Scholarship 11/14200-6 - Engenharia genética, Bioquímica de proteínas - BV FAPESP
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Functional studies of microbial hemicellulases with potential biotechnological application in biorefinery of hemicellulosic biomass

Grant number: 11/14200-6
Support Opportunities:Scholarships in Brazil - Master
Start date: March 01, 2012
End date: February 28, 2013
Field of knowledge:Biological Sciences - Biochemistry - Enzymology
Principal Investigator:Fábio Márcio Squina
Grantee:Zaira Bruna Hoffmam
Host Institution: Centro Nacional de Pesquisa em Energia e Materiais (CNPEM). Ministério da Ciência, Tecnologia e Inovação (Brasil). Campinas , SP, Brazil
Associated research grant:08/58037-9 - Library generation for biomass-conversion enzymes from soil metagenome, AP.BIOEN.JP

Abstract

The cane sugar is currently the option that yields higher biomass energy bioethanol per planted area. Alternatives have been sought to significantly increase ethanol production without increasing planted area, aiming a strong and renwable energy matrix for Brazil. The most promising is to produce ethanol from the biomass waste generated in the production of first generation alcohol. Therefore, it is necessary to hydrolyze the biomass and release fermentable sugars. However, given the recalcitrance of waste, is complex the task of proposing a process of hydrolysis that is efficient, inexpensive and does not generate toxic compounds that reduce the yield of the fermentation on a further step. In this sense, the complete hydrolysis of biomass depends on the synergistic action of several distinct catalytic activities, mainly cellulases and hemicellulases, with which is expected synergistic levels of income for saccharification of these complex polymers. Moreover, the presence of mesophilic and extremophilic enzymes in a multienzyme cocktail would guarantee activity on lignocellulosic substrates over a wide range of temperatures and pH. This implies versatility to the process of hydrolysis, and consequently, more significant results and lower costs for the process, considering that virtually all commercially available cocktail act in optimum temperatures varying from 50 to 55ºC, with the pH around 6.0. What limits the process of hydrolysis to the optimum conditions for the enzymes available, making it difficult to make changes in steps of the process in an attempt to optimize the production of ethanol in tests of hydrolysis followed by saccharification, or in steps with either low or costly returns for SSF (simultaneoussaccharification and fermentation), as growth and fermentation done by currently available yeast occurs in mild temperatures (different from optimum temperature of commercial cocktails).Based on these premises, this project proposes a study correlating structure and function of some key hemicellulases (4 hyperthermophilic and mesophilic hemicellulosic enzymes) to make a versatile hemicellulolytic complex, with different catalytic activities and active in a wide range of pH and temperature. The enzymes will be cloned in different expression systems and have their biochemical-kinetic parameters determined. These data will serve as basis for the construction of proteic chimeras from mesophilic and hyperthermophilic endoxylanase, with the aid of techniques, such as overlap PCR and extension end-to-end, and will be linked to carbohydrates binding motifs (CBM6) stemmed from the anaerobic etanolgenic bacteria (Clostridium thermocellum). The constructed gene will then be expressed and characterized. These complex of chimeric enzymes and catalytic modules (wild type) will behave the activities compared by various tests carried out in various conditions of pH, temperature and types of substrates. These enzymes produced will be biochemically (Vmax, Km, Kcat) and biophysically (circular dichroism, calorimetry DSC) studied in collaboration with other laboratories of this research center.Once these enzymes are produced, they will be studied in enzymatic mixtures combinations (in the complementation and supplementation cocktails enzyme) to analyse the extent of lingo-hemicellulosic compounds degradation in different conditions of preparation. The synergism of these components will be evaluated by experimental design based in mixtures with different combinations of these enzymes. These results may lead to a better combination of new cocktails that could contribute to increased income focused on the hydrolysis process of plant biomass and synthetic substrates. (AU)

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Scientific publications
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
MANDELLI, F.; BRENELLI, L. B.; ALMEIDA, R. F.; GOLDBECK, R.; WOLF, L. D.; HOFFMAM, Z. B.; RULLER, R.; ROCHA, G. J. M.; MERCADANTE, A. Z.; SQUINA, F. M.. Simultaneous production of xylooligosaccharides and antioxidant compounds from sugarcane bagasse via enzymatic hydrolysis. INDUSTRIAL CROPS AND PRODUCTS, v. 52, p. 770-775, . (13/03061-0, 08/58037-9, 11/14200-6, 12/18859-5)
HOFFMAM, ZAIRA B.; OLIVEIRA, LEANDRO C.; COTA, JUNIO; ALVAREZ, THABATA M.; DIOGO, JOS A.; NETO, MARIO DE OLIVEIRA; CITADINI, ANA PAULA S.; LEITE, VITOR B. P.; SQUINA, FABIO M.; MURAKAMI, MARIO T.; et al. Characterization of a Hexameric Exo-Acting GH51 alpha-l-Arabinofuranosidase from the Mesophilic Bacillus subtilis. MOLECULAR BIOTECHNOLOGY, v. 55, n. 3, p. 260-267, . (10/51890-8, 11/14200-6, 11/13242-7, 08/58037-9, 11/17658-3)
DIOGO, JOSE A.; HOFFMAM, ZAIRA B.; ZANPHORLIN, LETICIA M.; COTA, JUNIO; MACHADO, CARLA B.; WOLF, LUCIA D.; SQUINA, FABIO; DAMASIO, ANDRE R. L.; MURAKAMI, MARIO T.; RULLER, ROBERTO. Development of a chimeric hemicellulase to enhance the xylose production and thermotolerance. Enzyme and Microbial Technology, v. 69, p. 31-37, . (11/14200-6, 13/10443-7, 08/58037-9)
Academic Publications
(References retrieved automatically from State of São Paulo Research Institutions)
HOFFMAM, Zaira Bruna. Functional and structural studies of hemicellulases for potential biotechnological applications. 2013. Master's Dissertation - Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia Campinas, SP.