Maleate cis-trans isomerase mutant, and coding gene and application thereof

A technology of cis-trans isomerase and maleic acid, applied in the field of maleate cis-trans isomerase mutants, can solve the problems of many by-products, environmental pollution, and high cost of chemical synthesis

Inactive Publication Date: 2016-04-20
ANHUI BBCA FERMENTATION TECH ENG RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the production of fumaric acid is mostly prepared from maleic anhydride by petrochemical methods. However, the chemical synthesis method has problems such as high cost, many by-products and possible environmental pollution, which makes people gradually turn to the environmentally friendly biological method. to prepare fumaric acid

Method used

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  • Maleate cis-trans isomerase mutant, and coding gene and application thereof
  • Maleate cis-trans isomerase mutant, and coding gene and application thereof
  • Maleate cis-trans isomerase mutant, and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] The acquisition of embodiment 1 maleate cis-trans isomerase gene

[0041] Using 1 μg of Serratia marcescens (Serratiamarcescens) genomic DNA as a PCR reaction template, the forward primer MaiA-F was designed according to the sequence of SEQ ID NO. CCCAAGCTTATAAGCGCCGGACAG-3′, where the parts in italics are the enzyme cutting sites EcoRI and HindIII respectively. The PCR reaction was carried out in a total volume of 50 μl. The reaction conditions were 30 cycles of denaturation at 94°C for 5 min, denaturation at 94°C for 50 s, annealing at 58°C for 1 min, and extension at 72°C for 2 min. Take 3 μl of PCR amplification products for agarose gel electrophoresis verification, the results are as follows figure 1 shown. Take 100 μl of the PCR product for agarose gel electrophoresis, and recover the target fragment according to the steps of the gel recovery kit.

Embodiment 2

[0042] Embodiment 2 Construction of wild-type maleic acid cis-trans isomerase gene expression vector

[0043] In Example 1, the PCR product was digested with restriction endonucleases EcoRI and HindIII, and then ligated with the pET-24a (+) plasmid (purchased from Novagen) digested with EcoRI and HindIII endonucleases. The constructed The vector is called pET-MaiA, and then the pET-MaiA mixture is used to transform Escherichia coli DH5-α (purchased from promega company), and carry out sequence determination to extract the plasmid of the transformed cell library, and transform Escherichia coli BL21 (DE3) strain ( purchased from promega company).

Embodiment 3

[0044] Example 3 Error-prone PCR amplification of Serratia marcescens maleate cis-trans isomerase gene

[0045]Using the property that TaqDNA polymerase does not have 3'-5' proofreading function, the frequency of random mutations can be controlled at high magnesium ion concentration (5-9mmol / L) and different concentrations of dNTP (1.5-3.5mmol / L) , introduce random mutations into the target gene, and construct a mutation library. The optimal mutation rate in the experiment is about 0.6%.

[0046] Error-prone PCR reaction system (100μl):

[0047]

[0048] The PCR program was: pre-denaturation at 96°C for 4 minutes, denaturation at 94°C for 1 minute, annealing at 56°C for 1 minute, extension at 75°C for 2 minutes, 45 cycles of reaction, and finally extension at 75°C for 15 minutes. Take 5 μ l of the product and check it with agarose gel electrophoresis, the result is as follows: figure 2 shown. The PCR product was recovered by gel recovery method and stored at -20°C for ...

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Abstract

The invention relates to a protein mutant, particularly a maleate cis-trans isomerase, and a coding gene and application thereof. The amino acid sequence of the maleate cis-trans isomerase is disclosed as SEQ ID NO.4, or an amino acid sequence with equal functions, which is derived from SEQ ID NO.4 and subjected to substitution, deletion or addition of one or more amino acids. Compared with the wild type maleate cis-trans isomerase, the changes of the specific amino acid sequence of the mutant as follows: K51I (the 51st lysine is changed into isoleucine), R177S (the 177th arginine is changed into serine), and A212G (the 212th alanine is changed into glycine). The test verifies that the enzyme activity of the mutant is enhanced by 2.71 times as compared with the wild type maleate cis-trans isomerase.

Description

technical field [0001] The invention relates to protein mutants, in particular to a maleic acid cis-trans isomerase mutant. Background technique [0002] Fumaric acid, also known as fumaric acid and fumaric acid, is a naturally occurring organic acid. As an important four-carbon platform compound and fine chemical product, fumaric acid is widely used in food, medicine, chemical industry, coating, plasticizer and other fields. Fumaric acid can also produce some important compound raw materials with other industrial uses through biotransformation, such as L-aspartic acid, L-alanine and L-malic acid, etc. At present, the production of fumaric acid is mostly prepared from maleic anhydride by petrochemical methods. However, the chemical synthesis method has problems such as high cost, many by-products and possible environmental pollution, which makes people gradually turn to the environmentally friendly biological method. to prepare fumaric acid. [0003] Maleate cis-trans iso...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/90C12N15/61C12N15/70C12N1/21C12P7/46C12R1/19
CPCC12N9/90C12P7/46C12Y502/01001
Inventor 秦晴许鹏冯杰徐斌纪传侠马丽
Owner ANHUI BBCA FERMENTATION TECH ENG RES
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