Production of prebiotic oligosaccharides by novel enzymatic catalysis

Abstract

For tiden bliver der forsket meget i en bestemt gruppe af prebiotiske oligosaccharider kaldet HMO’er (mælke-oligosaccharider af human oprindelse), med det formål at fremstille og tilsætte dem til modermælkserstatning. Hvor prebiotika generelt anvendes til at modulere på et individs tarmflora, er HMO’erne ekstra interessante fordi de er stærkt medvirkende til at udvikle den første tarmflora der etableres i et spædbarn. Dette sker bl.a. igennem en stimulering af væksten af Bifidobacterium infantis, som er en gavnlig bakterie. HMO’er menes, foruden at være prebiotiske, at have en række gavnlige egenskaber som, i varierende grad, kan bakkes op videnskabeligt.Da kemisk syntese af kulhydrater er besværligt er der generelt en opfattelse af at det er nødvendigt at anvende biokemisk syntese til HMO-fremstilling og in vitro enzymatisk fremstilling er en populær metode. Det var således formålet med dette PhD-projekt at omfatte så mange aspekter af enzymatisk HMO production som muligt, med det formål at identificere muligheder for at forbedre enzymer, reaktionsbetingelser og processer.Til in vitro produktion af HMO’er bruges industrielle bi-produkter ofte som substrater for at reducere produktionsomkostningerne. For at anvende disse substrater er det, til gengæld ofte nødvendigt at tilvejebringe hydrolytiske enzymer med trans-glycosidaseaktivitet eller ideelt de sjældne trans-glycosidaser. Forskningsgruppen, BioEng, har tidligere udviklet en gen-manipuleret trans-sialidase der, som en del af dette projekt, blev videreudviklet og har sat barren endnu højre for konkkurerende enzymer. For at videreudvikle på dette enzym var der enighed om at ny viden ville være en nødvendighed og at sådan viden kunne opnås hvis en ny trans-sialidase kunne identificeres. Trans-sialidaser er dog, indtil videre, kun identificeret i en enkelt slægt. Ikke desto mindre blev en ny trans-sialidase identificeret som en del af dette PhD-projekt og det nye enzym var i stand til at syntetisere 3’SL samt et nyt trans-sialyleringsprodukt, 3SL, hvis egenskaber er ukendte.Med det mål at forbedre 3’SL fremstilling blev den anvendte proces taget nøje I betragtning, så svage led kunne identificeres og forbedres. Ved PhD-projektets begyndelse udførtes oprensning af 3’SL i en tretrins proces: Ultrafiltrering efterfulgt af kolonne-oprensning og separation af eluent. Som del af dette PhD-projekt blev kolonne-oprensning og eluent-separation erstattet af en innovativ anvendelse af nano-filtrering. Endvidere blev et integreret membran-system udviklet og anvendt til produktion af 3’SL.Fra PhD-projektets begyndelse var det klart at det I fremtiden vil være målet at syntetisere større HMO strukturer. For at dette kan lade sig gøre vil det være nødvendigt at identificere enzymer og substrater til formålet. Dette PhD-projekt bidrog også med fremgang i denne henseende idet to nye β-N-acetylhexosaminidaser blev identificeret ved hjælp af et DNA-bibliotek fremstillet af oprenset DNA fra en jordprøve. Begge enzymer blev med succes anvendt til at fremstille molekyler der er forstadier til HMO-backbones og som tidligere er anvendt til at fremstille disse.A group of prebiotic oligosaccharides known as human milk oligo-saccharides (HMOs) are currently receiving a lot of attention due to the prospect of their addition to infant formula. Whereas prebiotics in general are used as mediators for modulating the gut microbiome in human individuals, HMOs play an important role in development of this organ, where it contributes to the selective growth stimulation of the beneficial microorganism Bifidobacterium infantis. The effects of HMOs are not only prebiotic and a range of beneficial effects have been postulated, with varying amounts of scientific evidence backing them up.Since chemical synthesis of carbohydrates is extremely cumbersome, it is generally accepted that HMOs must be produced biochemically and enzymatic in vitro production is a popular strategy. Thus, the purpose of this PhD project was to encompass as many of the aspects of the enzymatic production of HMOs as possible, and identify opportunities to improve the enzymes, reaction efficiencies and processes involved.For enzymatic in vitro production of HMOs, industrial side stream products are often used as substrates to reduce the final product price. However, to use these substrates it is generally necessary to identify glycosyl hydrolases with trans-glycosidase activity or ideally rare trans-glycosidases. The BioEng group has previously developed a state of the art engineered trans-sialidase used for the synthesis of sialylated HMOs. Thus, synthesis of the simple genuine mono-sialylated HMO, 3’sialyllactose(3’SL), received particular attention in this PhD project. The BioEng state of the art trans-sialidase was, during this PhD project, further mutated, raising the bar for competing enzymes. For further improvement of the current leading enzyme, it was concluded that new knowledge would be required and that such knowledge could be provided by identification of novel trans-sialidases, which have, however, only been identified in a single genus. Never the less, as part of this PhD project a novel trans-sialidase was identified which was capable of producing 3’SL and a novel trans-sialylation product, 3SL, the properties of which are unknown.With the goal to further improve 3’SL production, the process strategy underwent scrutiny and weak points were identified and improved upon. At the start of the PhD project, 3’SL was purified in a three step process including ultrafiltration, with subsequent column chromatography and removal of eluent. As part of this PhD project, an innovative nanofiltration approach eliminated the necessity for column chromatography and eluent removal. Furthermore, by moving the HMO enzymatic synthesis to a membrane reactor, an integrated membrane system strategy was constructed and proof of concept was demonstrated.From the beginning of the PhD project, it was known that future endeavors would include the synthesis of larger HMO structures, for which enzymes and substrates for HMO backbone synthesis would be required. Progress in this aspect of HMO production was also achieved during this PhD project, as two novel β-N-acetylhexosaminidases were identified through screening of metagenomic libraries. Both enzymes were successfully used to produce HMO backbone precursors, which have previously been used for HMO backbone synthesis