Construction method, encoded protein and application of a thermostable malate dehydrogenase gene

Abstract

The invention discloses a method for constructing a heat-stable malate dehydrogenase gene, an encoded protein and its application. The malate dehydrogenase gene of the present invention uses the original expression plasmid gene sequence as a template and adopts designed mutation primers to carry out rapid Amplify the mutant plasmid by point mutation PCR, screen the resistance of transformed bacteria, construct a gene sequence capable of expressing malate dehydrogenase, and transfer the constructed mutant malate dehydrogenase gene to Escherichia coli DE3 strain for expression. The expressed malate dehydrogenase has the characteristics of higher thermal stability and higher activity than the malate dehydrogenase expressed in the wild type of the sample, and provides a more efficient resolution and purification enzyme for the production of D-malic acid.

Description

technical field [0001] The invention relates to the technical fields of biological genetic engineering and protein engineering, in particular to a method for constructing a heat-stable malate dehydrogenase gene, an encoded protein and its application. Background technique [0002] With the development of genetic engineering and protein engineering technology, enzymes with improved physical and chemical properties can be obtained by computer-aided design, site-directed mutation, structure extension mutation, hybrid enzyme and molecular directed evolution. Enzyme structure extension mutagenesis is a new method of evolutionary protein engineering research. It expands the protein structure by adding peptides at the C-terminus of the enzyme protein, thereby changing the high-level structure of the enzyme protein to achieve the purpose of improving the physical and chemical properties of the enzyme. [0003] In the natural state, malic acid (2-hydroxysuccinic acid) is a mixture of...

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Problems solved by technology

Among them, D-malic acid is an extremely important 4-carbon organic chiral source, which is widely used in the fields of chiral drugs, chiral additives, chiral auxiliary agents, etc., but in the production process of D-malic acid, due to the The other two forms of acid: L-malic acid and D,L-malic acid are mixed, resulting in a very low purification rate of D-malic acid. In order to improve the purification rate of D-malic acid, malate dehydrogenase (malate dehydrogenase, MDH) (EC 1.1.1.37) Due to its wide range of existence, high substrate specificity, and the reversible conversion between malic acid and oxaloacetic acid, it is widely used to split D, L-malic acid to obtain D-malic acid

[0004] When the applicant was studying malate dehydrogenase, it was found that the existing wild-type malate dehydrogenase used for splitting D,L-malic acid had poor heat resistance, and it took a long time to screen in the natural state , The process is cumbersome, which is not conducive to the application of malate dehydrogenase in the separation of D, L-malic acid and the purification of D-malic acid synthesized by other methods

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