Analysis Of Enzymes For 3R/3S-Acetoin Between 2,3-butanediol Conversion And Charcterization Of 2R,3R-Butanediol Dehydrogenase In Bacillus Sp.DL01

3R/3S-Acetoin?3R/3S-AC?and 2,3-Butanediol?2,3-BD?are important bulk chemicals used at different fields as food,dairy and pharmaceutical industries.Most of them are synthesized chemically,while the worldwide trend nowadays is depending on the green chemical to overcome the depletion of fuel fossils and avoid pollution of our environment.There are a lot number of microorganism utilized for 3R/3S-AC and 2,3-BD production,Bacillus species is one of them which showed a promising future for their production.Our aim in this study was determining the possible genes which may participate in3R/3S-AC and 2,3-BD formation from newly isolated Bacillus sp.DL01,then investigating the activity of Bacillus sp.DL01 towards the pathway of 3R/3S-AC and 2,3-BD formation then,focusing on identification and characterization of 2R,3R-Butanediol dehydrogenase?R-BDH?.The genes were isolated by PCR utilizing Bacillus velezensis SRCM 101413 genome to design the genes primers,each gene supposed for multiple alignments,then cloned into T-Vector for sequencing to get the complete sequence of each gene,also cloned into pET-28a?+?plasmid then transformed into E.coli BL21?DE3?for expression its protein.The total crude lysate activity was detected using substrates of 3R/3S-AC and 2,3-BD pathway.Isolated R-BDH activity was investigated under different conditions as pH,temperature,and metals ions.Also,the kinetic parameters towards different substrates were investigated using Line weaver-Burke plot.Successfully,we isolated the 5 genes[2R,3R-BDH?1041 bp?/2S,3S-BDH?843 bp?/meso-BDH?747 bp?/Alcohol dehydrogenase?1047 bp?/Glycerol dehydrogenase?1071 bp?],and each gene sequence displayed different similarities to other genes sequences from other microorganisms.From the total crude lysate activity the Bacillus sp.DL01 showed neither reduction nor oxidative activities towards 2S,3S-BD,while exhibited different activities towards Diacetyl?DA?,3R/3S-AC and 2R,3R-BD,and Meso-BD which confirmed the R-configuration enantioselectivity of Bacillus sp.DL01,so we focused in this study to identify and characterize the R-BDH.From the multiple alignments and the 3D structure,the R-BDH was strictly NAD⁺?H?and Zn⁺² dependent enzyme,showed reducing activities towards DA and 3R/3S-AC substrates using NADH as a coenzyme,and oxidizing activities with substrates 2R,3R-BD and Meso-BD using NAD⁺as a coenzyme,while no activity was detected with 2S,3S-BD which confirmed the R-configuration enantioselectivity as previously mentioned by the result of the total crude enzyme activity assay.The R-BDH exerted its catalytic reaction at temperature ranged from 25^°C to 65^°C.And at pH from pH 5.0 to pH 8.0.The metal ions have a great impact on the activity of R-BDH,Mn⁺² enhanced R-BDH to convert DA to 3R-AC recorded 120%activity.Furthermore,the R-BDH activity was increased obviously to 124.9%,114.2%and 138.5%by Cd²⁺when DA,3R/3S-AC and Meso-BD respectively,used as a substrate,while Fe⁺² enhanced the activity dramatically to 157.9%at 2R,3R-BD oxidation to 3R-AC.Kinetic parameters of the R-BDH showed that the lowest K_m value and the highest V_max,and K_cat values were for racemic 3R/3S-AC substrate,furthermore,the R-BDH showed low K_m values towards2R,3R-BD,and Meso-BD especially when compared with other reported R-BDHs.Thus,we considered that Bacillus sp.DL01 has R-configuration enantioselectivity,while it has low S-configuration enantioselectivity.The R-BDH showed high catalytic activity and provided an approach to regulate single chiral from 3R/3S-AC or 2,3-BD accumulation in bacteria,and can be utilized at biocatalysis and industrial scale.