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1 天然素材向けとなるきのこ由来酵素 ラッカーゼ の効率的生産方法 Effective laccase production method from mushroom as a natural product ( ) 1
2 (Lac)? Laccase (EC ) 4Cu (O 2 ) H 2 O Laccase 4 OH R Cu + Cu + Cu+ Cu + Lac (red) O 2 4e - +H + Laccase 4 Cu 2 + H 2 O O Cu 2 + Cu 2 +. Cu 2 + R Lac (ox) <Lac > (O 2 2 O) H 2 O O 2 Lac (red) Lac (ox) Med(red) Lac Med(ox) (ox) (red) *;Lac 2
3 Lac 産業利用の現状 <Lac > <Lac > ( ) ( ; Designer laccases: a vogue for high-potential fungal enzymes? Rodgers C.J Trends in Biotechnology vol. 28 (2) p63-72 () () () 3
4 < > (Lac) 4
5 白色腐朽菌の生産する Lac Lentinula edodes ( Trametes versicolor ) Pycnoporus cinnabarinus () リグニン構造 (Lac) 5
6 Lac 高生産株のスクリーニング (a) (U/ml) (b) Strain#ibrc05015 Strain#ibrc05017 Strain#ibrc05015 (U/ml) (mg/ml) Strain#ibrc05007 SR-1(Lentinula edodes) SR-1(Lentinula edodes) ABTS Strain# ibrc05015
7 オツネンタケモドキの特徴 Strain#ibrc05015 ITS (Polyporus brumalis) (7.72U/ml ABTS 25) (223mg/l 35) ( 0.9cm/day in MYPG agar medium ) Polyporus brumalis (;) (Polyporus brumalis)
8 オツネンタケモドキラッカーゼ (PbLac1) の精製 a) b) Table.1 Purification of PbLac1 Purification step Total protein Total Lcc Specific activity Purification Yield (mg) (unit) (unit/mg) (fold) (%) Culture filtrate Ultra centrifusion % ammonium HiLoad Phenyl HiLoad Sepharose Q PbLac1 PbLac1 a) Purified PbLac1 CBB b) Total Secreted proteins CBB
9 PbLac1 誘導剤の検討 Lac activity (U ml -1 ) No added Anicidine Catecol Guaiyacol Ferulic 2,5 acid xylidine Ethanol Galic acid H 2 O 2 Cu 2+ * Mn 2+ * Fe 2+ * PbLac1 * 0.25mM Cu 2+, Mn 2+ Fe 2+ 1% 9
10 各株間における銅添加による Lac 活性比較 Laccase activity (U/ml) SR-1 -Cu +Cu (ibrc05015) -Cu +Cu (MAFF420194) -Cu +Cu (MAFF460203) -Cu +Cu (MAFF460202) -Cu +Cu (MAFF460217) -Cu +Cu (MAFF20150) -Cu +Cu (MAFF420155) -Cu +Cu P. brumalis(ibrc
11 銅添加による ibrc05015lac 活性の誘導 A) Lac -Cu +Cu +Cu/-Cu C) *** CBB -Cu +Cu LacU * /ml (Max) 11.0 (day35) (day14) 14 Lac U/mg(Max) 92.0 (day35) (day14) 27 B) ** ( ABTS) -Cu +Cu **0.5mM CuSO 4 14 * ABTS ***0.5mM CuSO 4 14 PbLac1 PbLac1 11
12 PbLac1 特性評価 <PbLac1> 1) ph 2) 3) Lac < PbLac1> 1) 2) 3) P. brumalis 12
13 Pblac1 酵素特性 (1) a) b) 温度 ph の影響 Relative activity (%) c) Incubation ph d) Relative activity (%) Incubation Incubation Temperature ph ( Remaining activity (%) Incubation ph Remaining activity (%) Incubation Temperature ( PbLac1 ph pH
14 実用化試験 (1) PbLac1 による RBBR の脱色実験 A) RBBR (Remazol Brilliant Blue R) RBBR Polychlorinated biphenyl (PCB) RBBR 0.02% U PbLac1 +PbLac1 -PbLac1 RBBR PCB. Novotony et al. (1997) Folia Microbiol Decolorization rate (%) 0.5U PbLac1 2.5U PbLac1 5.0U PbLac1 Reaction Time (min) 難分解性環境汚染物質 PCB を分解出来ることが示唆された 14
