title: Characterization of an L-arabinose isomerase from Bacillus coagulans NL01 and its application for D-tagatose production.
abstract: L-arabinose isomerase (AI) is a crucial catalyst for the biotransformation of D-galactose to D-tagatose. In previous reports, AIs from thermophilic bacterial strains had been wildly researched, but the browning reaction and by-products formed at high temperatures restricted their applications. By contrast, AIs from mesophilic Bacillus strains have some different features including lower optimal temperatures and lower requirements of metallic cofactors. These characters will be beneficial to the development of a more energy-efficient and safer production process. However, the relevant data about the kinetics and reaction properties of Bacillus AIs in D-tagatose production are still insufficient. Thus, in order to support further applications of these AIs, a comprehensive characterization of a Bacillus AI is needed.
  
  
  
  The coding gene (1422 bp) of Bacillus coagulans NL01 AI (BCAI) was cloned and overexpressed in the Escherichia coli BL21 (DE3) strain. The enzymatic property test showed that the optimal temperature and pH of BCAI were 60 °C and 7.5 respectively. The raw purified BCAI originally showed high activity in absence of outsourcing metallic ions and its thermostability did not change in a low concentration (0.5 mM) of Mn(2+) at temperatures from 70 °C to 90 °C. Besides these, the catalytic efficiencies (k cat/K m) for L-arabinose and D-galactose were 8.7 mM(-1) min(-1) and 1.0 mM(-1) min(-1) respectively. Under optimal conditions, the recombinant E. coli cell containing BCAI could convert 150 g L(-1) and 250 g L(-1) D-galactose to D-tagatose with attractive conversion rates of 32 % (32 h) and 27 % (48 h).
  
  
  
  In this study, a novel AI from B. coagulans NL01was cloned, purified and characterized. Compared with other reported AIs, this AI could retain high proportions of activity at a broader range of temperatures and was less dependent on metallic cofactors such as Mn(2+). Its substrate specificity was understood deeply by carrying out molecular modelling and docking studies. When the recombinant E. coli expressing the AI was used as a biocatalyst, D-tagatose could be produced efficiently in a simple one-pot biotransformation system.
authors.affiliation: College of Chemical Engineering, Nanjing Forestry University, Nanjing, 210037, People's Republic of China.
authors.forename: Wending
authors.initials: W
authors.lastname: Mei
authors.affiliation: College of Chemical Engineering, Nanjing Forestry University, Nanjing, 210037, People's Republic of China.
authors.forename: Lu
authors.initials: L
authors.lastname: Wang
authors.affiliation: College of Forestry, Nanjing Forestry University, Nanjing, 210037, People's Republic of China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zang
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. hgouyj@njfu.edu.cn.|Key Laboratory of Forest Genetics & Biotechnology of the Ministry of Education, Nanjing, People's Republic of China. hgouyj@njfu.edu.cn.
authors.forename: Jia
authors.initials: J
authors.lastname: Ouyang
mesh_terms: D019747:Aldose-Ketose Isomerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000070000:Bacillus coagulans / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005690:Galactose / Q000737:chemistry*; D006601:Hexoses / Q000138:chemical synthesis*; D008956:Models, Chemical; D062105:Molecular Docking Simulation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013045:Species Specificity; D013379:Substrate Specificity; D013549:Sweetening Agents / Q000138:chemical synthesis
chemical_list: D006601:Hexoses; D011994:Recombinant Proteins; D013549:Sweetening Agents; D019747:Aldose-Ketose Isomerases; C019715:L-arabinose isomerase; C030192:tagatose; D005690:Galactose
keywords: Bacillus coagulans; Biotransformation; D-tagatose; L-arabinose isomerase