Glucose and xylose stimulation of a β-glucosidase from the thermophilic fungus Humicola insolens: A kinetic and biophysical study

Autor: Rodrigo Furriel Inocentes, Richard J. Ward, Rosa dos Prazeres Melo Furriel, Flavio Henrique Moreira Souza, João Atílio Jorge
Rok vydání: 2013
Předmět:
Zdroj: Repositório Institucional da USP (Biblioteca Digital da Produção Intelectual)
Universidade de São Paulo (USP)
instacron:USP
ISSN: 1381-1177
Popis: β-Glucosidases activated by glucose and xylose are uncommon yet intriguing enzymes that may enhance cellulose saccharification efficiency, and are of interest for application in bioethanol production processes. The molecular mechanisms of activation are completely unknown, and the aim of this study was the kinetic and biophysical characterization of the stimulation of a β-glucosidase from Humicola insolens by glucose and xylose. The effects of the monosaccharides were concentration dependent, where in a stimulatory range (0.1–50 mmol L−1), the activity increased up to 2-fold; in a stimulatory-inhibitory range (50–450 mmol L−1 glucose or 50–730 mmol L−1 xylose), the enzyme continued to be stimulated, but the activity was lower than maximal. Above 450 mmol L−1 glucose or 730 mmol L−1 xylose, increasing inhibition occurred. Dynamic light scattering confirmed that the enzyme is monomeric (54 kDa) and kinetic, intrinsic tryptophan fluorescence emission and far ultraviolet circular dichroism analyses indicated that the enzyme possesses a catalytic site (CS) and a modulator binding site (MS). Glucose or xylose binding to the MS induces conformational changes that stimulate the catalytic activity at the CS. Glucose and xylose may compete with the substrate for the CS while the substrate competes with the monosaccharides for binding to the MS. The stimulation of the enzymatic activity by glucose and xylose, which compete for the same sites on the enzyme molecule, is not synergistic. These data reveal allosteric interactions between the MS and the CS in H. insolens β-glucosidase that result in fine modulation of the catalytic activity by the monosaccharides. A kinetic model was developed that accurately described the experimental data for enzyme stimulation by glucose and/or xylose. Understanding the regulatory mechanisms of the enzyme activity, with the aid of kinetic models, may be useful for the application of the enzyme in cellulose hydrolysis processes.
Databáze: OpenAIRE
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