15 実用化試験 (1) PbLac1-Med 系による Poly R 478 の脱色実験 B) Poly R 478 Poly R-478 O NH NH Ac O NH -VA +VA +PbLac1 CH SO 3 H n Benzo[a]pyren Poly R-478 Benzo[a]pyren Field et al. (1992) Appl. Environ. Microbiol. 58; Absorbance ratio (A531/A362) Poly R % 5U/ml PbLac1 HN O N OH O N H O Violuric acid (VA) mm VA 0.5mM VA 1.0mM VA 2mM VA 5mM VA Reaction Time (min) 発癌性物質 Benzo[a]pyren を分解出来ることが示唆された 15
16 実用化試験 (2) PbLac1 による環境ホルモン (BPA) の分解 Bisphenol A (BPA)* *Bisphenol A A) PbLac1 BPA Bisphenol A * B) Relative activity of BPA (%) Blank +PbLac1 BPA(500ppm) PbLac1 (0.5U/ml) at 30ºC. 48hr, GC/MS BPA Relative activity of estrogen receptor activity (%) -PbLac1 +PbLac1 Blank PbLac1 は BPA のホルモン活性を減少させる 16
17 実用化試験 (3) PbLac1 の酸化還元電位 <*> Buffer; McIlvain buffer (ph5.0 ion intensity=0.3). lectrode;g/agcl E= mV vs. Ag/AgCl= mV v.s NHE *. <PbLac1> E=811mV vs. NHE NHE; normal hydrogen electrode < > Blue Copper redox protein 0v /mv Source Plastocyanin Spinach Azurin Pseudomonas eruginousa Stellacyanin Rhus vernicifera Psudoazurin Achrombactor cycloclastes Multi Copper Enzyme Bilirubin Oxidase Myrothecium verrucaria CotA Bacillus subtilis Laccase Rhus vernicifera Coprinus cinereus Trametes hirsute Trametes versicolor 811 Polyporus brumalis PbLac1 は既存の銅配位酵素の中で最も酸化還元電位が高い 17
18 高酸化還元電位 Lac の産業的利用例 SONY 0.5mW ( TrametesLac) PbLac1* T. versilcolor 780mV vs. NHE P. brumalis 811 mv vs. NHE *PbLac1 PbLac1 18
19 オツネンタケモドキラッカーゼの有用性 RBBR,olyR-478) BPA (E=811mV vs. NHE) PbLac1 (2009) IBRC
20 PbLac1 の評価 P. brumalis () T. Versicolor () P. Cinnabarinus () Ibrc05015() Ha1(New Zealand) ATCC200478(Australia) PbLac1 LACA Lcc PH mg/L CuPbLac mg/L 400mg/L-1.0g/L () mg/L Max 1.2g/L (EtOH) NR (not reported) P. Methanolica (12.6U/ml) P. pastoris ( mg/L) S. cerevisiae (2-9.95mg/L) Y. lipolytica (2.5mg/L) A.niger (70-80mg/L) P.pastoris (8mg/L) Y. lipolytica, NP( ), ( ) ) *PbLac1 (SONY) 0.73mV Pentachlorophenol (PCP) ) 高い酸化還元電位 高い蛋白質生産能を活かして産業化へ 20
21 A 21
22 <> <> Characterization of an extracellular laccase, PbLac1, purified from Polyporus brumalis. Keiko Nakade, Yuichi Sakamto, Yuko Nakagawa, Akira Yano and Toshitsugu Sato. Fungal Biology(2010): < > Purification and characterization of Polyporellus brumalius laccase (PbLac1). Keiko Nakade, Yuichi Sakamoto, Yuko Nakagawa, Akira Yano and Toshitsugu Sato. The 5 th Meeting of East Asia for Mushroom Science (Sep. 2008) Polyporus brumalis
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