Maltase protein of <i>Ogataea </i>(<i>Hansenula</i>) <i>polymorpha </i>is a counterpart to resurrected ancestor protein ancMALS of yeast maltases and isomaltases

Saccharomyces cerevisiae maltases use maltose, maltulose, turanose and maltotriose as substrates, isomaltases use isomaltose, α‐methylglucoside and palatinose and both use sucrose. These enzymes are hypothesized to have evolved from a promiscuous α‐glucosidase ancMALS through duplication and mutation of the genes. We studied substrate specificity of the maltase protein MAL1 from an earlier diverged yeast, Ogataea polymorpha (Op), in the light of this hypothesis. MAL1 has extended substrate specificity and its properties are strikingly similar to those of resurrected ancMALS. Moreover, amino acids considered to determine selective substrate binding are highly conserved between Op MAL1 and ancMALS. Op MAL1 represents an α‐glucosidase in which both maltase and isomaltase activities are well optimized in a single enzyme. Substitution of Thr200 (corresponds to Val216 in S. cerevisiae isomaltase IMA1) with Val in MAL1 drastically reduced the hydrolysis of maltose‐like substrates (α‐1,4‐glucosides), confirming the requirement of Thr at the respective position for this function. Differential scanning fluorimetry (DSF) of the catalytically inactive mutant Asp199Ala of MAL1 in the presence of its substrates and selected monosaccharides suggested that the substrate‐binding pocket of MAL1 has three subsites (–1, +1 and +2) and that binding is strongest at the –1 subsite. The DSF assay results were in good accordance with affinity (K (m)) and inhibition (K (i)) data of the enzyme for tested substrates, indicating the power of the method to predict substrate binding. Deletion of either the maltase (MAL1) or α‐glucoside permease (MAL2) gene in Op abolished the growth of yeast on MAL1 substrates, confirming the requirement of both proteins for usage of these sugars. © 2016 The Authors. Yeast published by John Wiley & Sons, Ltd

Preparation of onion-like multilayered particles comprising mainly poly(iso-butyl methacrylate)-block-polystyrene by two-step AGET ATRP

The role of dietary fiber in supporting healthy gut microbiota and overall well-being of the host has been revealed in several studies. Here, we show the effect of a bacterial polyfructan levan on the growth dynamics and metabolism of fecal microbiota in vitro by using isothermal microcalorimetry. Eleven fecal samples from healthy donors were incubated in phosphate-buffered defined medium with or without levan supplementation and varying presence of amino acids. The generation of heat, changes in pH and microbiota composition, concentrations of produced and consumed metabolites during the growth were determined. The composition of fecal microbiota and profile of metabolites changed in response to substrate (levan and amino acids) availability. The main products of levan metabolism were acetic, lactic, butyric, propionic and succinic acids and carbon dioxide. Associated growth of levan-degrading (e.g. Bacteroides) and butyric acid-producing (e.g. Faecalibacterium) taxa was observed in levan-supplemented media. The study shows that the capacity of levan and possibly also other dietary fibers/prebiotics to modulate the composition and function of colon microbiota can be predicted by using isothermal microcalorimetry of fecal samples linked to metabolite and consortia analyses

Quorum Sensing and Expression of Virulence in Pectobacteria

Quorum sensing (QS) is a population density-dependent regulatory mechanism in which gene expression is coupled to the accumulation of a chemical signaling molecule. QS systems are widespread among the plant soft-rotting bacteria. In Pectobacterium carotovorum, at least two QS systems exist being specified by the nature of chemical signals involved. QS in Pectobacterium carotovorum uses N-acylhomoserine lactone (AHL) based, as well as autoinducer-2 (AI-2) dependent signaling systems. This review will address the importance of the QS in production of virulence factors and interaction of QS with other regulatory systems in Pectobacterium carotovorum

Shelterwood systems in Estonian forests – review of studies and establishment of Järvselja study plot

Turberaie meetod on metsakasvatuslik võte, mille käigus raiutakse mets korduvate raiejärkudega. Nii säilitatakse osa vanast metsast, mis omakorda pakub turvet looduslikule uuendusele. Antud töös analüüsiti uuringute abil turberaiete kasutamist ning kujunemist Eestis. Kirjeldatakse erinevaid turberaieviise ning hinnatakse turberaietega kaasnevaid tegureid. Ühtlasi rajati turberaiete analüüsimiseks katseala. Leiti, et turberaied on olnud Eesti metsanduses kasutusel juba pikka aega. Kuid alles peale Teist maailmasõida hakkas selguma, et turberaiete kasutamine ei ole alati igal pool õigustatud. Kasutatud on turberaiete analoogseid meetodeid ning nende erinevaid kombinatsioone. Eelkõige tuleb turberaie valikul lähtuda puuliigist ning kasvukoha omadustest. Iga puistu on veidi erinev ning juhinduma peab puistu kohapealsest olukorrast lähtuvalt. Võib järeldada, et turberaied pakuvad metsa uuendamisel lageraie kõrval olulisi hüvesid ka tulevikus.Shelterwood method is a silvicultural technique by which a whole forest is cut in a series of partial cuttings. In this way some part of the old forest is maintained and in turns it offers shelter effect to natural regeneration. This paper analyzes the development and use of shelterwood cutting in Estonia by a number of researches. It describes a variety of shelterwood tehniques and evaluates shelterwood associated factors. There is also founded a test area to analyze shelterwood cutting. It was found that shelterwood cutting has been used in Estonian forestry for a long time. But it was after the Second World War when it began apparent that shelterwood cutting is not always justified in some places. Some methods that are similar to shelterwood and their various combinations has been used. In particular, choosing the appropriate shelterwood method should be according to tree species and site characteristics. Each stand is a bit different from another and therefore it must be guided according to sites local situation. It can be concluded that shelterwood cuttings are continuing to offer important benefits in the future as well

