Structural Insight into a Yeast Maltase—The BaAG2 from Blastobotrys adeninivorans with Transglycosylating Activity

An early-diverged yeast, Blastobotrys (Arxula) adeninivorans (Ba), has biotechnological potential due to nutritional versatility, temperature tolerance, and production of technologically applicable enzymes. We have biochemically characterized from the Ba type strain (CBS 8244) the GH13-family maltase BaAG2 with efficient transglycosylation activity on maltose. In the current study, transglycosylation of sucrose was studied in detail. The chemical entities of sucrose-derived oligosaccharides were determined using nuclear magnetic resonance. Several potentially prebiotic oligosaccharides with α-1,1, α-1,3, α-1,4, and α-1,6 linkages were disclosed among the products. Trisaccharides isomelezitose, erlose, and theanderose, and disaccharides maltulose and trehalulose were dominant transglycosylation products. To date no structure for yeast maltase has been determined. Structures of the BaAG2 with acarbose and glucose in the active center were solved at 2.12 and 2.13 Å resolution, respectively. BaAG2 exhibited a catalytic domain with a (β/α)8-barrel fold and Asp216, Glu274, and Asp348 as the catalytic triad. The fairly wide active site cleft contained water channels mediating substrate hydrolysis. Next to the substrate-binding pocket an enlarged space for potential binding of transglycosylation acceptors was identified. The involvement of a Glu (Glu309) at subsite +2 and an Arg (Arg233) at subsite +3 in substrate binding was shown for the first time for α-glucosidases.

Characterization of a Maltase from an Early-Diverged Non-Conventional Yeast Blastobotrys adeninivorans

Genome of an early-diverged yeast Blastobotrys (Arxula) adeninivorans (Ba) encodes 88 glycoside hydrolases (GHs) including two α-glucosidases of GH13 family. One of those, the rna_ARAD1D20130g-encoded protein (BaAG2; 581 aa) was overexpressed in Escherichia coli, purified and characterized. We showed that maltose, other maltose-like substrates (maltulose, turanose, maltotriose, melezitose, malto-oligosaccharides of DP 4‒7) and sucrose were hydrolyzed by BaAG2, whereas isomaltose and isomaltose-like substrates (palatinose, α-methylglucoside) were not, confirming that BaAG2 is a maltase. BaAG2 was competitively inhibited by a diabetes drug acarbose (Ki = 0.8 µM) and Tris (Ki = 70.5 µM). BaAG2 was competitively inhibited also by isomaltose-like sugars and a hydrolysis product—glucose. At high maltose concentrations, BaAG2 exhibited transglycosylating ability producing potentially prebiotic di- and trisaccharides. Atypically for yeast maltases, a low but clearly recordable exo-hydrolytic activity on amylose, amylopectin and glycogen was detected. Saccharomyces cerevisiae maltase MAL62, studied for comparison, had only minimal ability to hydrolyze these polymers, and its transglycosylating activity was about three times lower compared to BaAG2. Sequence identity of BaAG2 with other maltases was only moderate being the highest (51%) with the maltase MalT of Aspergillus oryzae.

Flavour chemical dynamics during fermentation of kombucha tea

Main physical and chemical properties, microbial population and flavour compounds of kombucha fermentation were dynamically analyzed. The results showed: the pH values decreased but total acidity increased in kombucha fermentation broth with fermentation time; the concentrations of tea polyphenols and free amino acids firstly increased in the initial stage and then decreased till the end of fermentation; total catechins and caffine in kombucha fermentation broth were degraded progressively with fermentation time. Only 21 volatile flavour compounds were identified in the initial kombucha fermentation broth but 56 volatile flavour compounds were identified in the 10 d fermentation. The largest group was acids among which there were 22 different types of acids compounds, accounting for up to 57.21% of all volatile flavour components. Bacteria were more abundant and diverse than yeasts in kombucha fermentation broth during fermentation. We separated and identified 8 main microbial communities in kombucha fermentation broth during kombucha fermentation; 6 bacteria belonged to Gluconacetobacter saccharivorans, Acetobacter sp., Gluconacetobacter sp., Gluconacetobacter europaeus, Acetobacter aceti and Lactobacillus fermentum, and 2 yeasts belonged to Saccharomyces cerevisiae and Arxula adeninivorans. According to the production of organic acids i.e., acetic acid, we envisaged that the predominant bacterial species identified were probably Acetobacter sp. and Acetobacter aceti after 5 days fermentation.