The method of integrated kinetics and its applicability to the exo-glycosidase-catalyzed hydrolyses of p-nitrophenyl glycosides

Anna S. Borisova; Sumitha K. Reddy; Dina R. Ivanen; Kirill S. Bobrov; Elena V. Eneyskaya; Georgy N. Rychkov; Mats Sandgren; Henrik Stålbrand; Michael L. Sinnott; Anna A. Kulminskaya; Konstantin A. Shabalin

doi:10.1016/j.carres.2015.03.021

The method of integrated kinetics and its applicability to the exo-glycosidase-catalyzed hydrolyses of p-nitrophenyl glycosides

Anna S. Borisova, Sumitha K. Reddy, Dina R. Ivanen, Kirill S. Bobrov, Elena V. Eneyskaya, Georgy N. Rychkov, Mats Sandgren, Henrik Stålbrand, Michael L. Sinnott, Anna A. Kulminskaya^*, Konstantin A. Shabalin

^*Corresponding author for this work

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Abstract

In the present work we suggest an efficient method, using the whole time course of the reaction, whereby parameters k_cat, K_m and product K_I for the hydrolysis of a p-nitrophenyl glycoside by an exo-acting glycoside hydrolase can be estimated in a single experiment. Its applicability was demonstrated for three retaining exo-glycoside hydrolases, β-xylosidase from Aspergillus awamori, β-galactosidase from Penicillium sp. and α-galactosidase from Thermotoga maritima (TmGalA). During the analysis of the reaction course catalyzed by the TmGalA enzyme we had observed that a non-enzymatic process, mutarotation of the liberated α-d-galactose, affected the reaction significantly.

Original language	English
Pages (from-to)	43-49
Number of pages	7
Journal	Carbohydrate Research
Volume	412
DOIs	https://doi.org/10.1016/j.carres.2015.03.021
Publication status	Published - 14 Aug 2015
MoE publication type	A1 Journal article-refereed

Funding

The work was supported by the Russian Foundation for Basic Research (project No. 12-08-00813-a ), a personal scholarship from the Swedish Institute to A.B., and personal fellowship of the Government of Leningrad District, St. Petersburg, Russia to G.R. NMR experiments were carried out using scientific equipment of the Center of Shared Usage “The analytical center of nano- and biotechnologies of Saint Petersburg State Polytechnical University” financially supported by the Ministry of Education and Science of the Russian Federation .

Keywords

Integrated kinetics
Mutarotation
Retaining glycoside hydrolase

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10.1016/j.carres.2015.03.021

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Borisova, A. S., Reddy, S. K., Ivanen, D. R., Bobrov, K. S., Eneyskaya, E. V., Rychkov, G. N., Sandgren, M., Stålbrand, H., Sinnott, M. L., Kulminskaya, A. A., & Shabalin, K. A. (2015). The method of integrated kinetics and its applicability to the exo-glycosidase-catalyzed hydrolyses of p-nitrophenyl glycosides. Carbohydrate Research, 412, 43-49. https://doi.org/10.1016/j.carres.2015.03.021

@article{001dbe0f04f641cfb9cb254d48594599,

title = "The method of integrated kinetics and its applicability to the exo-glycosidase-catalyzed hydrolyses of p-nitrophenyl glycosides",

abstract = "In the present work we suggest an efficient method, using the whole time course of the reaction, whereby parameters kcat, Km and product KI for the hydrolysis of a p-nitrophenyl glycoside by an exo-acting glycoside hydrolase can be estimated in a single experiment. Its applicability was demonstrated for three retaining exo-glycoside hydrolases, β-xylosidase from Aspergillus awamori, β-galactosidase from Penicillium sp. and α-galactosidase from Thermotoga maritima (TmGalA). During the analysis of the reaction course catalyzed by the TmGalA enzyme we had observed that a non-enzymatic process, mutarotation of the liberated α-d-galactose, affected the reaction significantly.",

keywords = "Integrated kinetics, Mutarotation, Retaining glycoside hydrolase",

author = "Borisova, {Anna S.} and Reddy, {Sumitha K.} and Ivanen, {Dina R.} and Bobrov, {Kirill S.} and Eneyskaya, {Elena V.} and Rychkov, {Georgy N.} and Mats Sandgren and Henrik St{\aa}lbrand and Sinnott, {Michael L.} and Kulminskaya, {Anna A.} and Shabalin, {Konstantin A.}",

