N-acetyl glutamic acid kinase mutant and application thereof

Abstract

The invention provides an N-acetyl glutamic acid kinase mutant capable of relieving the feedback inhibition effect of L-arginine and obviously increasing the catalytic activity and thermal stability and belongs to the field of bioengineering. A molecular docking technique and the structure analysis for N-acetyl glutamic acid kinase (CcNAGK) from corynebacterium crenatum CGMCC NO:0890 are firstly adopted for confirming that the E19 locus plays an important role in inhibiting the feedback of arginine and three key amino residue loci I74, F91 and K234 located on a substrate combining bag have significant impacts on enzymatic catalytic activity and heat stability; the encoded amino acid is replaced and the gene argB4 after compound mutation is introduced into the corynebacterium crenatum highly yielding arginine through pDXW10, so that the compounding rate of the arginine in corynebacterium crenatum can be obviously increased; lastly, after fermentation for 98h in a 5L fermentation tank, the yield of arginine is increased to 61.2g / L from original 43.2g / L, and the yield of the L-arginine is increased by 41.8%.

Description

technical field [0001] The invention relates to an N-acetylglutamic acid kinase mutant which completely relieves the feedback inhibition effect of L-arginine and significantly improves catalytic activity and thermal stability, and belongs to the technical field of bioengineering. Background technique [0002] L-arginine is one of the semi-essential basic amino acids required by the human body, and has a variety of unique physiological and pharmacological effects. It is an essential amino acid for the synthesis of cytoplasmic protein and nuclear protein; as the only source of ammonia, it participates in the synthesis of creatine; as an important intermediate in the urea cycle, it plays a role in eliminating excess ammonia in the liver, preventing excessive ammonia accumulation and causing poisoning ; It also has the function of regulating human immunity, can inhibit tumor growth, and promote wounded tissue healing. Moreover, arginine is the direct precursor of nitric oxide, ...

AI Technical Summary

Problems solved by technology

In order to further increase production, researchers gradually began to focus on the rate-limiting enzyme of L-arginine - N-acetylglutamate kinase (NAGK), by cloning the N-acetylglutamate kinase gene and introducing it into other strains for expression , but it is severely feedback-inhibited by the final product L-arginine. Although some researchers have used site-directed mutagenesis to alleviate the feedback inhibition of arginine, it still cannot be completely relieved, resulting in an increase in the production of L-arginine by fermentation. The acid ability of NAGK is weak, so to further increase the production of L-arginine, it is necessary to further release the feedback inhibition of NAGK by arginine; CcNAGK was overexpressed in Corynebacterium dentata, but the specific enzyme activity of CcNAGK was not high, that is, the catalytic activity of CcNAGK was weak, and the thermal stability was not good, that is, the half-life of CcNAGK was 36h at 30°C, while L-arginine The fermentation cycle is 96h

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