Fervidobacterium changbaicum Lip1: identification, cloning, and characterization of the thermophilic lipase as a new member of bacterial lipase family V.
A novel lipase gene encoded 315 amino acid residues was obtained using lipase-prospecting primers and genome walking from hyperthermophilic bacterium Fervidobacterium changbaicum CBS-1. Sequence alignment and phylogenetic analysis revealed this novel lipase is a new member of bacterial lipase family V. The recombinant enzyme F. changbaicum lipase 1 (FCLip1) showed maximum activity at 78 °C and pH 7.8. It displayed extreme thermostability at 70 °C and was also stable across a wide pH range from 6.0 to 12.0. Kinetic study demonstrated FCLip1 preferentially hydrolyzed middle-length acyl chains, especially p-nitrophenyl caprate and tricaprylin. With p-nitrophenyl caprate as a substrate, the enzyme exhibited a K(m) and k(cat) of 4.67 μM and 22.7/s, respectively. In addition, FCLip1 was resistant to various detergents and organic solvents. This enzyme is the first reported thermophilic lipase from bacterial family Thermotogaceae. Its extreme stability with respect to temperature and pH, along with its triglyceride hydrolysis activity, indicate that FCLip1 has high potential for future application.
Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun 130023, China.
Jingang
J
Cai
Yuan
Y
Xie
Bo
B
Song
Yanping
Y
Wang
Zuoming
Z
Zhang
Yan
Y
Feng
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D011994:Recombinant Proteins; D008049:Lipase