Optimized glutamine transaminase gene and leader sequence as well as secretory expression thereof

Abstract

The invention relates to a gene sequence optimized glutamine transaminase as well as a secretory expression method and application thereof. A streptomyces mobaraense-derived glutamine transaminase gene and a leader sequence thereof are reformed according to optimal codons of pichia pastorris to obtain an optimized glutamine transaminase gene and an optimized leader sequence thereof. A recombinant pichia pastoris system co-expressed by the optimized glutamine transaminase gene and the optimized leader sequence thereof is constructed, and a strain of high-expression recombinant glutamine transaminase is obtained by screening. The optimized glutamine transaminase gene and the optimized leader sequence thereof can be stably and efficiently expressed in the pichia pastoris, the activity of the expressed recombinant glutamine transaminase is up to 0.5 U / mL and is 4 times that of the original glutamine transaminase, and a foundation is laid for further industrial expanded production of the glutamine transaminase.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an optimized transglutaminase gene, an optimized transglutaminase leader sequence, secretion, expression and application thereof. Background technique [0002] Transglutaminase (Transglutaminase, TG) can catalyze the acyl transfer reaction between the γ-carboxamide group of the glutamine residue in the protein peptide chain and various acyl acceptors, and is an effective protein cross-linking agent. At present, the enzyme is most widely used in the meat industry to improve the structure and functional properties of protein and improve nutritional value. In recent years, TG has very broad application prospects in many fields such as biomedicine, tissue engineering, fixed-point protein cross-linking, textile and leather processing. [0003] The production strains of microbial transglutaminase (Microbial transglutaminase, MTG) are mainly Streptomyces, such as Streptomyc...

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Problems solved by technology

[0004] The gene expression of Streptomyces pro-MTG or MTG has been successfully achieved using a heterologous expression system, but the strategy of cloning and expressing the mature enzyme gene mtg to directly secrete the active enzyme MTG has not been successful

At present, there are two main strategies for expressing active MTG. One is to co-secrete and express pro-MTG and SAM-P45 protease in heterologous hosts, but this strategy may continue to degrade active TG due to the remaining protease activity in the fermentation broth. And cause adverse effects, and the subsequent removal of protease will increase the cost of separation and purification, so it is not suitable for industrialization requirements

The other is that Yurimoto et al., Li Pengfei et al. adopted the strategy of co-expressing the leader sequence (pro) and the mature transglutaminase (mtg) coding region of microbial-derived zymogen pro-MTG as two independent elements, successfully Achieved direct secretion and expression of active MTG, but the enzyme activity level is still low

However, Li Pengfei adopted a co-expression strategy different from Yurimoto's. By constructing pro and mtg gene expression cassettes separately, connecting them with Solution I ligase and placing them on an expression vector, the pro / MTG co-expression vector pAOα-pro-MTG was obtained. It also directly secretes and expresses active MTG, but the expression and enzyme activity levels are still low

An important advantage of the co-expression strategy is that active MTG can be obtained without protease activation. However, there are few reports on the co-expression of mtg and its pro

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