The Study On Enzyme Characterization And Application Of L-glutamate Oxidase

L-glutamate oxidase is a kind of flavoprotein enzyme that specifically catalyzes the oxidative deamination of L-glutamate with the formation of hydrogen peroxide(H2O2),ammonia and ?-ketoglutaric acid(?-KG).?-KG can be used as building block to synthesize amino acids and peptides drugs,and widely used in fine chemistry,food production,and pharmaceutical.Nowadays,there is few study on production of ?-KG by biotransformation,and the processes need complicated production steps with low productivity.Therefore,it has a high value in industry to achieve one-step production of ?-KG with high efficiency.In this study,four Streptomyces which may contain LGOX gene were found through sequence alignment in NCBI.A color development screening method was used to select the best LGOX-producing strain.According to color development,S.mobaraenis which presented a deeper color was chosen for futher syudy.The L-glutamate oxidase,SmLGOX,from S.mobaraensis,was overexpressed in Escherichia coli BL21(DE3)heterologously.After IPTG induction and purified,the SmLGOX showed a single band at a molecular weight(MW)of 64 kDa in SDS-PAGE.By measuring the biochemical characterization of SmLGOX,the maximum activity was observed at 35?,pH 6.0,and SmLGOX showed strict substrate specificity towards to L-glutamic acid.The kinetic arameters,Km and Vmax,were 9.28 ± 0.47 mmol/L and 158.98± 3.04 U mg-1 respectively.The protein expression of SmLGOX was low while expressed heterologously in E.coli.In order to improve the expression level of SmLGOX,we developed codon harmonization of SmLGOX gene according to the best approximates codon usage frequencies in E.coli.The protein levels from optimized-codon SmLGOX gene fragments were greater than levels from native genes.Along with the production of ?-KG,the generated H2O2 in the LGOX reaction may degrade ?-KG.Therefore,the KatE gene from E.coli K-12 MG 1655 expressing catalase was found to construct co-expressed plasmids with optimized SmLGOX gene.The recombinant strain could express both catalase and SmLGOX.The recombinant strain was used as whole-cell catalyst to optimize the whole cell transformation conditions including cell concentration,substrate concentration,temperature,pH,and time.The titer of ?-KG reached 77.35 g/L with conversion ratio 98.49%in 12 h at the condition of 30 g/L wet cells,100 g/L L-glutamate,pH 7.5 and 40?.This method achieved one-step production of ?-KG with high production efficiency and low production cost,and provided a new stratage for ?-KG production by biocatalysis.