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

Genome of an early-diverged yeast () () encodes 88 glycoside hydrolases (GHs) including two α-glucosidases of GH13 family. One of those, the -encoded protein (AG2; 581 aa) was overexpressed in , 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 AG2, whereas isomaltose and isomaltose-like substrates (palatinose, α-methylglucoside) were not, confirming that AG2 is a maltase.

Genome Mining of Non-Conventional Yeasts: Search and Analysis of Clusters and Proteins.

Genomic clustering of functionally related genes is rare in yeasts and other eukaryotes with only few examples available. Here, we summarize our data on a nontelomeric cluster of a non-conventional methylotrophic yeast () containing genes for α-glucosidase MAL1, α-glucoside permease MAL2 and two hypothetical transcriptional activators. Using genome mining, we detected clusters of varied number, position and composition in many other maltose-assimilating non-conventional yeasts from different phylogenetic groups.

Thermostability Measurement of an α-Glucosidase Using a Classical Activity-based Assay and a Novel Thermofluor Method.

α-glucosidases (including maltases and isomaltases) are enzymes which release glucose from a set of α-glucosidic substrates. Their catalytic activity, substrate specificity and thermostability can be assayed using this trait. Thermostability of proteins can also be determined using a high-throughput differential scanning fluorometry method, also named Thermofluor.

A Highly Active Endo-Levanase BT1760 of a Dominant Mammalian Gut Commensal Bacteroides thetaiotaomicron Cleaves Not Only Various Bacterial Levans, but Also Levan of Timothy Grass.

Bacteroides thetaiotaomicron, an abundant commensal of the human gut, degrades numerous complex carbohydrates. Recently, it was reported to grow on a β-2,6-linked polyfructan levan produced by Zymomonas mobilis degrading the polymer into fructooligosaccharides (FOS) with a cell surface bound endo-levanase BT1760. The FOS are consumed by B.

Repression vs. activation of MOX, FMD, MPP1 and MAL1 promoters by sugars in Hansenula polymorpha: the outcome depends on cell's ability to phosphorylate sugar.

A high-throughput approach was used to assess the effect of mono- and disaccharides on MOX, FMD, MPP1 and MAL1 promoters in Hansenula polymorpha. Site-specifically designed strains deficient for (1) hexokinase, (2) hexokinase and glucokinase, (3) maltose permease or (4) maltase were used as hosts for reporter plasmids in which β-glucuronidase (Gus) expression was controlled by these promoters. The reporter strains were grown on agar plates containing varied carbon sources and Gus activity was measured in permeabilized cells on microtitre plates.

Further study of the Hansenula polymorpha MAL locus: characterization of the alpha-glucoside permease encoded by the HpMAL2 gene.

The HpMAL2 gene of the MAL gene cluster of Hansenula polymorpha codes for a permease similar to yeast maltose and alpha-glucoside transporters. Genomic disruption of HpMAL2 resulted in an inability of cells to grow on maltose, sucrose, trehalose, maltotriose and turanose, as well as a lack of p-nitrophenyl-alpha-D-glucopyranoside (PNPG) transport. PNPG uptake was competitively inhibited by all these substrates, with Ki values between 0.

Clustering of MAL genes in Hansenula polymorpha: cloning of the maltose permease gene and expression from the divergent intergenic region between the maltose permease and maltase genes.

Hansenula polymorpha uses maltase to grow on maltose and sucrose. Inspection of genomic clones of H. polymorpha showed that the maltase gene HPMAL1 is clustered with genes corresponding to Saccharomyces cerevisiae maltose permeases and MAL activator genes orthologues.

Regulation of the Hansenula polymorpha maltase gene promoter in H. polymorpha and Saccharomyces cerevisiae1.

Hansenula polymorpha is an exception among methylotrophic yeasts because it can grow on the disaccharides maltose and sucrose. We disrupted the maltase gene (HPMAL1) in H. polymorpha 201 using homologous recombination.