Xilanases de leveduras e fungos leveduriformes e sua aplicação em processos de produção de bioetanol lignocelulósico e panificação

Abstract

The exploration of microbial diversity enables the discovery of new sources of enzymes with potential application in the biotechnology industry. The aim of this work was to select and characterize xylanolytic enzymes - produced by yeasts - and evaluate their applicability in biotechnological processes. In the first part of this study, yeast isolates associated to rotting wood and sugarcane bagasse were investigated for their ability to produce xylanolytic enzymes. Twenty-one yeast isolates showing xylanolytic activity on solid medium containing Beechwood xylan were grown in liquid medium, and the five isolates presenting the highest xylanolytic activity were characterized regarding the xylanase and -xylosidase activities using two inducing substrates: xylan and D -xylose. Xylan was the best inducer of xylanase activity (measured at 50°C) in Cryptococcus laurentii UFMG-HB-48, Scheffersomyces shehatae UFMG-HM-9.1a and Sugyiamaella smithiae UFMG-HM-80.1 (1.16, 0.69 and 0.50 U mL-1, respectively). The highest specific activity (11.05 U mg-1) was obtained by Cr. laurentii UFMG-HB-48 at 50°C using xylan as inducer. The highest level of extracellular -xylosidase activity found was induced by xylan in Su. smithiae UFMG-HM-80.1 (0.76 U mL-1). Upon induction of D-xylose, the highest level of intracellular -xylosidase activity was detected by Cr. laurentii UFMG-HB-48 at 50°C, and, when measured at 30°C, the highest intracellular -xylosidase activity was found by Candida tropicalis UFMG-HB-93a. This study identified a set of yeasts with xylan-hydrolyzing capacity, revealing the metabolic strategies for the assimilation of their degradation products (xylo- oligosaccharides and xylose). In this work, the yeast-like fungus Aureobasidium pullulans, known as a source of xylanolytic enzymes with high specific activity, has been used as a reference for the enzymatic assays. It was found that the strain A. pullulans UFMG-Bro-53 showed higher volumetric activity at 30°C, similar to that obtained at 50°C (21.7 U mL-1) and three times greater than the activity obtained at 30°C by A. pullulans Y-2311, a strain well characterized in the literature. The xylanolytic activities obtained with A. pullulans UFMG-Bro-53 were significantly higher than those obtained with the yeasts previously identified in this work. Therefore, this strain (A. pullulans UFMG-Bro-53) was further studied, particularly in the characterization of its xylanolytic potential. When induced by xylan, the extracellular extract of this strain showed a maximum xylanase activity at 40°C and pH 4.0-5.0. The most abundant fraction of the extract has an apparent molecular mass of 22kDa. Among the purified fractions showing xylanase activity, this fraction shows an optimal activity at 40°C and pH 4.0-5.0. The extracellular extract of A. pullulans UFMG-Bro-53 showed xylanolytic specificity without cellulase or protease activities. Thus, this extract has been used in biotechnological processes, as in the hydrolysis of hemicellulose (xylan) in lignocellulosic materials and baking processes. The extracellular extract of A. pullulans UFMG-Bro-53 was evaluated in the hydrolysis of xylan and pretreated wheat straw and in simultaneous saccharification and fermentation (SSF) for the production of lignocellulosic ethanol. The extract was more efficient in the hydrolysis of xylan and wheat straw when compared to the commercial hemicellulase HTec2, generating D-xylose yields five times higher with wheat straw at 10% solids content. When used in supplementation of the commercial cellulase CTec2, it reached a higher enzymatic hydrolysis yield than when using CTec2 without supplementation. In SSF experiments, at 35°C, using Beechwood xylan as substrate and yeast strain S. passalidarum UFMG-HMD-1.1, XBro extract acted efficiently in the degradation of xylan to xylose with a higher yield (estimated at 66%) compared to the hydrolysis performed separately (16%), reaching an ethanol concentration of 6.6 g L-1,corresponding to 0.2 g g-1 ethanol yield. When compared with the commercial hexose-fermenting yeast Saccharomyces cerevisiae (Ethanol Red), Spathaspora passalidarum UFMG-HMD-1.1, a hexose and pentose-fermenting yeast, promoted an improvement in the conversion yield of wheat straw into ethanol when supplemented with the XBro extract, once the xylanases present in the extract provided more xylose, which, in turn, could be fermented by S. passalidarum. The use of xylanolytic enzymes present in the extracellular extract of A. pullulans UFMG -Bro -53 was further analyzed in a baking process. In a dosage of 120 U (xylanase) kg-1 of flour of a baking mixture for bun production, it led to an increase in volume and a reduction in crumb firmness of bread, when compared to the negative control, i.e. without addition of enzymes, but reaching similar values to those obtained with the commercial xylanase Spring Mono, a glycoside hydrolase from family GH11. However, at dosages higher than 120 U kg-1 of flour, the extract appears to have a negative effect in bread properties, which seems to be associated with the fact that this extract contains family GH10 xylanases, in addition to family GH11 xylanases.