Bioprospection and construction of hemicellulases for the production of xylose from the xylooligosaccharides liquor and its use in the fermentative xylitol production

The plant biomasses are composed mainly of cellulose, hemicellulose and lignin. Cellulose and hemicellulose can be hydrolyzed to monomeric sugars (primarily glucose/C6 and xylose/C5) with cellulases and hemicellulases, respectively. Hemicellulases are enzymes of glycoside hydrolyses group, which catalyze the hemicellulose hydrolysis to xylose and other compounds. Hemicellulose represents around to 30% of the polysaccharide in the biomass of sugarcane bagasses. However, few studies have focused on hemicellulose saccharification for monomeric sugars production, as xylose, and then its complete conversion to value-added byproduct, such as bioethanol, organic acids and xylitol. This study aimed the selection of Bacillus sp. strains able to growth using xylooligosaccharide liquor from hydrothermal pretreatment of sugar cane bagass. Furthermore, a bifunctional enzyme (chimera) composed by an endo-xylanase and ?-xylosidase activities was constructed for the hydrolysis of xylooligosacharides to xylose and its conversion to xylitol using free and immobilized yeasts. After screening a library of 104 Bacillus sp. strains, 7 isolated were selected by the enzymatic index determination and their ability to growth in the liquor from pretreated sugarcane bagasse and hidrolyse the xylooligosacharides. The selected strains were identified by the RNAr 16S DNA sequence as being strains of Bacillus subtilis and Bacillus licheniformis. BH27 e BH93 strains showed the best results for growing and hydrolysis of xylooligosaccharides liquor and their ?-xylosidases (GH43) sequences were cloned, expressed and characterized completely. However, none of these sequences were used in the construction of the chimeric enzyme, because their biochemical parameters and xylose tolerance were lower or similar as already studied for ?-xylosidases. The chimeric enzyme was constructed using an endo-xylanase (M6) of directed evolution studies (previous works using mutagenesis) together of a ?-xylosidase from Bacillus subtilis ATCC 168. Although chimera presented a Kcat/KM lower than parental enzymes, it showed the ability to hydrolyze of 30% of xylooligosaccharides in the liquor to xylose for subsequent fermentation to xylitol. In the study of xylitol production from xylose by fermentation were obtained best results using the yeast Scherffersomyces stipitis and Candida guillermondii cultured in free form compared with immobilized cells. After 96 hours of fermentation of broth containing xylooligosaccharides liquor (non-detoxified) and enzymatically hydrolysed by chimera, the yeast Candida guillermondii produced 9.5 g /L of xylitol, showed 33% of conversion efficiency, a conversion factor of 0.3 g / g and a volumetric productivity of 0.1 g / L / hr (AU)