| Co-Investigator(Kenkyū-buntansha) |
WATANABE Takashi Ichinoseki National College of Technology, Chemical Engineering, Associate Professor, 物質化学工学科, 助教授 (90300516)
MURATA Takeomi Shizuoka University, Faculty of Agriculture, Associate Professor, 農学部, 助教授 (30273171)
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| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2005: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2004: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Research Abstract |
In a crude enzyme preparation of Trichoderma reesei, we found activities not only to transfer lactose (Lac) and N-acetyllactosamine (LacNAc) disaccharides to hydroxyl groups of various aglycons (R-OH) but also to catalyze the condensation reaction between those disaccharides and the aglycons. We have been suggesting the condensation enzyme are particular endo-B-1,4-glucanases, however, they have not been identified yet. In the study, we tried to purify the lactose condensation activity by serial column chromatographic methods to improve the condensation activity by protein engineering such as the site-directed mutagenesis.
The crude enzyme was purified by two kinds of anion exchange chromatography (HiTrap DEAE FF and UNO-Q1) after 70% ammonium sulfate saturated precipitation. The purified enzyme was homogeneous in SDS-PAGE and showed Lac condensing and disaccharide-releasing activities. The enzyme had Mr 54,000 and pI4.8-5.1, which are close to those of EGI. Several Lac condensing and d
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isaccharide-releasing activity were observed after chromato-focusing in Mono P column. The enzyme showed strong CMC'ase but no Avicel'ase activity, which is characteristic of endo-B-1,4-glucanase. Recombinant enzymes of EGI and EGII expressed in different host cells, gifted from Dr.Okada in Nagaoka University of Technology, were also tested for various activities. The rEGI expressed in A. oryzae showed both activity condensing Lac and releasing Lac and LacNAc while rEGII expressed in S. pombe showed none of them. In result, we concluded one of the disaccharides-condensing enzymes from T resei as EGI. The cDNA of EGI from T reesei was prepared by RT-PCR for protein engineering of EGI.
Dr.Yoshida et al. in Hirosaki University also found Lac condensation activity in culture broth of Aspergillus oryzae TB1 which is a mutant strain with highly expression of Ce1B, an endo-type cellulase. The homology of amino acid sequence is approximately 50% between EGI of T reesei and Ce1B of A. oryzae, both of which are endo-B-1,4-glucanases in Glycoside Hydrolase Family 7. We also tried to compare the dissacharides-releasing, transglycosylating, and condensing activities of CelB to these of T reesei EGI and rEGI. Less
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