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Structure-function relationship of a glucose- and xylose-stimulated beta-glucosidase from the thermophilic fungus Humicola insolens: directed evolution studies

Grant number: 11/22966-9
Support type:Scholarships in Brazil - Doctorate (Direct)
Effective date (Start): March 01, 2012
Effective date (End): February 28, 2017
Field of knowledge:Biological Sciences - Microbiology - Applied Microbiology
Principal researcher:Rosa dos Prazeres Melo Furriel
Grantee:Luana Parras Meleiro Garcia
Home Institution: Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (FFCLRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil

Abstract

Cellulose is one of the major components of agroindustrial by products and constitutes the most abundant renewable carbon source on earth. Nowadays, the enzymatic saccharification of cellulose raises a great interest, since it may generate undeniable environmental and economic benefits, particularly the viable production of second generation bioethanol. The enzymatic hydrolysis of cellulose involves the synergic action of endo-1,4-ß-glucanases, exo-1,4-ß-glucanases and 1,4-ß-glucosidases. Endo- and exo-glucanases act on cellulosic fibers, generating celloligosaccharides and cellobiose, which are converted to glucose by the ß-glucosidase. Considering that the glucanases are usually inhibited by cellobiose and celloligosaccharides, the ß-glucosidases are then responsible for the limiting step of the whole process. However, most known ß-glucosidases are also inhibited by glucose, generating a great interest in glucose tolerant enzymes. Recently, a ²-glucosidase from the thermophilic fungus Humicola insolens that is stimulated by glucose or xilose at concentrations up to 400 mmol L-1 was purified and characterized at our laboratory. This atypical feature, as well as its high thermal stability and high catalytic efficiency for cellobiose hydrolysis suggest that this enzyme may be interesting for efficient saccarification of lignocellulosic materials. However, it is an intracellular enzyme, produced at low levels, what hampers its large scale application. As a consequence, the gene for this ß-glucosidase was cloned and sequenced, and the enzyme was expressed in Escherichia coli in active form with high yield. The purified heterologous enzyme showed higher catalytic efficiency for cellobiose hydrolysis that the native one and maintained glucose and xylose stimulation, thus presenting an extraordinary potential to increase the economic viability of cellulose enzymatic saccharification. The main objective of this project is the study of the structure-function relationship of the glucose- and xylose-stimulated ß-glucosidase from H. insolens using directed evolution tools. The results may lead to the identification of the structural determinants of enzyme stimulation by the monosaccharides, yet contributing to clarify the intriguing mechanism of stimulation of an enzyme by its own reaction product. Moreover, based on the nucleotide sequences that codify for the regions involved in the stimulation by glucose/xylose, other ß-glucosidases with similar properties may be identified and/or constructed. Finally, we will also try to express H. insolens enzyme in Pichia pastoris, aiming to study the effect of glycosylation on biochemical, biophysical and structural properties of the enzyme, comparing the native enzyme and those expressed in E. coli and P. pastoris. (AU)

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