期刊
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
卷 393, 期 1, 页码 45-49出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.bbrc.2010.01.070
关键词
Metagenomic library; Compost; Family VIII alkaline esterase; Signal peptide; Short to medium length p-nitrophenyl ester
资金
- Ministry of Science and Technology
- Ministry of Agriculture and Forestry, Korea
- National Research Foundation of Korea [2005-042-F00013] Funding Source: Korea Institute of Science & Technology Information (KISTI), National Science & Technology Information Service (NTIS)
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 pl 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 p-lactam antibiotics ampicillin and nitrocefin, even though Est2K showed the highest similarity to EstC. (C) 2010 Elsevier Inc. All rights reserved.
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