Clonagem e expressão da enzima beta-glucosidase recombinante do fungo Aspergillus niger em Pichia pastoris

Abstract

Cellulolytic enzymes are produced by a variety of microorganisms. However the great majority have major constraints that hamper or hinder the process of enzyme production. In addition, most of the available commercial enzymatic consortia, although presenting the three groups of cellulases (Endoglucanases, Celobiohidrolase, β-glucosidase) for the complete degradation of the cellulose, have a low β-glucosidásica activity. Thus, it is necessary to supplement this enzyme with these enzyme preparations. To solve these constraints, the molecular biology allied to recombinant DNA technology is configured as a favorable tool in the enzymatic production, allowing the expression of only the enzymes of interest, minimizing or eliminating the production of interfering proteins. In this context, the production of recombinant β-glucosidase by the genetically modified microorganism Pichia pastoris, constitutes a great step to obtain this enzyme with an economically viable production and with great potential for industrial application. Therefore, recombinant β-glucosidases are potential tools in the production of bioethanol, and in the near future, can be successful in meeting the requirements of alternative and renewable energy sources. According to the growing importance of β-glucosidase in several applications, the present work aimed to clone and express the recombinant β-glucosidase enzyme of Aspergillus niger in the methylotrophic yeast P. pastoris. According to the results obtained, it was possible to demonstrate that the enzyme β-glucosidase of A. niger was efficiently expressed in yeast P. pastoris, as well as its secretion in the supernatant of the cell culture according to the analysis of immunodetection Colony Blotting. Based on the SDS-PAGE gel electrophoretic profile of the induction times of the β-glucosidase production of recombinant clones Mut+ (46) and clone Muts (22), the induction in liquid medium of the recombinant clones was successfully performed due to the presentation of the band corresponding to the β-glucosidase enzyme with molecular mass of approximately 103 kDa. In the Mut+ clone the best induction time for expression of the recombinant protein was 72 hours, in the Muts clone the best times were 72 hours and 96 hours. Therefore, the β-glucosidase activity of the recombinant Muts clone was 12,517.33 U. L-1.