Improving activity and thermostability of GH 62 a-L-arabinofuranosidase by directed evolution

• Grant number: 22/04557-9
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: October 03, 2022
• End date: May 02, 2023
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: Rosana Goldbeck Coelho
• Grantee: Manoela Martins
• Supervisor: Marc Ostermeier
• Host Institution: Faculdade de Engenharia de Alimentos (FEA). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
• Institution abroad: Johns Hopkins University (JHU), United States
• Associated to the scholarship: 20/02871-2 - Bifunctional chimeric protein of endoxylanase/arabinofuranosidase for agro-industrial wastes hydrolysis and xylooligosaccharides production, BP.DR

Abstract

Thermostability is an important parameter to measure the feasibility of enzymes for industrial applications. Generally, higher thermostable enzymes are more desirable in industry as they may adapt to harsh conditions, decrease reaction time, and reduce risk of microbial contamination. Particularly for lignocellulose depolymerization, several hemicellulases acting synergistically must present good stability under the same operational conditions. However, most natural enzymes show poor thermostability, which restricts their application. Protein engineering, including directed evolution, is a powerful approach to improve enzyme properties. In this project, error-prone PCR and cassette mutagenesis will be used to build a library of enzymes variants of GH 62 arabinofuranosidase. The enzymatic activity and melting temperature of the mutants will be assayed to select and analyze the sequence and structure of the variants that exhibit higher stability. The major interest to improve the thermostability of GH 62 is due to its excellent substrate specificity and strong synergism with endoxylanases for the depolymerization of arabinoxylan, the second most abundant polysaccharide found in agro-industrial field, to enhance prebiotic xylo-oligosaccharides production. (AU)