Department of Food and Applied Biosciences, Faculty of Agriculture, Yamagata University, 1-23 Wakaba-machi, Tsuruoka 997-8555, Japan.
Department of Food and Applied Biosciences, Faculty of Agriculture, Yamagata University, 1-23 Wakaba-machi, Tsuruoka 997-8555, Japan.
J Biosci Bioeng. 2014 Oct;118(4):392-5. doi: 10.1016/j.jbiosc.2014.04.003. Epub 2014 May 21.
In this study, the biochemical properties of the recombinant tannase from Aspegillus oryzae were compared with those of the native enzyme. Extracellular native tannase was purified from a commercial enzyme source. Recombinant tannase highly expressed in Pichia pastoris was prepared as an active extracellular protein. Purified native and recombinant tannases produced smeared bands with apparent molecular masses of 45-80 kDa and 45-75 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After N-deglycosylation, the native enzyme yielded molecular masses of 33 kDa and 30 kDa, whereas the recombinant enzyme yielded molecular masses of 34 kDa and 30 kDa. Purified native and recombinant tannases had an optimum pH of 4.0-5.0 and 5.0, respectively, and were stable up to 40°C. After N-deglycosylation, both enzymes exhibited reduced thermostability. Catalytic efficiencies of both purified enzymes were greater with natural substrates, such as (-)-catechin, (-)-epicatechin, and (-)-epigallocatechin gallates, than those with synthetic substrates, such as methyl, ethyl, and propyl gallates. However, there were no activities against the methyl esters of ferulic, p-coumaric, caffeic, and sinapic acids, which indicate feruloyl esterase activity, or the ethyl, propyl, and butyl esters of 4-hydroxybenzoic acid, which indicate paraben hydrolase activity.
在这项研究中，比较了米曲霉来源的重组单宁酶和天然酶的生化特性。从商业酶源中纯化了胞外天然单宁酶。通过毕赤酵母表达系统高表达了重组单宁酶，并制备成活性的胞外蛋白。纯化的天然和重组单宁酶经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）后分别产生表观分子量为 45-80 kDa 和 45-75 kDa 的弥散条带。经 N-去糖基化后，天然酶的分子量分别为 33 kDa 和 30 kDa，而重组酶的分子量分别为 34 kDa 和 30 kDa。纯化的天然和重组单宁酶的最适 pH 值分别为 4.0-5.0 和 5.0，且在 40°C 以下稳定。经 N-去糖基化后，两种酶的热稳定性均降低。与合成底物（如甲基、乙基和丙基没食子酸酯）相比，两种纯化酶对天然底物（如（-）-儿茶素、（-）-表儿茶素和（-）-表没食子儿茶素没食子酸酯）的催化效率更高。然而，它们对阿魏酸、对香豆酸、咖啡酸和芥子酸的甲酯（表明具有酯酶活性），以及对 4-羟基苯甲酸的乙酯、丙酯和丁酯（表明具有对羟苯甲酸酯酶活性）没有活性。
