and heavy pollution of chemical synthesis limit its popularization and application in various fields. With the continuous development of biotechnology, the construction of microbial cell factories to synthesize 5-ALA by fermentation has the advantages of low cost and no pollution, and has gradually become a research hotspot. There are two main pathways for the synthesis of 5-ALA in organisms, namely the C4 pathway and the C5 pathway. Among them, t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P13/00C12R1/19
CPCC12N9/1096C12N15/70C12P13/005C12Y206/01044C12N2800/101
Inventor 王智文洪坤强陈涛
Owner TIANJIN UNIV
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