Expression Of Thermostable Glucose Dehydrogenase And Its Application In The Biosynthesis Of L-tert-leucine

Glucose Dehydrogenase(GDH,EC1.1.1.47)belong to short chain dehydrogenase/reductase(SDRs).It has four identical subunits(28.2 x 4 kDa)that catalyze the oxidation of D-glucose to D-gluconolactone while reducing NAD(P)+ to NAD(P)H.In recent years,the research on GDH has made considerable progress,and GDH has been applied in many fields such as coenzyme regeneration,clinical testing and biofuel cells.The early GDH source is mainly extracted from the fresh liver of animals,and now most of them are fermented by microorganism.At present,the domestic GDH production is not high,mainly rely on imports.This paper studies a mutant GDH-DN46 of GDH from Bacillus megaterium IWG3,which exhibits significantly enhanced thermal stability and alkali resistance,with no catalytic activity loss regardless of the presence NaCl or not.NAD(P)+ must be required as the electron acceptor in the oxidoreductase-catalyzed reaction,which great limits the oxidoreductase in industrial catalysis application due to its expensive cost and unstability.In this paper,mutant GDH-DN46 was successfully expressed in Escherichia coli BL21,GDH activity was improved with optimizing the expression conditions,and its application was studied as the coenzyme regeneration system in the biosynthesis of L-tert-leucine.At the same time,we designed a high-throughput screening platform that could screen GDH mutants using biomimetic coenzyme.The main research of the paper is as follows:1.The recombinant expression plasmid pET28a-GDH of GDH was constructed to be highly expressed in E.coli BL21,the GDH activity reached 0.95 U/mg.By optimizing expression conditions of the recombinant E.coli BL21(pET28a-GDH),the enzyme activity reached 1.52 U/mg and increased by 60%compared with the initial activity.2.The enzymatic properties of GDH were studied,and the optimum temperature of GDH activity was 50?.The enzyme activity increased 30%compared with the 30?.The optimum pH of GDH activity was 7.0.This high-stability enzyme is helpful for clinical trials and the food industry.3.The expression plasmids pET28a-GDH and pET20b-LeuDH were constructed,respectively,and transformed into E.coli BL21 to obtain the double plasmid expression system E.coli BL21(pET28a-GDH/pET20b-LeuDH).Compared with E.coli BL21(pET20b-LeuDH)whole cell catalyzed TMP production,the conversion rate of L-tert-leucine were increased by 3 times using double-plasmid whole cells that the plasmids pET28a-GDH was used as the coenzyme NADH regeneration system and other plasmids as catalysis enzyme.4.The high-throughput screening method of GDH mutant using the biomimetic coenzyme was studied and contructed.Through the study of chromogenic reaction between different dyes with coenzyme NADH,the result showed the optimal high-throughput screening dye is nitrogen blue four triazole(NBT).The colorimetric plate composition of the screening platform was:Tryptone 1%;Yeast extract 0.5%;NaCl 1%;Agar 1.8%;NBT 0.1 g/1;PMS 0.01 mM;Kan 0.05 mM;IPTG 0.1 mM;Glucose 1 mM;NMN + 0.05 MM.Through the heat treatment of E.coli BL21(pET28a-GDH)70 ? for 1 hours,the intracellular coenzyme NADH can be decormposed,and the heat treatment can effectively avoid the interference of the intracellular coenzyme NADH in the colorimetic screening process.