Effects of the pectinolytic activity of pectin methyl esterase-CBM3 chimeras on the saccharification of different biomass feedstocks

Abstract

Lignocellulosic biomass is a major renewable resource that is readily available, and is a suitable source as a raw material for biofuel production. The efficiency of 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 through the improvement of the catalytic efficiency of lignocellulolytic enzymes involved in plant cell walls hydrolysis by bioprospection of new enzymes and improvement of sugarcane saccharification using recombinant enzymes to supplement commercial cocktails. During the post-doctoral project associated with the present proposal (FAPESP 2013/14454-3) supplementation of commercial cocktails by pectinolytic enzymes has been shown to improve the saccharification of pre-treated sugarcane bagasse. Specifically, our previous studies have shown that the supplementation of the commercial cocktail ACCELLERASE®1500 with recombinant Pectin methyl esterase (PME) increased hydrolysis of hydrothermal pretreated sugarcane bagasse by 20% and by 80% using delignified bagasse. As the next step, we are interested in evaluating the effects of cocktail supplementation against different biomass feedstocks (sugarcane bagasse, maize residues and Miscanthus) after different pretreatments with the pectinases arabinanase, galactanase, endopolygalacturonase and pectin methyl esterase (cloned and expressed as part of the post-doctorate FAPESP project: 2013/14454-3) and a collection of pectin methyl esterase-CBMs chimeras. Pectinases generally do not include CBM domains, and we propose to investigate the influence of the fusion of CBMs at various positions on Pectin methyl esterase structure to create PME chimeras containing CBM3a and/or CBM35. In addition to evaluation of these constructs as supplements for commercial cocktails, these chimeric enzymes will also help understand the structural relation between pectin and cellulose (CBM3a) or galacturonides and glucuronoxylan (CBM35) in the plant cell wall. For the analysis of effects of pectinases and pectinase-CBM chimeras on biomass saccharification, monosaccharide release will be evaluated by HPAEC in a 96-sample high-throughput format. (AU)