Levaansukraasi Lsc3 ja endo-levanaasi BT1760 iseloomustamine ja rakendatavus uudsete prebiootikumide tootmises

Väitekirja elektrooniline versioon ei sisalda publikatsiooneToidus sisalduvatest süsivesikutest on kiudained inimese soolebakteritele kõige sobilikumad. Need toimivad prebiootikumidena: jõuavad seedumata jämesoolde ja lagundatakse inimesele kasulike bakterite, nt bifidobakterite ja laktobatsillide toimel. Prebiootikume lisatakse näiteks imikute piimasegudele ja müüakse toidulisandina poes ja apteegis. Populaarseimad neist on fruktoosi polümeer inuliin ja selle hüdrolüüsil saadud frukto-oligosahhariidid (FOS). Sigurist eraldatud inuliini lubatakse kasutada toidus 2002. aastast. Lisaks inuliinile on looduses olemas ka teise sidemetüübiga fruktoosi polümeeri – levaani. Levaani sünteesivad peamiselt bakterid, kuid ka mõned taimed. Levaanil ja levaani-tüüpi FOS-l on näidatud tugevat prebiootilist toimet, kuid tootmiskulude kõrge hinna tõttu on neid veel vähe uuritud. Antud doktoriväitekiri keskendub kahele biotehnoloogiliselt olulisele bakteriaalsele ensüümile: levaansukraasile ja endo-levanaasile, mille abil on võimalik levaani-tüüpi fruktaane toota tavalisest lauasuhkrust. Näitasime, et taimepatogeeni Pseudomonas syringae levaansukraas Lsc3 on üks efektiivsemaid levaansukraase. Ta sünteesib sahharoosist produktide segu: tekib inuliini- ja levaani-tüüpi FOS-e ning ka levaani. Ebasoovitavaks kõrvalproduktiks on suur kogus glükoosi, mis takistab FOS-ide segu kasutamist prebiootikumina ilma eelneva puhastuseta. Levaani ensümaatilise hüdrolüüsiga on võimalik toota levaani-tüüpi FOS-e nende bioloogiliste efektide uurimiseks. Levaani lagundavaks ensüümiks sobib hästi inimese jämesoolebakteri Bacteroides thetaiotaomicron endo-levanaas BT1760. Näitasin, et BT1760 ’tükeldas’ efektiivselt nii taimset kui ka bakteriaalseid levaane. Endo-levanaasi 3D struktuur näitas, et ensüümi substraaditasku on sügava kausi kujuline ning et pikk levaaniahel peab painduma kausi põhja, et oleks võimalik esimese lõike tegemine.Diet containing a sufficient amount of food fibre acts prebiotically – supports the growth of beneficial probiotic bacteria. Prebiotics are functional food ingredients, which pass the stomach unchanged and in the gut serve as energy and carbon source for probiotic bacteria such as bifidobacteria and lactobacilli. The most popular currently available prebiotics are inulin – a polymeric fructan extracted from chicory roots, and fructo-oligosaccharide (FOS) preparations produced from inulin by enzymatic hydrolysis. There is also another natural fructan – levan – which is synthesized mostly by bacteria. Levan and respective FOS have shown even higher prebiotic effect than those of inulin, but high production cost of these preparations hinders their study and utilization as a prebiotic supplement. Our workgroup contributes extensively to synthesis and development of fructose-based novel prebiotics, focusing mainly on levan-type fructans. For that, we have been using two enzymatic approaches: i) synthesis of levan-type fructans using a levansucrase, and ii) hydrolysis of levan into FOS using an endo-levanase. Levansucrase Lsc3 from a plant-associated bacterium Pseudomonas syringae pv. tomato was shown by us as a highly active and stable catalyst which makes it a perfect candidate for levan synthesis. Along with levan, levansucrase produces also FOS. Unfortunately, the mixture of FOS is full of glucose as an obligatory side-product of levansucrase reaction, and it contains both, inulin- and levan-type FOS. Isolation of levan-type FOS from this mixture is complicated. We showed that a feasible way for levan-type FOS production is the hydrolysis of polymeric levan using the endo-levanase BT1760 from a human gut commensal Bacteroides thetaiotaomicron. The endo-levanase was able to hydrolyse every tested levan, preferring levan of plant origin. From the crystal structure analysis of BT1760, we concluded that levan chain should be bent into the substrate-binding pocket of the enzyme to enable the hydrolysis at the bottom of the cavity.https://www.ester.ee/record=b5258768~S

A hyperpromiscuous antitoxin protein domain for the neutralization of diverse toxin domains

