Molecular Modification Of Glutamate Dehydrogenase From Bacillus Subtilis

Oxidoreductases are an important kind of biocatalysts widely used in many important biocatalytic reactions as cofactor regeneration enzyme.The enzymes commonly used include glucose dehydrogenase,formate dehydrogenase,glutamate dehydrogenase and so on.Among them glutamate dehydrogenase（GDH）catalyzed the synthesis ofα-ketoglutaric acid from glutamic acid and its reversible reaction,accompanied by the regeneration of NAD（P）⁺and NAD（P）H.In this study,its pH and stability were modified to further improve its efficiency in redox reaction.Main results are as follows:（1）In this study,the gene encodings glutamate dehydrogenase（GDH）from 8 different strains were cloned and expressed in Escherichia coli.We selected NADH-dependent BsGDH from Bacillus subtilis 168 with a higher enzyme activity as the research object.The molecular properties showed that BsGDH can catalyze oxidative deamination reaction and reductive amination reaction.The optimal pHs of oxidative direction and reductive direction were recorded at slightly alkaline pH8.0 and pH7.5,respectively.However,the enzyme exhibited poor stability above pH 7.0.Only 46%of the enzyme activity was left after 35 h in pH 8.0.The enzyme is completely inactivated after half an hour at pH 8.5.The optimum temperatures for oxidative direction and reductive direction of BsGDH were 60℃and 65℃,respectively.BsGDH exhibited poor thermal stability.After 2 h of incubation over 55℃,the enzyme activity of oxidative direction was completely lost and the enzyme activity of reductive direction was only 14%conserved.（2）To improve the economy of BsGDH in redox reaction,firstly based on protein surface charge engineering,the enzyme was molecularly modified in order to adjust its catalytic optimum pH to neutral environment with high stability.We selected 8 mutation sites by ROSETTA supercharge software analysis and found the optimal pH for oxidative deamination activities of N16D、K218D and M277D were reduced from 8.0 to 7.0,simultaneously the activities were increased by 2.9 folds,5.4 folds and 3.9 folds respectively.However,the enzymatic activity of the combined mutant was completely lost.In addition,we got the mutation sites by aligning the amino acid sequences of BsGDH and other 8thermophilic sources of GDH.We obtained the mutant BsGDH_{E27F+S70V+S305E} with significantly improved thermal stability.The enzyme activity remained 48%after 16 h in50℃,but WT was inactive after 3 h.（3）BsGDH was employed as the main enzyme to catalyze the synthesis of α-ketoglutarate from glutamic acid.Firstly,we researched the effect of cofactor concentration on the catalysis of BsGDH,and it was found that the higher the concentration of NAD⁺/NADH and the faster the reaction rate.In order to reduce tne addition of NAD⁺/NADH,we constructed a cofactor recycling system with NADH oxidase from B.subtilis and BsGDH.Compared with the single-enzyme conversion of BsGDH,this cofactor recycling system accelerated the reaction that BsGDH catalyzes the synthesis ofα-ketoglutarate.According to the previous analysis of the enzymatic properties of BsGDH,the optimum catalytic pH of BsNOX is 9.0.In order to adjust its optimum pH,we designed 16 mutation sites by the same method of ROSETTA software.Then we got a mutant BsNOX_N20D+N116E that had its optimum pH reduced to 7.0 and the enzymatic activity of was 2.9 folds WT.The combination of BsGDH_K218D+BsNOX_N20D+N116E could achieve a conversion rate of about 90%in 45 min.（4）We explored the application prospect of BsGDH in NAD⁺/NADH regeneration.Firstly,we constructed a cofactor recycling system with BsGDH and leucine dehydrogenase from Bacillus cereus（BcLDH）.It was found that the BsGDH_K218D-BcLDH system could yield60.47 g·L^-1 of L-phenylglycine,but the single-enzyme conversion of BcLDH yield 10.8 g·L^-1of L-phenylglycine,which explains BsGDH_{E27F+S70V+I331P} can be effectively used in NADH regeneration.In addition,we constructed a cofactor recycling system with BsGDH and acetoin reductase from B.subtilis（BsAR/BDH）.The single-enzyme conversion of BsAR/BDH only yield 14.53 g·L^-1 of acetoin;the BsGDH_{E27F+S70V+I331P}-BsAR/BDH system could yield 30.93 g·L^-1 of acetoin,which explains BsGDH can be effectively used in NAD⁺regeneration.