title: Kinetic characterization of recombinant Bacillus coagulans FDP-activated l-lactate dehydrogenase expressed in Escherichia coli and its substrate specificity.
abstract: Bacillus coagulans is a homofermentative, acid-tolerant and thermophilic sporogenic lactic acid bacterium, which is capable of producing high yields of optically pure lactic acid. The l-(+)-lactate dehydrogenase (l-LDH) from B. coagulans is considered as an ideal biocatalyst for industrial production. In this study, the gene ldhL encoding a thermostable l-LDH was amplified from B. coagulans NL01 genomic DNA and successfully expressed in Escherichia coli BL21 (DE3). The recombinant enzyme was partially purified and its enzymatic properties were characterized. Sequence analysis demonstrated that the l-LDH was a fructose 1,6-diphosphate-activated NAD-dependent lactate dehydrogenase (l-nLDH). Its molecular weight was approximately 34-36kDa. The Km and Vmax values of the purified l-nLDH for pyruvate were 1.91±0.28mM and 2613.57±6.43μmol(minmg)(-1), respectively. The biochemical properties of l-nLDH showed that the specific activity were up to 2323.29U/mg with optimum temperature of 55°C and pH of 6.5 in the pyruvate reduction and 351.01U/mg with temperature of 55°C and pH of 11.5 in the lactate oxidation. The enzyme also showed some activity in the absence of FDP, with a pH optimum of 4.0. Compared to other lactic acid bacterial l-nLDHs, the enzyme was found to be relatively stable at 50°C. Ca(2+), Ba(2+), Mg(2+) and Mn(2+) ions had activated effects on the enzyme activity, and the enzyme was greatly inhibited by Ni(2+) ion. Besides these, l-nLDH showed the higher specificity towards pyruvate esters, such as methyl pyruvate and ethyl pyruvate.
authors.affiliation: College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, People's Republic of China.
authors.forename: Ting
authors.initials: T
authors.lastname: Jiang
authors.affiliation: College of Forest Resources Environment, Nanjing Forestry University, Nanjing 210037, People's Republic of China.
authors.forename: Yanbing
authors.initials: Y
authors.lastname: Xu
authors.affiliation: College of Forest Resources Environment, Nanjing Forestry University, Nanjing 210037, People's Republic of China.
authors.forename: Xiucheng
authors.initials: X
authors.lastname: Sun
authors.affiliation: College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, People's Republic of China.
authors.forename: Zhaojuan
authors.initials: Z
authors.lastname: Zheng
authors.affiliation: College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, People's Republic of China. Electronic address: hgouyj@njfu.edu.cn.
authors.forename: Jia
authors.initials: J
authors.lastname: Ouyang
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005635:Fructosediphosphates / Q000378:metabolism; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008670:Metals; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D005635:Fructosediphosphates; D008670:Metals; D011994:Recombinant Proteins; D007770:L-Lactate Dehydrogenase; C029063:fructose-1,6-diphosphate
keywords: Bacillus coagulans; Enzymatic properties; Fructose 1,6-diphosphate; Pyruvate esters; l-Lactate dehydrogenase