Genome Mining of Non-Conventional Yeasts: Search and Analysis of MAL 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 MAL cluster of a non-conventional methylotrophic yeast Ogataea (Hansenula) polymorpha containing genes for &alpha;-glucosidase MAL1, &alpha;-glucoside permease MAL2 and two hypothetical transcriptional activators. Using genome mining, we detected MAL clusters of varied number, position and composition in many other maltose-assimilating non-conventional yeasts from different phylogenetic groups. The highest number of MAL clusters was detected in Lipomyces starkeyi while no MAL clusters were found in Schizosaccharomyces pombe and Blastobotrys adeninivorans. Phylograms of &alpha;-glucosidases and &alpha;-glucoside transporters of yeasts agreed with phylogenesis of the respective yeast species. Substrate specificity of unstudied &alpha;-glucosidases was predicted from protein sequence analysis. Specific activities of Scheffersomycesstipitis &alpha;-glucosidases MAL7, MAL8, and MAL9 heterologously expressed in Escherichia coli confirmed the correctness of the prediction&mdash;these proteins were verified promiscuous maltase-isomaltases. &alpha;-Glucosidases of earlier diverged yeasts L. starkeyi, B. adeninivorans and S. pombe showed sequence relatedness with &alpha;-glucosidases of filamentous fungi and bacilli

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 ap-plicable 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 deter-mined. 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.This article belongs to the Special Issue: Fungal Enzymes 2021</p

Fructan Enzymes in Microbes and Plants : Structure, Function, and Product Formation

Fructans—fructose-based oligo- or polysaccharides—are de novo synthesized from sucrose by transfructosylating enzymes of microorganisms and plants. Fructan-producing enzymes belong to glycoside hydrolase families 32 and 68. Levansucrases, inulosucrases, some invertases, sucrose:sucrose 1-fructosyl transferases, fructan:fructan 1-fructosyl transferases, sucrose:fructan 6-fructosyl transferases and β-fructofuranosidases synthesize independently or in cascades a wide variety of fructose-containing saccharides. Fructans from different sources often differ in the linkage between the monosaccharide residues and the degree of polymerization. This chapter reviews the literature on fructan-metabolizing enzymes from bacteria, haloarchaea, yeasts, filamentous fungi, mono- and dicot plants. The focus is mostly on the product spectra of the enzymes, structure-function relationships that determine substrate specificities, and on the enzymatic production of fructans or fructo-oligosaccharides that at later stages may lead to practical applications

High-Throughput Assay of Levansucrase Variants in Search of Feasible Catalysts for the Synthesis of Fructooligosaccharides and Levan

Bacterial levansucrases polymerize fructose residues of sucrose to β-2,6 linked fructans—fructooligosaccharides (FOS) and levan. While β-2,1-linked FOS are widely recognized as prebiotics, the health-related effects of β-2,6 linked FOS are scarcely studied as they are not commercially available. Levansucrase Lsc3 (Lsc-3) of Pseudomonas syringae pv. tomato has very high catalytic activity and stability making it a promising biotechnological catalyst for FOS and levan synthesis. In this study we evaluate feasibility of several high-throughput methods for screening and preliminary characterization of levansucrases using 36 Lsc3 mutants as a test panel. Heterologously expressed and purified His-tagged levansucrase variants were studied for: (1) sucrose-splitting activity; (2) FOS production; (3) ability and kinetics of levan synthesis; (4) thermostability in a Thermofluor assay. Importantly, we show that sucrose-splitting activity as well as the ability to produce FOS can both be evaluated using permeabilized levansucrase-expressing E. coli transformants as catalysts. For the first time we demonstrate the key importance of Trp109, His113, Glu146 and Glu236 for the catalysis of Lsc3. Cost-effective and high-throughput methods presented here are applicable not only in the levansucrase assay, but have a potential to be adapted for high-throughput (automated) study of other enzymes

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 &alpha;-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, &alpha;-methylglucoside) were not, confirming that BaAG2 is a maltase. BaAG2 was competitively inhibited by a diabetes drug acarbose (Ki = 0.8 &micro;M) and Tris (Ki = 70.5 &micro;M). BaAG2 was competitively inhibited also by isomaltose-like sugars and a hydrolysis product&mdash;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