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| Author(s): |
Luana Parras Meleiro
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Ribeirão Preto. |
| Institution: | Universidade de São Paulo (USP). Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (PCARP/BC) |
| Defense date: | 2017-02-14 |
| Examining board members: |
Rosa dos Prazeres Melo Furriel;
Marcos Silveira Buckeridge;
María Eugenia Guazzaroni;
Sandro Roberto Marana;
Mario Tyago Murakami
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| Advisor: | Rosa dos Prazeres Melo Furriel |
| Abstract | |
One of the prerequisites for the economically viable production of ethanol from the lignocellulosic biomass is the development of efficient and inexpensive processes of enzymatic hydrolysis of cellulose and hemicellulose. The enzymes are responsible for high percentages of hydrolysis costs, since it is necessary to use high enzymatic loads for acceptable yields due to inhibition of lignocellulolitic enzymes by products, which is intensified by the use of high initial concentrations of biomass. One of the strategies to improve efficiency and decrease the costs of hydrolysis is the identification of more efficient enzymes with great attention to those that are tolerant and/or stimulated by the reaction products. In this context, protein engineering is a powerful tool for the improvement and understanding of the structure-function relationship of these enzymes. The present work aimed to evaluate the effect of glycosylation on the biochemical characteristics of glucose and xylose-stimulated -glucosidase from Humicola insolens, comparing the native enzyme and the recombinant enzymes expressed in Escherichia coli (Bglhi) and Pichia pastoris (BglhiPp) and study the structure-function relationship through directed evolution techniques aiming the understanding of the mechanisms involved in the stimulation of the enzyme by the monosaccharides. With regard to glycosylation, the expression and characterization of BglhiPp allowed to evaluate that the main characteristics influenced by different carbohydrate contents in the enzyme were optimum temperature and thermostability. The study of directed evolution culminated in the generation of 4 mutants with pattern of stimulation by glucose and xylose different from Bglhi (used as control). All mutants contain one of the two substitutions (D237V and N235S) grouped around the aglycone binding sites (+1 and +2). The kinetic and transglycosylation data allowed us to suggest that the mechanism of stimulation of these enzymes involves allosteric interactions, modulation of the hydrolysis and transglycosylation routes, and competition between substrate and monosaccharides by binding to the subsites in active site. The mutation D237V (present in mutants 4-12D and 5-7H) favored the hydrolysis route over that of transglycosylation and pNP-glucosidase activity, but not cellobiase activity, was stimulated by xylose. The substitution N235S (present in mutants 1-6D and 5-7C) abolished the preference for hydrolysis or transglycosylation and cellobiase activity, but not pNP-glucosidase activity, was strongly inhibited by xylose. In addition, both mutations decreased the tolerance of the enzymes by the monosaccharides. These results showed that fine modulation of Bglhi and mutant enzymes activities by glucose and/or xylose is regulated by the relative affinities of the glycone and aglycone subsites for the substrates and the free monosaccharides. The changes in the topology and physicochemical properties of the +1/+2 aglycone sites of the mutants have been proposed to rationalize the kinetic and transglycosylation data. (AU) | |
| FAPESP's process: | 11/22966-9 - Structure-function relationship of a glucose- and xylose-stimulated beta-glucosidase from the thermophilic fungus Humicola insolens: directed evolution studies |
| Grantee: | Luana Parras Meleiro Garcia |
| Support Opportunities: | Scholarships in Brazil - Doctorate (Direct) |