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Effects of the pectinolytic activity of Endopolygacturonase-CBMs chimeras on the saccharification of different biomass feedstocks

Grant number: 19/26891-5
Support type:Scholarships abroad - Research Internship - Doctorate
Effective date (Start): June 29, 2020
Effective date (End): October 28, 2020
Field of knowledge:Biological Sciences - Biochemistry - Chemistry of Macromolecules
Principal Investigator:Richard John Ward
Grantee:Sibeli de Carli
Supervisor abroad: Simon Mcqueen-Mason
Home Institution: Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (FFCLRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
Local de pesquisa : University of York, England  
Associated to the scholarship:17/13734-3 - Engineering of an endopolygalacturonase by insertion of different Carbohydrate Binding Modules (CBMs): chimerogenesis and proximity effects, BP.DR

Abstract

Lignocellulosic biomass is a readily available and abundant renewable resource, and is a suitable source of raw material for biofuel production. However, the enzymatic hydrolysis of biomass polysaccharides to fermentable sugars is impaired due to the high complexity of the plant cell wall matrix. We have been working to improve the efficiency of enzymatic degradation of lignocellulosic biomass with protein engineering strategies to enhance the catalytic performance of lignocellulolytic enzymes involved in plant cell wall hydrolysis, and using the modified recombinant enzymes to supplement commercial cocktails. During the doctoral project associated with the present proposal (FAPESP 2017/13734-3), we have demonstrated that supplementation of a commercial enzyme cocktail with an endopolygalacturonase (EndoPG) improved the saccharification of sugarcane bagasse. Specifically, these previous studies have shown that the supplementation of the commercial cocktail Celluclast 1.5L with a recombinant EndoPG increased hydrolysis of sugarcane bagasse about 10% at 24h. The EndoPG has been fused with several carbohydrate-binding modules (CBMs) in order promote binding to different wall components and enhance activity. To this end, we have generated a collection of 14 EndoPG-CBMs chimeras, and as the next step we are interested in evaluating the effects of cocktail supplementation with these EndoPG-CBMs chimeras against a range of abundant biomass feedstocks (including sugarcane bagasse, maize residues, wheat straw and rice straw) after different pretreatments. The effects of pectinase and pectinase-CBM chimeras on biomass saccharification, monosaccharide release will be evaluated using HPAEC in a semi-automated 96-sample high-throughput format. Pectinases generally do not include CBM domains, and this project will investigate the effects of the fusion of CBMs at various positions in the endopolygalacturonase structure on the hydrolysis of complex substrates. In addition to evaluation of the EndoPG-CBMs as supplements for commercial cocktails, these results will also help understand the spatial organization of pectin with respect to other polysaccharides in the plant cell wall.