title: Molecular cloning and characterization of a novel family VIII alkaline esterase from a compost metagenomic library.
abstract: A metagenomic library was constructed from completely fermented compost using a fosmid vector. From a total of 23,400 clones, 19 esterase-positive clones were selected on LB plates containing 1% glyceryl tributyrate as the substrate. The esterase gene of an esterase-positive clone, est2K, was on an ORF of 1299 bp and encoded a protein of 432 amino acids. Est2K had a SMTK motif and was a family VIII esterase. Unlike most family VIII esterases, Est2K had a signal peptide of 27 amino acids. The molecular mass and pI of the mature Est2K was calculated to be 44,668 Da and 4.48, respectively. The amino acid sequence of Est2K showed 72% identity with that of EstC, an esterase of an uncultured bacterium from leachate. The purified Est2K was optimally active at pH 10.0 and 50 degrees C. Est2K was stable in the presence of 30% methanol and exhibited a 2.4-fold higher activity in the presence of 5% methanol than in the presence of 1% isopropanol. Est2K preferred short to medium length p-nitrophenyl esters, especially p-nitrophenyl butyrate, as the substrate. Est2K did not hydrolyze beta-lactam antibiotics ampicillin and nitrocefin, even though Est2K showed the highest similarity to EstC.
authors.affiliation: Department of Agricultural Chemistry, Sunchon National University, Suncheon 540-742, Republic of Korea.
authors.forename: Yong Ho
authors.initials: YH
authors.lastname: Kim
authors.forename: Eun Ju
authors.initials: EJ
authors.lastname: Kwon
authors.forename: Sung Kyum
authors.initials: SK
authors.lastname: Kim
authors.forename: Yu Seok
authors.initials: YS
authors.lastname: Jeong
authors.forename: Jungho
authors.initials: J
authors.lastname: Kim
authors.forename: Han Dae
authors.initials: HD
authors.lastname: Yun
authors.forename: Hoon
authors.initials: H
authors.lastname: Kim
mesh_terms: D003001:Cloning, Molecular; D015723:Gene Library; D054892:Metagenome; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000235:genetics*; D012987:Soil; D012988:Soil Microbiology
chemical_list: D012987:Soil; D010727:Phosphoric Diester Hydrolases