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Expression and purification of recombinants xylanolytic enzymes and study of their activities against sugar cane extracted arabinoxylans

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Carolina Victal Garbelotti
Total Authors: 1
Document type: Doctoral Thesis
Press: Ribeirão Preto.
Institution: Universidade de São Paulo (USP). Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (PCARP/BC)
Defense date:
Examining board members:
Richard John Ward; Fernando Batista da Costa; Luiz Alberto Beraldo de Moraes; Wanderley Dantas dos Santos
Advisor: Richard John Ward

Brazil maintains a strong agricultural base, with a long tradition ofsugar cane cultivation in the region of Ribeirão Preto. The search for more sustainable and renewable alternative energy sources have encouraged studies focussed on obtaining biofuels and other valuable chemicals using sugars derived from the hydrolysis of lignocellulosic biomass. With this goal, carbohydrate active enzymes have also attracted attention as a means of sustainable and specific hydrolysis, avoiding chemical waste and unwanted by-products. However, plant cell wall recalcitrance has hindered the industrial application of enzymatic hydrolysis, and overcoming this obstacle requires a better comprehension of the plant cell wall structure, efficient biomass pre-treatments and improved enzyme cocktails. In this work, we have studied the enzymatic hydrolysis of three arabinoxylan fractions from sugar cane variety SP80-3280 extracted from delignified stem either with sodium chlorine only, or after alkaline treatment or after DMSO extraction. Combinations of recombinant hemicellulolytic enzymes used included the glycosyl hydrolases xylanases GH10 and GH11, arabinofuranosidase GH43, and α-glucuronidase GH67 together with acetyl xylan esterase CE4 and feruloil esterase CE1. Hydrolysate analyses of total reducing sugar were correlated with mass spectrometry coupled with hydrophobic interaction liquid chromatography and metabolomics analysis tools. It was observed that the arabinoxylans from the different extraction methods had distinct structures and organization, resulting in altered enzymatic digestion profiles and hydrolysis products. The alkaline-treated arabinoxylan was most accessible to complete hydrolysis by this set of enzymes in comparison to the more recalcitrant polysaccharides in the chlorite and DMSO extracts. Furthermore, combinatory enzymatic assays with the four hydrolases and acetyl xylan esterase did not provide evidence of synergistic effects but demonstrated cooperativity between xylanases and the accessory enzymes. Although feruloyl esterase treatment did not provide evidence of phenolic decorations in any of the arabinoxylans, a detailed biochemical e biophysical characterization demonstrated the optimum activity of this enzyme at 60°C and pH 6.5 and a catalytic efficiency of about 390 s -1.mM-1 against the synthetic substrate ethyl ferulate. The enzyme was stable at high temperature, and active against feruloylated oligosaccharides from wheat bran arabinoxylan showing the great potential of the enzyme for industrial uses. (AU)

FAPESP's process: 18/25664-2 - Expression and purification of recombinants xylanolitics enzymes and study of their activities against sugar cane extracted arabinoxylans
Grantee:Carolina Victal Garbelotti
Support Opportunities: Scholarships in Brazil - Doctorate (Direct)