The α-glucosidase encoded by the aglA gene of Aspergillus niger is a secreted enzyme belonging to family 31 of glycoside hydrolases. This enzyme has a retaining mechanism of action and displays transglycosylating activity that makes it amenable to be used for the synthesis of isomaltooligosaccharides (IMOs). We have expressed the aglA gene in Saccharomyces cerevisiae under control of a galactose-inducible promoter. Recombinant yeast cells expressing the aglA gene produced extracellular α-glucosidase activity about half of which appeared cell bound whereas the other half was released into the culture medium. With maltose as the substrate, panose is the main transglycosylation product after 8 h of incubation, whereas isomaltose is predominant after 24 h. Isomaltose also becomes predominant at shorter times if a mixture of maltose and glucose is used instead of maltose. To facilitate IMO production, we have designed a procedure by which yeast cells can be used directly as the catalytic agent. For this purpose, we expressed in S. cerevisiae gene constructs in which the aglA gene is fused to glycosylphosphatidylinositol anchor sequences, from the yeast SED1 gene, that determine the covalent binding of the hybrid protein to the cell membrane. The resulting hybrid enzymes were stably attached to the cell surface. The cells from cultures of recombinant yeast strains expressing aglA-SED1 constructions can be used to produce IMOs in successive batches.

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Isomaltooligosaccharides (IMOS) are sugars with health promoting properties that make them relevant for the pharmaceutical and food industries. IMOS have ample chemical diversity achieved by different α-glucosidic linkages and polymerization degrees, forming linear, branched and cyclic structures. Enzymatic synthesis of these compounds can be carried out by glycoside hydrolases (GHs) with transglycosylating activity. Different substrates are used for the synthesis: combinations of disaccharides and monosaccharides, or polymeric carbohydrates such as starch or dextran, which are converted to IMOS by a combination of hydrolysis and transglucosylation. In this review, the structural features of different enzyme families (GH31, GH13, GH70, GH57 and GH66) involved in IMOS synthesis are analysed. Focus is placed on structural traits that affect substrate and product specificity, and on the relative efficiency of transglucosylation and hydrolysis. Information resulting from site-directed mutagenesis and sequence alignments complements structural data to understand the role of specific residues in the performance of the enzymes. Altogether, these studies provide a frame of knowledge which may be used to design new enzymes with improved properties

We describe a procedure by which filter paper is digested with a cellulolytic enzyme preparation, obtained from Trichoderma reesei cultivated under solid state fermentation conditions and then fermented by a recombinant Saccharomyces cerevisiae strain. The yeast strain produces a β-glucosidase encoded by the BGL1 gene from Saccharomycopsis fibuligera that quantitatively and qualitatively complements the limitations that the Trichoderma enzyme complex shows for this particular activity. The supplemental β-glucosidase activity fuels the progression of cellulose hydrolysis and fermentation by decreasing the inhibitory effects caused by the accumulation of cellobiose and glucose. Fermentation of filter paper by this procedure yields ethanol concentrations above 70 g/L.

Fermentation of agave sap, also known as exudate, has become an ancestral practice throughout Ecuadorian Andean. In Cayambe, located in this region, grows Agave americana L., which is recollected, and its sap is fermented. The agave-based fermented beverage, locally named "tzawar mishki", exhibits variable features, mainly ethanol concentration. In this work, fermentation conditions of agave sap were studied to enhance ethanol yield. Two thermal treatments for raw exudate were evaluated, pasteurization at boiling point for 30 minutes and sterilization at 121°C for 15 minutes; fermentation temperature, 30°C and room (around 18°C); and two yeast strains. Thermal pretreatments have a positive impact on reducing sugars and sucrose concentration. In the first case, an increase of 76 % and 30 % has been reported, while sucrose concentration quadrupled and doubled in pasteurized and sterilized samples, respectively. The highest ethanol concentration (63,31 g/L) and the best yield (66,21 %) were accomplished through agave sap pasteurized and fermented for 96 hours at 30°C. Negligible differences have been evidenced in ethanol and other volatile compounds content between the two yeast strains evaluated. Keywords: agave sap, ethanol, fermentation, fermentable sugars, yeast