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26 kDa endochitinase from barley seeds: Real-time monitoring of the enzymatic reaction and substrate binding experiments using electrospray ionization mass spectrometry

Paper ID Volume ID Publish Year Pages File Format Full-Text
24524 43522 2009 10 PDF Available
Title
26 kDa endochitinase from barley seeds: Real-time monitoring of the enzymatic reaction and substrate binding experiments using electrospray ionization mass spectrometry
Abstract

A 26 kDa 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.

Keywords
GlcNAc, N-acetylglucosamine; (GlcNAc)n, β-1,4-linked GlcNAc oligosaccharide with a polymerization degree of n; HEWL, hen egg white lysozyme; GlcN, glucosamine; (GlcN)n, β-1,4-linked GlcN oligosaccharide with a polymerization degree of nChitinase; Kinetic
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26 kDa endochitinase from barley seeds: Real-time monitoring of the enzymatic reaction and substrate binding experiments using electrospray ionization mass spectrometry
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 143, Issue 4, 25 September 2009, Pages 274–283
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us