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

Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt

Standard

26kDa endochitinase from barley seeds: real-time monitoring of the enzymatic reaction and substrate binding experiments using electrospray ionization mass spectrometry. / Dennhart, Nicole; Weigang, Linda M M; Fujiwara, Maho; Fukamizo, Tamo; Skriver, Karen; Letzel, Thomas.

I: Journal of Biotechnology, Bind 143, Nr. 4, 2009, s. 274-83.

Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt

Harvard

Dennhart, N, Weigang, LMM, Fujiwara, M, Fukamizo, T, Skriver, K & Letzel, T 2009, '26kDa endochitinase from barley seeds: real-time monitoring of the enzymatic reaction and substrate binding experiments using electrospray ionization mass spectrometry', Journal of Biotechnology, bind 143, nr. 4, s. 274-83. https://doi.org/10.1016/j.jbiotec.2009.08.003

APA

Dennhart, N., Weigang, L. M. M., Fujiwara, M., Fukamizo, T., Skriver, K., & Letzel, T. (2009). 26kDa endochitinase from barley seeds: real-time monitoring of the enzymatic reaction and substrate binding experiments using electrospray ionization mass spectrometry. Journal of Biotechnology, 143(4), 274-83. https://doi.org/10.1016/j.jbiotec.2009.08.003

Vancouver

Dennhart N, Weigang LMM, Fujiwara M, Fukamizo T, Skriver K, Letzel T. 26kDa endochitinase from barley seeds: real-time monitoring of the enzymatic reaction and substrate binding experiments using electrospray ionization mass spectrometry. Journal of Biotechnology. 2009;143(4):274-83. https://doi.org/10.1016/j.jbiotec.2009.08.003

Author

Dennhart, Nicole ; Weigang, Linda M M ; Fujiwara, Maho ; Fukamizo, Tamo ; Skriver, Karen ; Letzel, Thomas. / 26kDa endochitinase from barley seeds: real-time monitoring of the enzymatic reaction and substrate binding experiments using electrospray ionization mass spectrometry. I: Journal of Biotechnology. 2009 ; Bind 143, Nr. 4. s. 274-83.

Bibtex

@article{ecc3d130d5b011dea1f3000ea68e967b,
title = "26kDa 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.",
author = "Nicole Dennhart and Weigang, {Linda M M} and Maho Fujiwara and Tamo Fukamizo and Karen Skriver and Thomas Letzel",
note = "Keywords: Chitinase; Kinetic analysis; ESI-MS (electrospray ionization mass spectrometry); Catalytic residue; Substrate binding",
year = "2009",
doi = "10.1016/j.jbiotec.2009.08.003",
language = "English",
volume = "143",
pages = "274--83",
journal = "Journal of Biotechnology",
issn = "0168-1656",
publisher = "Elsevier",
number = "4",

}

RIS

TY - JOUR

T1 - 26kDa endochitinase from barley seeds: real-time monitoring of the enzymatic reaction and substrate binding experiments using electrospray ionization mass spectrometry

AU - Dennhart, Nicole

AU - Weigang, Linda M M

AU - Fujiwara, Maho

AU - Fukamizo, Tamo

AU - Skriver, Karen

AU - Letzel, Thomas

N1 - Keywords: Chitinase; Kinetic analysis; ESI-MS (electrospray ionization mass spectrometry); Catalytic residue; Substrate binding

PY - 2009

Y1 - 2009

N2 - 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.

AB - 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.

U2 - 10.1016/j.jbiotec.2009.08.003

DO - 10.1016/j.jbiotec.2009.08.003

M3 - Journal article

C2 - 19665502

VL - 143

SP - 274

EP - 283

JO - Journal of Biotechnology

JF - Journal of Biotechnology

SN - 0168-1656

IS - 4

ER -

ID: 15923373