title: A thermostable S-adenosylhomocysteine hydrolase from Thermotoga maritima: properties and its application on S-adenosylhomocysteine production with enzymatic cofactor regeneration.
abstract: S-adenosylhomocysteine (SAH) is an effective sedative, a good sleep modulator, and a new anticonvulsant. SAH can be synthesized from adenosine and homocysteine by using microbial S-adenosylhomocysteine hydrolase (SAHase). The extremely thermostable SAHase and lactate dehydrogenase (LDH) from Thermotoga maritima were successfully overexpressed in Escherichia coli, and purified by heat treatments. The SAHase exhibited the highest activity at 85 °C and pH 8.0 with a specific activity of 6.2 U/mg when NAD concentration was 1mM. However, optimal SAHase reaction conditions shifted to 100 °C and pH 11.2, and its specific activity increased to 36.8 U/mg after NAD concentration was raised to 8mM. Biosynthesis of SAH at 85 °C largely increased the adenosine solubility which was a limiting factor for improving the titer of product. At 85 °C and pH 8.0, 24 μmol of SAH was obtained when 0.5mg of SAHase was applied to a 10 ml reaction mixture. The SAH production was further increased to 153 μmol by adding LDH and pyruvate into the reaction mixture for NAD regeneration. Therefore, extremely thermostable enzymes SAHase and LDH from T. maritima form an efficient NAD consumption and regeneration system for SAH biosynthesis. This method has great potential for industrial-scale enzymatic production of SAH.
authors.affiliation: Department of Life Sciences, Nanjing Normal University, Nanjing, Jiangsu 210023, People's Republic of China.
authors.forename: Guojun
authors.initials: G
authors.lastname: Qian
authors.affiliation: Department of Life Sciences, Nanjing Normal University, Nanjing, Jiangsu 210023, People's Republic of China.
authors.forename: Caiping
authors.initials: C
authors.lastname: Chen
authors.affiliation: Department of Life Sciences, Nanjing Normal University, Nanjing, Jiangsu 210023, People's Republic of China.
authors.forename: Rong
authors.initials: R
authors.lastname: Zhou
authors.affiliation: Department of Life Sciences, Nanjing Normal University, Nanjing, Jiangsu 210023, People's Republic of China.
authors.forename: Yanbin
authors.initials: Y
authors.lastname: He
authors.affiliation: Biofuels Institute, School of Environment, Jiangsu University, Zhenjiang, Jiangsu 212013, People's Republic of China. Electronic address: weilanshao@gmail.com.
authors.forename: Weilan
authors.initials: W
authors.lastname: Shao
mesh_terms: D043383:Adenosylhomocysteinase / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D007218:Industrial Microbiology; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D009243:NAD / Q000096:biosynthesis / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012435:S-Adenosylhomocysteine / Q000378:metabolism*; D013696:Temperature; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D009243:NAD; D012435:S-Adenosylhomocysteine; D007770:L-Lactate Dehydrogenase; D043383:Adenosylhomocysteinase
keywords: Lactate dehydrogenase; NAD regeneration; S-adenosylhomocysteine; S-adenosylhomocysteine hydrolase; Thermotoga maritima