Production and characterization of Sfbgli-CBM, a chimeric protein with beta-glycosidic and cellulose binding domains

• Grant number: 11/51903-5
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: January 01, 2012
• End date: February 28, 2014
• Field of knowledge: Biological Sciences - Biochemistry - Enzymology
• Principal Investigator: Sandro Roberto Marana
• Grantee: Larissa Martins Gonçalves
• Host Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Enzymatic hydrolysis of cellulose depends on the combined action of endoglucanases, cellobiohydrolases and b-glucosidases. In the "initial steps" endoglucanases and cellobiohydrolases attack the cellulose fibers generating the disaccharide cellobiose, which in the "final stage" is hydrolyzed by b-gtycosidases. However, the rate of cellulose hydrolysis decreases during the reaction, resulting in productivity loss for this process. One of the factors that cause this problem is the inhibitory effect of cellobiose on endoglucanase and cellobiohydrolases. This project aims to test a new strategy for reducing the cellobiose inhibition of endoglucanase and cellobiohydrolases through the production and characterization of a specially designed chimeric protein. This chimeric protein combines the b-glucosidase of Spodoptera frugiperda (Sfbgli) and "cellulose binding domain (CBM)" of the endoglucanase EngXCA from Xhantomonas axonopodis pv citri (CBM). The CBM will direct this chimeric protein to the surface of cellulose fibers, where the domain formed by Sfbgli will reduce the local concentration of cellobiose, removing the inhibitory effect of this disaccharide directly in the micro-environment of action of the endoglucanases and cellobiohydrolases. Thus, in theory, this chimeric enzyme may help to reduce falling rate of the enzymatic hydrolysis of cellulose. The expression vector that encodes Sfbgli-CBM has been built. Moreover, the activity of cellulose-binding CBM has been demonstrated. Based on this starting point, this project will seek production of the chimeric enzyme in the soluble form in E. coli, the characterization of its binding activity on crystalline cellulose and characterization of its activity upon cellobiose. After that, the ability of Sfbgli-CBM reduce inhibition upon cellobiohidrolase I will be tested. (AU)