Directed evolution and biochemical characterization of the 1,3-1,4-B-Glucanase from Bacillus subtilis

• Grant number: 11/23842-1
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: April 01, 2012
• End date: October 31, 2013
• Field of knowledge: Biological Sciences - Biochemistry - Enzymology
• Principal Investigator: Richard John Ward
• Grantee: Gustavo Avelar Molina
• Host Institution: Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (FFCLRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Associated research grant: 10/18850-2 - Identification, characterization and engineering of plant cell wall degrading enzymes, AP.TEM

Abstract

The 1,3-1,4-B-Glucanases are enzymes that catalyze the hydrolysis of B-D-Glucan, a homopolysaccharide of D-glucose residues united by B-glycosidic linkages, which is found in many cereals, plants and fungi, being present in paper pulp produced by paper industries. However, the process commonly used by these industries for bleaching of pulp involves a sequence of stages of chlorination, leading to the formation of a wide range of organochlorine compounds of diverse structure, called "chlorolignins", which present toxicity to various aquatic organisms and high resistance to microbial degradation. For this reason, we work with the improvement of 1,3-1,4-B-Glucanase, to be included in a mixture of several enzymes capable of catalyzing the hydrolysis of other polysaccharides present in paper pulp, which may replace the traditional industrial processes to perform this step. With that, this project has basically three main objectives: The first is the directed evolution of 1,3-1,4-B-Glucanase from Bacillus subtilis in order to obtain more efficient strains of this enzyme, through the use of various molecular biology techniques; the second is the characterization of the main biochemical properties of evolved enzymes by assessing the activity as a function of temperature and thermotolerance tests; the third is the study of structure-function relationship of this enzyme to better understand and rationalize the role of mutations in this relationship. (AU)