tailieunhanh - Báo cáo khoa hoc : Chitin oligosaccharide binding to a family GH19 chitinase from the moss Bryum coronatum

Substrate binding of a family GH19 chitinase from a moss species, Bryum coronatum(BcChi-A, 22 kDa), which is smaller than the 26 kDa family GH19 barley chitinase due to the lack of several loop regions (‘loop-less’), was investigated by oligosaccharide digestion, thermal unfolding experiments and isothermal titration calorimetry (ITC). | IFEBS Journal Chitin oligosaccharide binding to a family GH19 chitinase from the moss Bryum coronatum Takayuki Ohnuma1 Morten S0rlie2 Tatsuya Fukuda1 Noriko Kawamoto1 Toki Taira3 and Tamo Fukamizo1 1 Department of Advanced Bioscience Kinki University Nara Japan 2 Department of Chemistry Biotechnology and Food Science Norwegian University of Life Sciences As Norway 3 Department of Bioscience and Biotechnology Faculty of Agriculture University of the Ryukyus Okinawa Japan Keywords binding subsite family GH19 chitinase isothermal titration calorimetry oligosaccharides thermodynamics Correspondence T. Fukamizo Department of Advanced Bioscience Kinki University 3327-204 Nakamachi Nara 631-8505 Japan Fax 81 742 73 8976 Tel 81 742 73 8237 E-mail fukamizo@ M. Sorlie Department of Chemistry Biotechnology and Food Science Norwegian University of Life Sciences PO Box 5003 N-1432 As Norway Fax 47 64 96 59 01 Tel 47 64 96 59 02 E-mail Received 26 May 2011 revised 27 July 2011 accepted 9 August 2011 doi Substrate binding of a family GH19 chitinase from a moss species Bryum coronatum BcChi-A 22 kDa which is smaller than the 26 kDa family GH19 barley chitinase due to the lack of several loop regions loopless was investigated by oligosaccharide digestion thermal unfolding experiments and isothermal titration calorimetry ITC . Chitin oligosaccharides b-1 4-linked oligosaccharides of N-acetylglucosamine with a polymerization degree of n GlcNAc n n 3-6 were hydrolyzed by BcChi-A at rates in the order GlcNAc 6 GlcNAc 5 GlcNAc 4 GlcNAc 3. From thermal unfolding experiments using the inactive BcChi-A mutant BcChi-A-E61A in which the catalytic residue Glu61 is mutated to Ala we found that the transition temperature Tm was elevated upon addition of GlcNAc n n 2-6 and that the elevation ATm was almost proportional to the degree of polymerization of GlcNAc n. ITC experiments provided the thermodynamic parameters for .