Heterologous production and functional characterization of a beta-fructocuranosidase identified in a cDNA library of Sphenophorus levis, a sugarcane pest

• Grant number: 11/19886-3
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Effective date (Start): December 01, 2011
• Effective date (End): November 30, 2012
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Flavio Henrique da Silva
• Grantee: Rafael Pedezzi
• Host Institution: Centro de Ciências Biológicas e da Saúde (CCBS). Universidade Federal de São Carlos (UFSCAR). São Carlos , SP, Brazil
• Associated research grant: 98/14138-2 - Center for Structural Molecular Biotechnology, AP.CEPID

Abstract

Brazil is world renowned for having an efficient methodology for the production of ethanol. The national industry uses sugarcane as raw material, generating interest in investing and innovative technologies to increase yield of sugarcane cultivation. The beetle Sphenophorus levis is among the sugarcane pests lacking effective control methods. Transcriptome approach aiming the better understanding of the insect digestive arsenal, revealed the presence of a gene sharing high similarity with invertase (beta-fructofuranosidase), an enzyme that catalyzes the hydrolysis of sucrose into glucose and fructose. The invertase emerges as an important alternative in the production of fructose syrup, replacing the acid hydrolysis, which is inefficient and generates waste products. In this work we intend to carry out the construction of the expression vector containing the pPICZalpha whit beta-fructofuranosidase gene. After the heterologous expression and purification of the protein in the model organism Pichia pastoris, having protein in its pure form, we will proceed to the kinetics characterization of the enzyme using sucrose substrate. The enzymatic characterization results will be compared to the known enzymes in order to evaluate using of the recombinant protein in industrial processes as an alternative to other enzymes and acid hydrolysis. In addition, insect tissue-specific expression will be assayed using Real-Time PCR in order to check developmental stages and tissue location of beta-fructofuranosidase transcripts, allowing evaluation of its importance to the insect physiology. Considering that S. levis generates large losses to the sugarcane crop, information at the molecular level of its digestive arsenal may generates subsidies for the development of biotechnological methods in order to control this pest. (AU)