Studies On Heterologous Expression Of Glutamic Acid Decarboxylase Genes, Optimization Of Preparation Conditions For GABA And Gene Site-directed Mutagenesis

γ-aminobutyric acid（GABA）has many important physiological functions,widely used in the agricultural,food and pharmaceutical industries.In addition,GABA can also be used as an important raw material for engineering plastics,so GABA has important economic value.In organisms,GABA is formed from the irreversibleα-decarboxylation of L-glutamic acid catalyzed by Glutamate decarboxylase（GAD）.In recent years,researchers have mainly cloned GAD-encoding genes from acid-resistant Lactobacillus brevis,and achieved heterologous expression in E.coli,etc.,and improved GABA production through genetic engineering and optimization of fermentation conditions.However,its limited yield and the narrow pH range of GAD still limit the extensive application of GABA in industrial and agricultural production.Therefore,it is necessary to carry out work from multiple angles and explore more genetic resources to overcome the above shortcomings.To this end,this paper mainly carried out the following research work:（1）Firstly,several glutamate decarboxylase putative ecoding genes were cloned and heterologous expression in Escherichia coli The glutamate decarboxylase putative ecoding gene was cloned from the Lactobacillus brevis ATCC367 and Mycobacterium smegmatis MC²155 genomes by PCR.The putative genes were named gad _A,gad _Band gad _Ms,respectively,they were inserted into E.coli expression plasmid p ET28a to construct prokaryotic expression plasmids,the results recombinant plasmids were named p ET28a-gad _Ms,p ET28a-gad _Aand p ET28a-gad _B,respectively.The above expression recombinant plasmids were introduced into E.coli BL21,ITPG induced expression.SDS-PAGE analysis showed that the putative genes gad _Msand gad _Bwere successfully expressed in the soluble form,while gad _Awas not successfully expressed.（2）Secondly,the optimized conditions（temperature,reaction time,ion and PLP concentration）for preparationγ-aminobutyric acid（GABA）by recombinant enzyme GAD_Mswere investigated.The results showed that the enzyme showed highest conversion efficiency at 36℃,pH 4.6,0.01 m M PLP,3-4 M（NH₄）₂SO₄.（3）Thirdly,a three-dimensional structure of glutamic acid decarboxylase of M.smegmatis was constructed through homology modeling using 1pmm,glutamate decarboxyla of E.coli as template.And the structure and molecular docking were analysed;Molecular simulation analysis of the point mutations glutamate decarboxylase were conduct;（4）Finally,studies on site directed mutagenesis was carried out,anda mutant with increased pH adaptation range was obtained.The mutant V299I,mutant R301H/V302I and mutant T211I were constructed by site-directed mutagenesis.The transformation activity of each mutant was analyzed.The results showed that the pH adaptation range of T211I was significantly broadened and had higher transformation activity at pH 5.8.