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Characterization of the starch surface binding site on Bacillus paralicheniformis α-amylase

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2020
accepted_ijbm2020.pdf (1.736Mb)
Authors
Božić, Nataša
Rozeboom, Henriëtte J.
Lončar, Nikola
Šokarda Slavić, Marinela
Janssen, Dick B.
Vujčić, Zoran
Article (Accepted Version)
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Abstract
α-Amylase from Bacillus paralicheniformis (BliAmy), belonging to GH13_5 subfamily of glycoside hydrolases, was proven to be a highly efficient raw starch digesting enzyme. The ability of some α-amylases to hydrolyze raw starch is related to the existence of surface binding sites (SBSs) for polysaccharides that can be distant from the active site. Crystallographic studies performed on BliAmy in the apo form and of enzyme bound with different oligosaccharides and oligosaccharide precursors revealed binding of these ligands to one SBS with two amino acids F257 and Y358 mainly involved in complex formation. The role of this SBS in starch binding and degradation was probed by designing enzyme variants mutated in this region (F257A and Y358A). Kinetic studies with different substrates show that starch binding through the SBS is disrupted in the mutants and that F257 and Y358 contributed cumulatively to binding and hydrolysis. Mutation of both sites (F257A/Y358A) resulted in a 5-fold lower ef...ficacy with raw starch as substrate and at least 5.5-fold weaker binding compared to the wild type BliAmy, suggesting that the ability of BliAmy to hydrolyze raw starch with high efficiency is related to the level of its adsorption onto starch granules.

Keywords:
Crystal structure / Mutant / Starch / Surface binding site / α-Amylase
Source:
International Journal of Biological Macromolecules, 2020, 165, A, 1529-1539
Publisher:
  • Elsevier
Funding / projects:
  • Ministry of Education, Science and Technological Development, Republic of Serbia, Grant no. 200026 (University of Belgrade, Institute of Chemistry, Technology and Metallurgy - IChTM) (RS-200026)
  • Ministry of Education, Science and Technological Development, Republic of Serbia, Grant no. 200168 (University of Belgrade, Faculty of Chemistry) (RS-200168)
Note:
  • This is the peer-reviewed version of the article: N. Božić, H.J. Rozeboom, N. Lončar, et al., Characterization of the starch surface binding site on Bacillus paralicheniformis α-amylase, International Journal of Biological Macromolecules, 2020, 165, A, 1529-1539, DOI: https://doi.org/10.1016/j.ijbiomac.2020.10.025
  • Published version: https://cer.ihtm.bg.ac.rs/handle/123456789/3728

DOI: 10.1016/j.ijbiomac.2020.10.025

ISSN: 0141-8130

PubMed: 33058974

WoS: 000600768200144

Scopus: 2-s2.0-85092737032
[ Google Scholar ]
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URI
https://cer.ihtm.bg.ac.rs/handle/123456789/3736
Collections
  • Radovi istraživača / Researchers' publications
Institution/Community
IHTM
TY  - JOUR
AU  - Božić, Nataša
AU  - Rozeboom, Henriëtte J.
AU  - Lončar, Nikola
AU  - Šokarda Slavić, Marinela
AU  - Janssen, Dick B.
AU  - Vujčić, Zoran
PY  - 2020
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/3736
AB  - α-Amylase from Bacillus paralicheniformis (BliAmy), belonging to GH13_5 subfamily of glycoside hydrolases, was proven to be a highly efficient raw starch digesting enzyme. The ability of some α-amylases to hydrolyze raw starch is related to the existence of surface binding sites (SBSs) for polysaccharides that can be distant from the active site. Crystallographic studies performed on BliAmy in the apo form and of enzyme bound with different oligosaccharides and oligosaccharide precursors revealed binding of these ligands to one SBS with two amino acids F257 and Y358 mainly involved in complex formation. The role of this SBS in starch binding and degradation was probed by designing enzyme variants mutated in this region (F257A and Y358A). Kinetic studies with different substrates show that starch binding through the SBS is disrupted in the mutants and that F257 and Y358 contributed cumulatively to binding and hydrolysis. Mutation of both sites (F257A/Y358A) resulted in a 5-fold lower efficacy with raw starch as substrate and at least 5.5-fold weaker binding compared to the wild type BliAmy, suggesting that the ability of BliAmy to hydrolyze raw starch with high efficiency is related to the level of its adsorption onto starch granules.
PB  - Elsevier
T2  - International Journal of Biological Macromolecules
T1  - Characterization of the starch surface binding site on Bacillus paralicheniformis α-amylase
VL  - 165
IS  - A
SP  - 1529
EP  - 1539
DO  - 10.1016/j.ijbiomac.2020.10.025
ER  - 
@article{
author = "Božić, Nataša and Rozeboom, Henriëtte J. and Lončar, Nikola and Šokarda Slavić, Marinela and Janssen, Dick B. and Vujčić, Zoran",
year = "2020",
abstract = "α-Amylase from Bacillus paralicheniformis (BliAmy), belonging to GH13_5 subfamily of glycoside hydrolases, was proven to be a highly efficient raw starch digesting enzyme. The ability of some α-amylases to hydrolyze raw starch is related to the existence of surface binding sites (SBSs) for polysaccharides that can be distant from the active site. Crystallographic studies performed on BliAmy in the apo form and of enzyme bound with different oligosaccharides and oligosaccharide precursors revealed binding of these ligands to one SBS with two amino acids F257 and Y358 mainly involved in complex formation. The role of this SBS in starch binding and degradation was probed by designing enzyme variants mutated in this region (F257A and Y358A). Kinetic studies with different substrates show that starch binding through the SBS is disrupted in the mutants and that F257 and Y358 contributed cumulatively to binding and hydrolysis. Mutation of both sites (F257A/Y358A) resulted in a 5-fold lower efficacy with raw starch as substrate and at least 5.5-fold weaker binding compared to the wild type BliAmy, suggesting that the ability of BliAmy to hydrolyze raw starch with high efficiency is related to the level of its adsorption onto starch granules.",
publisher = "Elsevier",
journal = "International Journal of Biological Macromolecules",
title = "Characterization of the starch surface binding site on Bacillus paralicheniformis α-amylase",
volume = "165",
number = "A",
pages = "1529-1539",
doi = "10.1016/j.ijbiomac.2020.10.025"
}
Božić, N., Rozeboom, H. J., Lončar, N., Šokarda Slavić, M., Janssen, D. B.,& Vujčić, Z.. (2020). Characterization of the starch surface binding site on Bacillus paralicheniformis α-amylase. in International Journal of Biological Macromolecules
Elsevier., 165(A), 1529-1539.
https://doi.org/10.1016/j.ijbiomac.2020.10.025
Božić N, Rozeboom HJ, Lončar N, Šokarda Slavić M, Janssen DB, Vujčić Z. Characterization of the starch surface binding site on Bacillus paralicheniformis α-amylase. in International Journal of Biological Macromolecules. 2020;165(A):1529-1539.
doi:10.1016/j.ijbiomac.2020.10.025 .
Božić, Nataša, Rozeboom, Henriëtte J., Lončar, Nikola, Šokarda Slavić, Marinela, Janssen, Dick B., Vujčić, Zoran, "Characterization of the starch surface binding site on Bacillus paralicheniformis α-amylase" in International Journal of Biological Macromolecules, 165, no. A (2020):1529-1539,
https://doi.org/10.1016/j.ijbiomac.2020.10.025 . .

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