title: Expression and Characterization of a Novel Thermo-Alkalistable Lipase from Hyperthermophilic Bacterium Thermotoga maritima.
abstract: A gene coding for lipase (Tm1350) from the hyperthermophilic bacterium Thermotoga maritima MSB8 was cloned and overexpressed by Escherichia coli. The enzyme can degrade substrates with both short and long acyl chain lengths. The apparent Km and Vmax values for p-nitrophenyl butyrate were 8 mM and 333 U/mg, respectively. The enzyme displayed optimal activity at pH 7.5 and 70 °C, maintained 66 % of the original activity after 8 h of incubation, and its half-lives at pHs 9 and 10 were 8 and 1 h. The activity of Tm1350 was stimulated up to 131 or 151 % of the original activity by incubating with 4 M urea or 20 % (v/v) methanol, and 90.1 or 70.2 % of the activity was maintained after 8 h incubation of the enzyme in 20 or 75 % (v/v) of the methanol, showing potential for biodiesel production. The activity of the enzyme without cysteine residue was stimulated up to 618 and 550 % of the original activity by incubating with dithiothreitol (DTT) and reduced glutathione (GSH) at a concentration of 1 mM. However, the circular dichroism spectra of the enzyme have no obvious change after DTT treatment. It is speculated that DTT interacts with potential residues in some key active sites without influence of structure.
authors.affiliation: Institute of Life Sciences, Jiangsu University, Zhenjiang, 212013, China.
authors.forename: Rui
authors.initials: R
authors.lastname: Tian
authors.forename: Huayou
authors.initials: H
authors.lastname: Chen
authors.forename: Zhong
authors.initials: Z
authors.lastname: Ni
authors.forename: Qing
authors.initials: Q
authors.lastname: Zhang
authors.forename: Zhongge
authors.initials: Z
authors.lastname: Zhang
authors.forename: Tianxi
authors.initials: T
authors.lastname: Zhang
authors.forename: Chunxia
authors.initials: C
authors.lastname: Zhang
authors.forename: Shengli
authors.initials: S
authors.lastname: Yang
mesh_terms: D000468:Alkalies / Q000494:pharmacology*; D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004229:Dithiothreitol / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability / Q000187:drug effects; D005978:Glutathione / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007477:Ions; D008049:Lipase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008670:Metals / Q000494:pharmacology; D000432:Methanol / Q000494:pharmacology; D008969:Molecular Sequence Data; D011487:Protein Conformation; D019163:Reducing Agents / Q000494:pharmacology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012997:Solvents / Q000494:pharmacology; D013501:Surface-Active Agents / Q000494:pharmacology; D013696:Temperature; D020124:Thermotoga maritima / Q000201:enzymology*
chemical_list: D000468:Alkalies; D007477:Ions; D008670:Metals; D019163:Reducing Agents; D012997:Solvents; D013501:Surface-Active Agents; D004492:Edetic Acid; D008049:Lipase; D005978:Glutathione; D004229:Dithiothreitol; D000432:Methanol