title: Selection and characterization of forest soil metagenome genes encoding lipolytic enzymes.
abstract: A metagenome is a unique resource to search for novel microbial enzymes from the unculturable microorganisms in soil. A forest soil metagenomic library using a fosmid and soil microbial DNA from Gwangneung forest, Korea, was constructed in Escherichia coli and screened to select lipolytic genes. A total of seven unique lipolytic clones were selected by screening of the 31,000-member forest soil metagenome library based on tributyrin hydrolysis. The ORFs for lipolytic activity were subcloned in a high copy number plasmid by screening the secondary shortgun libraries from the seven clones. Since the lipolytic enzymes were well secreted in E. coli into the culture broth, the lipolytic activity of the subclones was confirmed by the hydrolysis of p-nitrophenyl butyrate using culture broth. Deduced amino acid sequence analysis of the identified ORFs for lipolytic activity revealed that 4 genes encode hormone-sensitive lipase (HSL) in lipase family IV. Phylogenetic analysis indicated that 4 proteins were clustered with HSL in the database and other metagenomic HSLs. The other 2 genes and 1 gene encode non-heme peroxidase-like enzymes of lipase family V and a GDSL family esterase/lipase in family II, respectively. The gene for the GDSL enzyme is the first description of the enzyme from metagenomic screening.
authors.affiliation: Bio-Organic Science Division, Korea Research Institute of Chemical Technology, Daejeon, Korea.
authors.forename: Kyung Sik
authors.initials: KS
authors.lastname: Hong
authors.forename: He Kyoung
authors.initials: HK
authors.lastname: Lim
authors.forename: Eu Jin
authors.initials: EJ
authors.lastname: Chung
authors.forename: Eun Jin
authors.initials: EJ
authors.lastname: Park
authors.forename: Myung Hwan
authors.initials: MH
authors.lastname: Lee
authors.forename: Jin-Cheol
authors.initials: JC
authors.lastname: Kim
authors.forename: Gyung Ja
authors.initials: GJ
authors.lastname: Choi
authors.forename: Kwang Yun
authors.initials: KY
authors.lastname: Cho
authors.forename: Seon-Woo
authors.initials: SW
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D016680:Genome, Bacterial; D015698:Genomic Library; D008049:Lipase / Q000737:chemistry* / Q000235:genetics*; D008969:Molecular Sequence Data; D010802:Phylogeny; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012988:Soil Microbiology; D002787:Sterol Esterase / Q000235:genetics
chemical_list: D002787:Sterol Esterase; D008049:Lipase