The Study Of 2,3-butanediol Dehydrogenase From Bacillus Thuringiensis

Marine bacteria are considered to be one of the most promising sources for the development of new compounds.The special nature of the marine environment makes marine microbes and their metabolites diverse,and is also an important place to find and discover active substances.Among them,Bacillus thuringiensis contains a large number of genes encoding 2,3-butanediol dehydrogenase.2,3-butanediol dehydrogenase as a key enzyme can reversibly catalyze the formation of acetoin from 2,3-butanediol.At present,the study of 2,3-butanediol dehydrogenase is still at the development stage.Accordingly,there is an ongoing need to develop new and efficient strain and genes.The research of catalytic properties of 2,3-butanediol dehydrogenase has the important academic value and the project application value.In this study,an uncharacterized gene encoding a(2R,3R)-2,3-butanediol dehydrogenase(BtBDH)from Bacillus thuringiensis was screened and analyzed by bioinformatics in NCBI.It was cloned and expressed in Escherichia coli BL21(DE3).Recombinant BtBDH was successfully purified by ion chromatograph of Q Sepharose?Fast Flow.The oxidative properties and reduction properties properties of BtBDH was tested.The optimum conditions for the enzymatic reaction of BtBDH were determined.Finally,the 2,3-butanediol dehydrogenase was immobilized on graphene oxide by hydrophobic interaction and the immobilized catalytic properties were initially explored.Results:A novel butanediol dehydrogenase,BtBDH,was identified from Bacillus thuringiensis subsp.Kurstaki ACCC 10066 and its enzymatic properties were characterized.This novel BtBDH is a genuine(2R,3R)-2,3-butanediol dehydrogenase and solely NADH/NAD~+dependent dehydrogenase which belongs to medium chain dehydrogenase family.The optimum pH and temperature for the oxidation activity of BtBDH are 10.0 and 50?C,respectively,and the values for the reduction activity are 7.5 and 35?C,respectively.In addition,it exhibits remarkable stability over a wide pH range(6–10)and temperatures up to 70?C.BtBDH showed good stability after storage for 3 month at 4?C.Meso-2,3-butanediol and(2R,3R)-2,3-butanediol was screened the preferred substrate which showed a high activity and enantioselectivity for BtBDH while was not active for(2S,3S)-2,3-butanediol.It has higher reducing activity for acetoin and diacetyl,and its oxidation activity is higher than that of reducing activity,which proves its potential potential for the production of acetoin.The immobilization of 2,3-butanediol dehydrogenase by graphene oxide has high immobilization efficiency.The immobilized enzyme retained certain activity after five reuses containously.This study not only expanded the research scope of(2R,3R)-2,3-butanediol dehydrogenase,but also provided a basis for the directed evolution of enantiomerically pure 2,3-butanediol.