title: Biochemical characterization of Aspergillus oryzae native tannase and the recombinant enzyme expressed in Pichia pastoris.
abstract: 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.
authors.affiliation: Department of Food and Applied Biosciences, Faculty of Agriculture, Yamagata University, 1-23 Wakaba-machi, Tsuruoka 997-8555, Japan.
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Mizuno
authors.affiliation: Department of Food and Applied Biosciences, Faculty of Agriculture, Yamagata University, 1-23 Wakaba-machi, Tsuruoka 997-8555, Japan.
authors.forename: Yoshihito
authors.initials: Y
authors.lastname: Shiono
authors.affiliation: Department of Food and Applied Biosciences, Faculty of Agriculture, Yamagata University, 1-23 Wakaba-machi, Tsuruoka 997-8555, Japan. Electronic address: tkoseki@tds1.tr.yamagata-u.ac.jp.
authors.forename: Takuya
authors.initials: T
authors.lastname: Koseki
mesh_terms: D001236:Aspergillus oryzae / Q000737:chemistry* / Q000201:enzymology; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002392:Catechin / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D005656:Fungal Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D002392:Catechin; C045651:epigallocatechin gallate; D002265:Carboxylic Ester Hydrolases; C022711:tannase
keywords: Aspergillus oryzae; Esterase; Substrate specificity; Tannase