Studies on cellulases for the construction of biorefinary from cellulose

Research Project

Project/Area Number	24580120
Research Category	Grant-in-Aid for Scientific Research (C)
Allocation Type	Multi-year Fund
Section	一般
Research Field	Applied microbiology
Research Institution	Ishikawa Prefectural University
Principal Investigator	KUMAGAI HIDEHIKO 石川県立大学, 生物資源環境学部, 教授 (70027192)
Co-Investigator(Kenkyū-buntansha)	YAMAMOTO Kenji 石川県立大学, 生物資源環境学部, 教授 (70109049) KATAYAMA Takane 石川県立大学, 寄附講座教授 (70346104)
Project Period (FY)	2012-04-01 – 2015-03-31
Project Status	Completed (Fiscal Year 2014)
Budget Amount *help	¥5,460,000 (Direct Cost: ¥4,200,000、Indirect Cost: ¥1,260,000) Fiscal Year 2014: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000) Fiscal Year 2013: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000) Fiscal Year 2012: ¥2,470,000 (Direct Cost: ¥1,900,000、Indirect Cost: ¥570,000)
Keywords	バイオリファイナリー / リニューワブル資源 / セルラーゼ / β-グルコシダーゼ / 耐熱性セルラーゼ / 酵素耐熱性向上変異 / 耐熱性酵素発現耐熱性酵母 / β-グルコシダーゼＸ線構造解析 / β‐グルコシダーゼ / β‐グルコシダーゼＸ線構造解析 / 未利用資源 / セルロース / 耐熱性 / b-グルコシダーゼ / バイオエタノール / Kluyveromyces / 糖化同時発酵
Outline of Final Research Achievements	We developed ventilation-mediated simultaneous ethanol fermentation and recovery system with a thermo-tolerant yeast strain, K. marxianus. Running the system, apparently pure ethanol (28 g) was recovered from cellobiose (100g) by growing recombinant expressing β-glucosidase from a thermo-tolerant fungus. To create much more thermo-stable enzyme, we mutagenized its gene and obtained five mutant enzymes showing higher thermo-stability than the wild type enzyme. These mutations were integrated in one enzyme molecule and the mutant enzyme showed 10℃ higher Tm value,than that of wild type enzyme. The mutant enzyme showed the same specific activity on its substrates with that of wild type enzyme. The transformant with this mutant enzyme gene showed 1.3times higher productivity of ethanol compared with the strain having wild type β-glucosidase gene. The mutant enzyme was crystallized and the crystalline structure was determined at the 2.7Å resolution.