Molecular cloning and characterization of a thermostable lipase from deep-sea thermophile Geobacillus sp. EPT9

• Published in: World journal of microbiology & biotechnology Vol. 31; no. 2; pp. 295 - 306
• Main Authors: Zhu, Yanbing, Li, Hebin, Ni, Hui, Xiao, Anfeng, Li, Lijun, Cai, Huinong
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer-Verlag 01.02.2015; Springer Netherlands; Springer Nature B.V
• Subjects: Amino acids; Analysis; Applied Microbiology; Bacillus; Bacillus (bacteria); Bacteria; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biochemistry; Bioengineering; Biomedical and Life Sciences; Biosynthesis; Biotechnology; Catalysis; Catalysts; Catalytic Domain; Cloning; Cloning, Molecular - methods; Deep sea; E coli; Environmental Engineering/Biotechnology; Enzymatic activity; enzyme activity; Enzyme Stability; Enzymes; Escherichia coli; Food; Gene expression; gene overexpression; Genes; Genetic engineering; Geobacillus; Geobacillus - enzymology; Geobacillus - genetics; Laboratories; Life Sciences; Lipase; Lipase - chemistry; Lipase - genetics; Lipase - metabolism; Microbiology; Microorganisms; Models, Molecular; molecular cloning; molecular weight; open reading frames; Original Paper; Phylogeny; Protein Structure, Secondary; Proteins; Recombinant; Residues; serine; Software; Structural Homology, Protein; Studies; Substrate Specificity; thermal stability; Thermophiles; thermophilic microorganisms; Thermostability; Characterization; Lipase; Expression
• DOI: 10.1007/s11274-014-1775-0
• ISSN: 0959-3993

A gene (1,254 bp) encoding a lipase was identified from a deep-sea hydrothermal field thermophile Geobacillus sp. EPT9. The open reading frame of this gene encoded 417 amino acid residues. The gene was cloned, overexpressed in Escherichia coli, and the target protein was purified to homogeneity. The purified recombinant enzyme presented a molecular mass of 44.8 kDa. When p-nitrophenyl palmitate was used as a substrate, the recombinant lipase was optimally active at 55 °C and pH 8.5. The recombinant enzyme retained 44 % residual activity after incubation at 80 °C for 1 h, which indicated that Geobacillus sp. EPT9 lipase was thermostable. Homology modeling of strain EPT9 lipase was developed with the lipase from Bacillus sp. L2 as a template. The core structure exhibits an α/β-hydrolase fold and the typical catalytic triad might consist of Ser142, Asp346, and His387. The enzymatic activity of EPT9 lipase was inhibited by addition of phenylmethylsulfonyl fluoride, indicating that it contains serine residue, which plays an important role in the catalytic mechanism.