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Engineering increased thermostability in the thermostable GH-10 xylanase from Thermoascus aurantiacus, by epPCR and site-directed mutagenesis

Grant number: 14/04272-8
Support Opportunities:Scholarships abroad - Research Internship - Doctorate
Effective date (Start): June 10, 2014
Effective date (End): December 09, 2014
Field of knowledge:Biological Sciences - Biochemistry - Molecular Biology
Principal Investigator:Roberto da Silva
Grantee:Angelica Rodrigues de Souza
Supervisor: Jeffrey A. Mertens
Host Institution: Instituto de Biociências, Letras e Ciências Exatas (IBILCE). Universidade Estadual Paulista (UNESP). Campus de São José do Rio Preto. São José do Rio Preto , SP, Brazil
Research place: National Center for Agricultural Utilization Research (NCAUR), United States  
Associated to the scholarship:11/22461-4 - Improved thermal stability of Thermoascus aurantiacus GH10 xylanase, expressed in E. coli, by directed evolution and rational design: Production, purification, biochemical and physico-chemical characterization of the enzyme., BP.DR


The enzymatic saccharification of lignocellulosic biomass and biobleaching paper pulp are attractive strategies for the bioprocess industry. Xylanases, which hydrolyze the b-1,4 glycosidic bond in the xylan polymer, have been applied heavily in these sectors. However, these processes require enzymes that tolerate conditions of high environmental stress such as extremes of temperature or pH. The wild xylanase (xynA) produced by thermophilic fungus Thermoascus aurantiacus shows high optimum activity (75°C) and remains stable in wide temperature range (from 35°C to 75°C). Thus, the xylanase enzyme from T. aurantiacus is a good candidate for rational protein engineering studies to further improve and understand temperature and pH sability, for example. During execution of the Ph.D. project some problems were incurred and motivated the construction of a new work plan. In the Ph.D. project, the xynA gene was cloned into vector YPGK-1, expressed in S. cerevisiae and confirmed by zymogram activity. However the expression levels in the culture medium were very low making it difficult to produce enough enzyme to perform enzymatic assays and thermoinactivation studies. Sequencing of xynA gene, cloning in S. cerevisiae, revealed the presence of mutations in the native enzyme that may explain the reduced expression and activity, when compared to P. pastoris expression. These results were discussed with Jeffrey A. Mertens, Ph.D. (USDA - Peoria - USA), an expert in protein engineering and recombinant protein expression in E. coli and yeast, who is interested in the project. Potential projects to overcome the expression issues and enzyme improvement through mutagenesis were proposed and are the motivation of this request for a sandwich stage abroad: rebuilt the original xynA gene (wild) by reamplifying native sequence, cloning the xynA gene into E. coli vector; then the plasmid will be used as template for construction of mutants libraries, with the goal of increasing thermal tolerance and activity. But if the enzyme (native and mutated) are expressed in the insoluble fraction, an alternative strategy will be developed, so in this case the pPIC9-xynA will be used as template for construction of mutants libraries. The mutants will be obtained by directed evolution of the enzymes, using multiple rounds of mutations by different techniques such as error-prone PCR (epPCR) and/or site-directed mutation (SDM). In SDM, specific amino acids in the xynA sequence will be changed to verify the impact in relation to thermal stability and activity of the enzyme, being tested different strategies and mutagenic xylanase will be selected for thermostability testing. This project proposal internship abroad will be carried out in partnership with a major research center, Bioenergy Research Unit of United States Department of Agriculture (USDA), in Peoria, Illinois, which will provide a major contribution to the execution of this work. (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)
DE SOUZA, ANGELICA R.; DE ARAUJO, GABRIELA C.; ZANPHORLIN, LETICIA M.; RULLER, ROBERTO; FRANCO, FERNANDA C.; TORRES, FERNANDO A. G.; MERTENS, JEFFREY A.; BOWMAN, MICHAEL J.; GOMES, ELENI; DA SILVA, ROBERTO. Engineering increased thermostability in the GH-10 endo-1,4-beta-xylanase from Thermoascus aurantiacus CBMAI 756. International Journal of Biological Macromolecules, v. 93, n. A, p. 20-26, . (14/02080-4, 14/04272-8, 10/12624-0, 11/17658-3)

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