Production of prebiotic oligosaccharides by novel enzymatic catalysis

(30/11/2018) Production of prebiotic oligosaccharides by novel enzymatic catalysis 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 transglycosidase 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 monosialylated HMO, 3’sialyllactose(3’SL), received particular attention in this PhD project. The BioEng state of the art transsialidase 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.