Identification of a GDSL-motif carboxylester hydrolase from rice bran (Oryza sativa L.)

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• 作者：Shigeki Hamada ; ^{sty564@affrc.go.jp} ; Yoichi Hasegawa ; Yasuhiro Suzuki
• 关键词：Oryza sativa ; Rice bran ; GDSL-motif esterase ; Enzyme purification
• 刊名：Journal of Cereal Science
• 出版年：2012
• 出版时间：March 2012
• 年：2012
• 卷：55
• 期：2
• 页码：100-105
• 全文大小：671 K

文摘

A major esterase (designated OsEST1) showing high activity using 1-naphthyl acetate as a substrate was identified from rice bran and purified approximately 239-fold to near-homogeniety. The purified enzyme migrated as a single polypeptide band on native and SDS-polyacrylamide gels and had a molecular mass of 25?kDa under denaturing conditions. Analysis of its tryptic peptides by MALDI-TOF-MS and subsequent data mining identified a corresponding cDNA OsEST1 consisting of 714 nucleotides and encoding a 238 amino acid protein. Analysis of its primary sequence indicated that OsEST1 is a GDSL-motif carboxylester hydrolase belonging to the SGNH protein subfamily in containing the putative catalytic triad of Ser¹¹, Asp¹⁸⁷, and His¹⁹⁰. OsEST1 showed the highest catalytic activity at approximately pH 8.0-8.5 and at 45?¡ãC with K_m and V_max values for 1-naphthyl acetate of 172?¦ÌM and 63.7?¦Ìmol/min/mg protein, respectively. However, OsEST1 showed no activity with triacylglycerol. Alignment of the primary sequence of OsEST1 and other rice GDSL-motif esterases/lipases showed that OsEST1 aligns with a specific family of plant SGNH esterases involved in response to dehydration and cuticle formation. These results suggest that OsEST1 is not a lipase but an esterase activity which has some other function in rice, especially during seed development.