Isolation and characterization of a thermostable esterase from a metagenomic library.
A novel esterase gene was isolated by functional screening of a metagenomic library prepared from an activated sludge sample. The gene (est-XG2) consists of 1,506 bp with GC content of 74.8 %, and encodes a protein of 501 amino acids with a molecular mass of 53 kDa. Sequence alignment revealed that Est-XG2 shows a maximum amino acid identity (47 %) with the carboxylesterase from Thermaerobacter marianensis DSM 12885 (YP_004101478). The catalytic triad of Est-XG2 was predicted to be Ser₁₉₂-Glu₃₁₃-His₄₁₂ with Ser₉₂ in a conserved pentapeptide (GXSXG), and further confirmed by site-directed mutagenesis. Phylogenetic analysis suggested Est-XG2 belongs to the bacterial lipase/esterase family VII. The recombinant Est-XG2, expressed and purified from Escherichia coli, preferred to hydrolyze short and medium length p-nitrophenyl esters with the best substrate being p-nitrophenyl acetate (K(m) and k(cat) of 0.33 mM and 36.21 s⁻¹, respectively). The purified enzyme also had the ability to cleave sterically hindered esters of tertiary alcohols. Biochemical characterization of Est-XG2 revealed that it is a thermophilic esterase that exhibits optimum activity at pH 8.5 and 70 °C. Est-XG2 had moderate tolerance to organic solvents and surfactants. The unique properties of Est-XG2, high thermostability and stability in the presence of organic solvents, may render it a potential candidate for industrial applications.
Key Lab of Molecular Biophysics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, 430074, People's Republic of China.
Hua
H
Shao
Li
L
Xu
Yunjun
Y
Yan
D000595:Amino Acid Sequence; D001709:Biotechnology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D004950:Esterases / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015723:Gene Library; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008049:Lipase / Q000145:classification / Q000235:genetics; D056186:Metagenomics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009596:Nitrophenols / Q000378:metabolism; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012722:Sewage / Q000737:chemistry; D013379:Substrate Specificity; D013696:Temperature
D009596:Nitrophenols; D011994:Recombinant Proteins; D012722:Sewage; C008642:4-nitrophenyl acetate; D004950:Esterases; D008049:Lipase