note = "Funding Information: The work was supported by the Russian Foundation for Basic Research (project No. 12-08-00813-a ), a personal scholarship from the Swedish Institute to A.B., and personal fellowship of the Government of Leningrad District, St. Petersburg, Russia to G.R. NMR experiments were carried out using scientific equipment of the Center of Shared Usage “The analytical center of nano- and biotechnologies of Saint Petersburg State Polytechnical University” financially supported by the Ministry of Education and Science of the Russian Federation . Publisher Copyright: {\textcopyright} 2015 Elsevier Ltd. All rights reserved. Copyright: Copyright 2015 Elsevier B.V., All rights reserved.",

year = "2015",

month = aug,

day = "14",

doi = "10.1016/j.carres.2015.03.021",

language = "English",

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journal = "Carbohydrate Research",

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Borisova, AS, Reddy, SK, Ivanen, DR, Bobrov, KS, Eneyskaya, EV, Rychkov, GN, Sandgren, M, Stålbrand, H, Sinnott, ML, Kulminskaya, AA & Shabalin, KA 2015, 'The method of integrated kinetics and its applicability to the exo-glycosidase-catalyzed hydrolyses of p-nitrophenyl glycosides', Carbohydrate Research, vol. 412, pp. 43-49. https://doi.org/10.1016/j.carres.2015.03.021

TY - JOUR

T1 - The method of integrated kinetics and its applicability to the exo-glycosidase-catalyzed hydrolyses of p-nitrophenyl glycosides

AU - Borisova, Anna S.

AU - Reddy, Sumitha K.

AU - Ivanen, Dina R.

AU - Bobrov, Kirill S.

AU - Eneyskaya, Elena V.

AU - Rychkov, Georgy N.

AU - Sandgren, Mats

AU - Stålbrand, Henrik

AU - Sinnott, Michael L.

AU - Kulminskaya, Anna A.

AU - Shabalin, Konstantin A.

N1 - Funding Information: The work was supported by the Russian Foundation for Basic Research (project No. 12-08-00813-a ), a personal scholarship from the Swedish Institute to A.B., and personal fellowship of the Government of Leningrad District, St. Petersburg, Russia to G.R. NMR experiments were carried out using scientific equipment of the Center of Shared Usage “The analytical center of nano- and biotechnologies of Saint Petersburg State Polytechnical University” financially supported by the Ministry of Education and Science of the Russian Federation . Publisher Copyright: © 2015 Elsevier Ltd. All rights reserved. Copyright: Copyright 2015 Elsevier B.V., All rights reserved.

PY - 2015/8/14

Y1 - 2015/8/14

N2 - In the present work we suggest an efficient method, using the whole time course of the reaction, whereby parameters kcat, Km and product KI for the hydrolysis of a p-nitrophenyl glycoside by an exo-acting glycoside hydrolase can be estimated in a single experiment. Its applicability was demonstrated for three retaining exo-glycoside hydrolases, β-xylosidase from Aspergillus awamori, β-galactosidase from Penicillium sp. and α-galactosidase from Thermotoga maritima (TmGalA). During the analysis of the reaction course catalyzed by the TmGalA enzyme we had observed that a non-enzymatic process, mutarotation of the liberated α-d-galactose, affected the reaction significantly.

AB - In the present work we suggest an efficient method, using the whole time course of the reaction, whereby parameters kcat, Km and product KI for the hydrolysis of a p-nitrophenyl glycoside by an exo-acting glycoside hydrolase can be estimated in a single experiment. Its applicability was demonstrated for three retaining exo-glycoside hydrolases, β-xylosidase from Aspergillus awamori, β-galactosidase from Penicillium sp. and α-galactosidase from Thermotoga maritima (TmGalA). During the analysis of the reaction course catalyzed by the TmGalA enzyme we had observed that a non-enzymatic process, mutarotation of the liberated α-d-galactose, affected the reaction significantly.

KW - Integrated kinetics

KW - Mutarotation

KW - Retaining glycoside hydrolase

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U2 - 10.1016/j.carres.2015.03.021

DO - 10.1016/j.carres.2015.03.021

M3 - Article

C2 - 26005928

AN - SCOPUS:84929649802

SN - 0008-6215

VL - 412

SP - 43

EP - 49

JO - Carbohydrate Research

JF - Carbohydrate Research

ER -

The method of integrated kinetics and its applicability to the exo-glycosidase-catalyzed hydrolyses of p-nitrophenyl glycosides

Abstract

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Keywords

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