Toxin–antitoxin (TA) gene pairs are ubiquitous in microbial chromosomal genomes and plasmids as well as temperate bacteriophages. They act as regulatory switches, with the toxin limiting the growth of bacteria and archaea by compromising diverse essential cellular targets and the antitoxin counteracting the toxic effect. To uncover previously uncharted TA diversity across microbes and bacteriophages, we analyzed the conservation of genomic neighborhoods using our computational tool FlaGs (for flanking genes), which allows high-throughput detection of TA-like operons. Focusing on the widespread but poorly experimentally characterized antitoxin domain DUF4065, our in silico analyses indicated that DUF4065-containing proteins serve as broadly distributed antitoxin components in putative TA-like operons with dozens of different toxic domains with multiple different folds. Given the versatility of DUF4065, we have named the domain Panacea (and proteins containing the domain, PanA) after the Greek goddess of universal remedy. We have experimentally validated nine PanA-neutralized TA pairs. While the majority of validated PanA-neutralized toxins act as translation inhibitors or membrane disruptors, a putative nucleotide cyclase toxin from a Burkholderia prophage compromises transcription and translation as well as inducing RelA-dependent accumulation of the nucleotide alarmone (p)ppGpp. We find that Panacea-containing antitoxins form a complex with their diverse cognate toxins, characteristic of the direct neutralization mechanisms employed by Type II TA systems. Finally, through directed evolution, we have selected PanA variants that can neutralize noncognate TA toxins, thus experimentally demonstrating the evolutionary plasticity of this hyperpromiscuous antitoxin domain

Porphyromonas gingivalis fimbrial protein Mfa5 contains a von Willebrand factor domain and an intramolecular isopeptide

The Gram-negative bacterium Porphyromonas gingivalis is a secondary colonizer of the oral biofilm and is involved in the onset and progression of periodontitis. Its fimbriae, of type-V, are important for attachment to other microorganisms in the biofilm and for adhesion to host cells. The fimbriae are assembled from five proteins encoded by the mfa1 operon, of which Mfa5 is one of the ancillary tip proteins. Here we report the X-ray structure of the N-terminal half of Mfa5, which reveals a von Willebrand factor domain and two IgG-like domains. One of the IgG-like domains is stabilized by an intramolecular isopeptide bond, which is the first such bond observed in a Gram-negative bacterium. These features make Mfa5 structurally more related to streptococcal adhesins than to the other P. gingivalis Mfa proteins. The structure reported here indicates that horizontal gene transfer has occurred among the bacteria within the oral biofilm

Innovationskapacitet för att leda och organisera hållbar samhällsutveckling - erfarenheter och rekommendationer från innovationsplattformarna

Projektet Nationell samverkan mellan innovationsplattformar för hållbara attraktiva städer har sedan november 2016 arbetat med kunskapsutbyte och lärande mellan de sex städerna Borås, Göteborg, Kiruna, Lund, Malmö och Stockholm som ingår i Vinnovasatsningen “Utveckling av innovationsplattformar för hållbara attraktiva städer”. Alla städer har, på lite olika sätt, tagit sig an utmaningen att bygga innovationskapacitet och hitta vägar runt hinder för innovation för hållbar stadsutveckling.Tillsammans har städerna utvecklat unik kunskap om lokala samhällsutmaningars komplexitet och på vilket sätt innovationsarbete kan främja en hållbar utveckling. Fokus för arbetet har varit gränsöverskridande samarbete och på vilket sätt gränsgångare, ledarskap, organisationskultur och stödjande strukturer kan bygga kapacitet och mobilisera en omställning för hållbar utveckling. Med andra ord hur man kan utveckla innovationskapacitet och innovationsledning för hållbar samhällsutveckling i städer.Redan i halvtidsrapporten Leda och organisera innovation för hållbara städer och samhällen -erfarenheter från innovationsplattformarna 2017, redovisades viktiga erfarenheter med konkreta exempel från de olika städernas innovationsplattformar. I rapporten beskrivs också några av de samhällsomvälvande hållbarhetsutmaningar som gör behovet av radikalt nya lösningar ocharbetssätt akut. Rapporten argumenterade för att många av utmaningarna är så komplexa att det behövs ett helhetsperspektiv som inkluderar samverkande aktörer från både regionala instanser, den statliga nivån och nationella och internationella nätverk, för att möjliggöra innovation och förändring på det lokala planet. Den här rapporten bygger vidare på dessa erfarenheter, med en rad slutsatser och exempel som kan användas på kommunal nivå, men den innehåller också ett förslag på en agenda för nationella och regionala aktörer.Under det senaste året, med bland annat alarmerande klimatrapporter, fortsatt ökade sociala klyftor, en extrem värmebölja som stressade samhällssystemen och politisk turbulens både nationellt och internationellt har vår övertygelse stärkts än mer; innovationsplattformar är centrala för att utifrån sina unika lokala förutsättningar utforska och utveckla lösningar för hur vi ska klara av samhällets hållbarhetsutmaningar

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

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