title: Functional proteomic analysis of rice bran esterases/lipases and characterization of a novel recombinant esterase.
abstract: An esterase from rice ( Oryza sativa ) bran was identified on two-dimensional gel using 4-methylumbelliferyl butyrate as a substrate. The esterase cDNA (870 bp) encoded a 289 amino acid protein (designated OsEST-b) and was expressed in Escherichia coli . The molecular weight of recombinant OsEST-b (rOsEST-b) was 27 kDa, as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Biochemical characterization demonstrated that rOsEST-b was active over a broad temperature range (optimum at 60 °C) and preferred alkaline conditions (optimum at pH 9.0). The rOsEST-b showed maximum activity toward p-nitrophenyl butyrate (C(4)) among various p-nitrophenyl esters (C(4)-C(18)), indicating that rOsEST-b is an esterase for short-chain fatty acids. The kinetic parameters under optimal conditions were K(m) = 27.03 μM, k(cat) = 49 s(-1), and k(cat)/K(m) = 1.81 s(-1) μM(-1). The activity of rOsEST-b was not influenced by ethylenediaminetetraacetic acid, suggesting that it is not a metalloenzyme. The amino acid sequence analysis revealed that OsEST-b had a conserved pentapeptide esterase/lipase motif but that the essential active site serine (GXSXG) was replaced by cysteine (C). These results suggest that OsEST-b is distinct from traditional esterases/lipases and is a novel lipolytic enzyme in rice bran.
authors.affiliation: Department of Food Science and Biotechnology, National Chung Hsing University , Taichung 402, Taiwan.
authors.forename: Hsu-Han
authors.initials: HH
authors.lastname: Chuang
authors.forename: Po-Ting
authors.initials: PT
authors.lastname: Chen
authors.forename: Wun-Nai
authors.initials: WN
authors.lastname: Wang
authors.forename: Yu-Ting
authors.initials: YT
authors.lastname: Chen
authors.forename: Jei-Fu
authors.initials: JF
authors.lastname: Shaw
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D004950:Esterases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D008049:Lipase / Q000032:analysis*; D008969:Molecular Sequence Data; D012275:Oryza / Q000201:enzymology*; D040901:Proteomics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D012639:Seeds / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D018076:DNA, Complementary; D011994:Recombinant Proteins; D004950:Esterases; D008049:Lipase