부산대학교 도서관

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.jbiotec.2009.08.003 Byline: Nicole Dennhart (a), Linda M.M. Weigang (a), Maho Fujiwara (b), Tamo Fukamizo (b), Karen Skriver (c), Thomas Letzel (a) Keywords: Chitinase; Kinetic analysis; ESI-MS (electrospray ionization mass spectrometry); Catalytic residue; Substrate binding Abbreviations: GlcNAc, N-acetylglucosamine; (GlcNAc).sub.n , [beta]-1,4-linked GlcNAc oligosaccharide with a polymerization degree of n; HEWL, hen egg white lysozyme; GlcN, glucosamine; (GlcN).sub.n , [beta]-1,4-linked GlcN oligosaccharide with a polymerization degree of n Abstract: A 26kDa endochitinase from barley seeds was enzymatically characterized exclusively by electrospray ionization mass spectrometry (ESI-MS). At first, oligosaccharide hydrolysis catalyzed by the barley chitinase was monitored in real-time by ESI-MS. The reaction time-course obtained by ESI-MS monitoring was found to be consistent with the data obtained earlier by HPLC, and the quantitative profile was successfully simulated by kinetic modeling of the enzymatic hydrolysis. It is obvious that the real-time monitoring method by ESI-MS allows a faster and cheaper determination of the chitinase activity with unlabeled substrate. Further, the enzymatic activity of the E67Q mutant of the barley chitinase was analyzed and the role of Glu67 was discussed comparing the mass spectra of enzyme protein obtained in native and in denatured conditions. Then it was determined that the observed loss of the enzymatic activity in E67Q is definitely caused by a point mutation of Glu67 but not due to partial unfolding of the mutated enzyme. Finally, association constants of enzyme-oligosaccharide complexes were calculated from Scatchard plots obtained by mass spectra. The binding free energy values obtained for E67Q were found to be comparable to those previously obtained in liquid phase, but less dependent upon the chain length of the oligosaccharides. To our knowledge, this study is the first enzymatic characterization of chitinase exclusively by such an innovative ESI-MS system. Author Affiliation: (a) Analytical Research Group, Chair of Biopolymer Chemistry, Department for Basic Life Sciences, Technische Universitat Munchen, Weihenstephaner Berg 3, 85354 Freising-Weihenstephan, Germany (b) Department of Advanced Bioscience, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan (c) Department of Molecular Biology, University of Copenhagen, 2A Aster Farimagsgade 1353 Copenhagen K, Denmark Article History: Received 3 December 2008; Revised 30 July 2009; Accepted 3 August 2009