| Summary: | 博士 === 臺灣大學 === 農業化學研究所 === 98 === Activated sludge in a swine wastewater treatment facility comprises a complicated microbiological community. Based on BLASTN analysis of cloned 16S rRNA genes, we found that most of these communities (90%) were of uncultivated bacteria. It is highly possible that we may to discover new biocatalyst genes from activated sludge samples collected from such facilities using the metagenomic approach. The metagenomic library was constructed using a plasmid vector and DNA extracted directly from samples of activated sludge. The average insert size was approximately 5.1 kb. A total of 12 unique and lipolytic clones were obtained using the tributyrin plate assay. The rate of discovering lipolytic clones in this study was as high as 0.31%. Molecular analysis revealed that most of the 16 putative lipolytic enzymes showed 28–55% identity with non-redundant protein sequences in the database. Briefly, our study demonstrated that activated sludge was an ideal bioresource for isolating new lipolytic enzymes. Two esterase genes est6 and est13 were studied in depth for their highly novel amino acid sequences compared to the database. The est6 gene with 729-bp DNA encoded a 242-amino acid protein (designated Est6), and the est13 gene with 1326-bp DNA encodes a 441-amino acid protein (designated Est6). Most of the closely related proteins of both esterases were uncharacterized and conceptually translated from whole genome sequencing data of microorganisms. Multiple sequence alignments and phylogenetic analysis of Est6 and Est13 showed that Est6 and Est13 belonged to family VI and family II esterases/lipases, respectively. In addition, both esterase genes could be over expressed in their soluble form in Escherichia coli and then purified to a tag-free, homogeneous form by affinity chromatography. The purified Est6 in pH 7.0 or pH 8.0 buffers was active as a monomer, but the purified Est13 in Tris–HCl buffer (pH 8.0) was active as a dodecamer. Est6 showed significant regioselectivity in spectrophotometric analysis. The optimum temperature and pH for Est6 and Est13 were pH 8.0, 45°C, and pH 8.0, 35°C, respectively, when using p-nitrophenyl acetate as a substrate. In particular, the Est13 esterase was active with 45%－72% of maximal activity across low temperature ranges of 5°C－20°C. Both esterases were stable over wide temperature and pH ranges, and they exhibited activity in the presence of organic solvents, cations, or detergents. Because Est6 and Est13 possess noteworthy properties, they have the potential to be developed for biotechnological applications.
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