Clonagem e expressão do gene engXCA de Xanthomonas campestri pv. campestris em Saccharmyces cerevisiae e estudos das endoglucanases nativa e recombinante

• Ano de defesa: 2014
• Autor(a) principal: Pivetta, Daniele Heiras [UNESP]
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Estadual Paulista (Unesp)
• Palavras-chave em Português: Biotecnologia; Etanol; Saccharomyces cerevisiae; Petróleo; Biologia molecular; Oil
• Link de acesso: http://hdl.handle.net/11449/110372

The dependence on highly polluting fossil sources of energy, such as oil and oil products, has led the society to look for alternatives for renewable fuel use. Current researches indicate as emerging technology the production of fuel ethanol from lignocellulosic biomass waste. With the development and the establishment of new technologies for obtaining second-generation ethanol, cellulases may become the first class of enzymes marketed and industrially used worldwide. Despite being produced by a range of microorganisms, there are still limitations that hinder the process to obtain these enzymes. In order to overcome some of these difficulties, molecular biology appears as a favorable enzyme production tool. Since cloning and expression of cellulase genes in yeasts represents a interesting strategy for using these enzymes in simultaneous saccharification and fermentation (SSF) process. Within this context, this study aimed the construction of a yeast strain capable of expressing a bacterial cellulase for future testing of SFS. To this end, engXCA gene was isolated from Xanthomonas campestris pv. campestris (Xcc), and a gene fragment coding for the signal peptide of the alpha factor Sc (MFalpha). The latter was connected upstream to engXCA gene, resulting in MFalpha-engXCA merger. This chimera was linked to vector pYES2 Saccharomyces cerevisiae (Sc) and the recombinant expression endoglucanase was secreted into the culture medium. The strain that showed the best production of the recombinant enzyme was Sc BJ3501. When growing for 36 h at 30°C, in rich medium YPGal + CMC (carboxymethyl cellulose) 1.0%. The reaction conditions were evaluated for both native and Xcc recombinant endoglucanases and the best conditions for enzymatic activities were pH 5.5 and 65°C. Regarding stability, recombinant and native endoglucanases were more stable at pH 5.0 and maintained a high thermostability up to 55 °C when incubated for 1 hour.