1.

論文

論文
片岡, 邦重 ; Kataoka, Kunishige ; Tanaka, Kazuhiro ; Sakai, Yoko ; Sakurai, Takeshi
出版情報: Protein expression and purification.  41  pp.77-83,  2005-05-01.  Elsevier
URL: http://hdl.handle.net/2297/1732
概要: Bilirubin oxidase (BO) from Myrothecium verrucaria (authentic BO) catalyzing the oxidation of bilirubin to biliverdine was overexpressed in the methylotrophic yeast, Pichia pastoris. The cDNA encoding BO was cloned into the P. pastoris expression vector pPIC9K under the control of the alcohol oxidase 1 promoter and its protein product was secreted using the Saccharomyces cerevisiae α-mating factor signal sequence. The productivity of recombinant BO (rBO) in P. pastoris was approximately 5000 U/L of culture broth, being about 2.5- and 250-fold higher than rBO expressed in Aspergillus oryzae and S. cerevisiae, respectively. The calculated molecular mass of rBO consisting of 538 amino acids was 60,493 kDa, however, that of SDS–PAGE was 66 kDa because of non-native type N-linked sugar chains. The spectroscopic properties of rBO were typical of multicopper oxidase containing four Cu ions per protein molecule. The specific activity to oxidize bilirubin was 57 U/mg, having a value about twice that of authentic BO and rBO expressed in A. oryzae. Moreover, the thermostability of rBO expressed in P. pastoris was significantly high compared to the authentic BO previously reported. Accordingly, a heterologous expression system of rBO to meet clinical and industrial needs was constructed 続きを見る
2.

論文

論文
片岡, 邦重 ; Kataoka, Kunishige ; Tanizawa, Katsuyuki
出版情報: Journal of molecular catalysis b enzymatic.  23  pp.299-309,  2003-09-01.  Elsevier
URL: http://hdl.handle.net/2297/1731
概要: The residues L40, A113, V291, and V294, in leucine dehydrogenase (LeuDH), predicted to be involved in recognition of the substrate side chain, have been mutated on the basis of the molecular modeling to mimic the substrate specificities of phenylalanine (PheDH), glutamate (GluDH), and lysine dehydrogenases (LysDH). The A113G and A113G/V291L mutants, imitating the PheDH active site, displayed activities toward -phenylalanine and phenylpyruvate with 1.6 and 7.8% of kcat values of the wild-type enzyme for the preferred substrates, -leucine and its keto-analog, respectively. Indeed, the residue A113, corresponding to G114 in PheDH, affects the volume of the side-chain binding pocket and has a critical role in discrimination of the bulkiness of the side chain. Another two sets of mutants, substituting L40 and V294 of LeuDH with the corresponding residues predicted in GluDH and LysDH, were also constructed and characterized. Emergence of GluDH and LysDH activities in L40K/V294S and L40D/V294S mutants, respectively, indicates that the two corresponding residues in the active site of amino acid dehydrogenases are important for discrimination of the hydrophobicity/polarity of the aliphatic substrate side chain. All these results demonstrate that the substrate specificities of the amino acid dehydrogenases can be altered by protein engineering. The engineered dehydrogenases are expected to be used for production and detection of natural and non-natural amino acids. 続きを見る
3.

論文

論文
片岡, 邦重 ; Kataoka, Kunishige ; Nakai, Masami ; Yamaguchi, Kazuya ; Suzuki, Shinnichiro
出版情報: Biochemical and Biophysical Research Communications.  250  pp.409-413,  1998-01-01.  Elsevier
URL: http://hdl.handle.net/2297/1733
概要: The gene coding for the 109-amino-acid, non-glycosylated form of mavicyanin was synthesized and expressed inEscherichia coli.The recombinant protein refolded fromE. coliinclusion bodies was purified and characterized. Its spectroscopic properties are fully identical to those of mavicyanin isolated from zucchini, even in the absence of its carbohydrate moiety. The blue cooper center of mavicyanin strongly binds three ligands (2His and Cys) as well as many blue copper proteins. To disclose the fourth ligand of mavicyanin, Met was substituted for Gln95 by site-directed mutagenesis. The replacement changes from a rhombic EPR signal to an axial one and exhibits the quite similar absorption and CD spectra to those of plastocyanin. The midpoint potential of Gln95→Met mavicyanin shows the positive shift of 187 mV compared with the recombinant protein, being close to the values of plastocyanins. The differences of the spectroscopic and electrochemical properties between mavicyanin and its mutant demonstrate that the fourth ligand of mavicyanin is Gln95. 続きを見る
4.

論文

論文
片岡, 邦重 ; Kataoka, Kunishige
出版情報: 平成25(2013)年度 科学研究費補助金 基盤研究(C) 研究成果報告書.  2011-2013  pp.5p.-,  2014-06-02.  金沢大学理工研究域物質化学系
URL: http://hdl.handle.net/2297/00052200
概要: バイオ燃料電池の実用化に必須である白金代替酸素還元触媒の創成を目的として,酸素の4電子還元反応を触媒するマルチ銅オキシダーゼをターゲットに,高反応性酵素の探索と既存酵素のタンパク質工学的高機能化を行った。探索研究では,嫌気性細菌のラッカーゼ と,昆虫のクチクラ硬化ラッカーゼ遺伝子の発現系を構築し,前者が既存酵素と全く異なる性質を持つ事を明らかにした。さらに,微生物由来の既存酵素をベースとしたタンパク質工学的改変により,酸素還元活性,電極反応性の向上に成功した。<br />研究課題/領域番号:23580131, 研究期間(年度):2011-2013 続きを見る
5.

論文

論文
片岡, 邦重 ; Kataoka, Kunishige
出版情報: 平成21(2009)年度 科学研究費補助金 基盤研究(C) 研究成果報告書 = 2009 Fiscal Year Final Research Report.  2007-2009  pp.4p.-,  2010-05-17.  金沢大学理工研究域物質化学系
URL: http://hdl.handle.net/2297/00052201
概要: プロトンポンプの分子進化過程を明らかにするために,一酸化窒素還元酵素(NOR)の異種発現系を構築し,ヘムb3-FeB複核活性中心を構成するFeB配位残基に変異を導入した。Glu190, Glu194及びGlu259を対象にAla, Asp, Gln置換変異体を作製したところ,何れの変異体も複核中心の形成が阻害され,これらの残基がNORの活性中心形成に重要であることが明らかになった。また,NORの生理的電子供与体を検索し,3種類の可溶性Cytcを単離することに成功した。<br />研究課題/領域番号:19570125, 研究期間(年度):2007-2009 続きを見る
6.

論文

論文
片岡, 邦重 ; Kataoka, Kunishige
出版情報: 平成29(2017)年度 科学研究費補助金 挑戦的萌芽研究 研究成果報告書.  2015-04-01 - 2018-03-31  pp.5p.-,  2018-06-26.  金沢大学理工研究域物質化学系
URL: http://hdl.handle.net/2297/00052199
概要: 二臭化インディゴの分子構造をもつ貝紫は,紀元前1,600 年以前から使用されている希少な動物性天然染料である。本研究では,アカニシガイの鰓下腺から貝紫色素前駆体の臭素化反応を触媒するブロモペルオキシダーゼ(BPO)の精製を試みた。BPOは鰓 下腺に微量しか存在していないため,均一精製には至らなかったが,膜画分に存在するBPOの活性発現に,可溶性活性化タンパク質が必要であることを明らかにした。2成分で構成されるBPOはこれまでに報告例のない新規の酵素である。<br />研究課題/領域番号:15K14709, 研究期間(年度):2015-04-01 - 2018-03-31 続きを見る