Synthesis, detection and quantification of inulooligosaccharides and fructooligosaccharides by extracellular and intracellular inulinase and fructosyltransferase enzymes isolated from coprophilous fungi.
Ojwach, Jeff David Okinda.
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Exploration of fungal biodiversity capable of producing fructosyltransferase and inulinase enzymes in significant amounts is crucial for the production of oligofructans. Indigenous coprophilous fungi are predominantly sustainable bioresources, harbouring novel enzymes with potential industrial and biotechnological applications. Fructosyltransferase (Ftase) and inulinase are gaining considerable attention due to their capability to synthesise biofunctional nutraceuticals with low calories and health benefits when ingested in recommended dosages. Hence, due to several health benefits associated with prebiotics, bioprospecting for coprophilous fungi as unique bioresources of fructosyltransferase and inulinase was imperative. The present study therefore focused on the collection of herbivore dung from various terrestrial habitats in KwaZulu-Natal Province, South Africa whereby sixty-one (61) indigenous coprophilous fungal strains were isolated after repeated purification to monoculture. The axenic fungal strains were identified using morpho-taxonomic keys and molecular identification by 18S rDNA sequencing where Neocosmospora spp, Trichoderma spp., Aspergillus spp and Fusarium spp. were dominant. The fungal strains were subsequently assessed for their ability to produce extracellular and intracellular Ftase and inulinase enzymes. During the preliminary screening, the culture filtrate was examined for transfructosylating and hydrolytic activity using 2,3,5-triphenyl tetrazolium chloride (TTC) as a chromogenic marker and Lugol’s iodine solution, respectively. Zones of hydrolysis on 30 fungal isolates were observed on the TTC assay plates in diameters ranging from 15 mm to 30 mm, representing high extracellular Ftase activity. The formation of clear zones following addition of iodine solution on inulin rich media indicated the presence of inulinolytic activity. Secondary screening involved DNS assays of eight (8) isolates that secreted high concentrations of Ftase while six (6) different fungal strains showed <50 % inulinase: invertase ratio. The final screening step was tertiary screening where products of biocatalysis were qualitatively detected by thin layer chromatography to visualize saccharide spots of fructooligosaccharides and inulooligosaccharides. HPLC analysis of Ftase and inulinase reaction products revealed and further confirmed that coprophilous fungi harbour fructosyltransferase and inulinase enzymes. The crude extracellular fructosyltransferase enzyme was partially purified by 9.3-fold with a yield of 7.3 % and a specific activity of 2465.5 U mg-1 after a three-step procedure involving (NH4)2SO4 fractionation, dialysis and ion exchange chromatography. The apparent molecular weight of this Ftase was estimated by SDS-PAGE to be approximately 70 kDa. Zymogram analysis under non-reducing conditions showed the enzyme migrating as a polydisperse aggregate yielding broad band of approximately 100 kDa. The enzyme further exhibited an enhanced activity at a broad pH range of 4.0 – 8.0 and optimal activity at a temperature range of 40 °C – 80 °C, while the enzyme was stable at pH 8.0 and between 40 °C – 60 °C, respectively. Under these conditions, the enzyme remained stable retaining 95 % residual activity after incubation for 6 h. The presence of metal ions such as Hg2+ and Ag2+ inhibited Ftase activity while, Ca2+, Mg2+ and K+ at 1 mM increased the enzyme activity, with stabilization observed with Na+, Zn2+ and Cu2+. With sucrose as the substrate, the enzyme kinetics fitted the Michaelis-Menten model. The Km, Vmax and kcat values were 2.076 mM, 4.717 μmole min-1, and 4.7 min-1, respectively with a catalytic efficiency of 2.265 μmole min-1. In vitro antioxidant potential of FOS by 1,1 - diphenyl-2-picryl hydroxyl (DPPH) assay, ferric reducing antioxidant power (FRAP) assay and nitric oxide (NO) radical inhibition yielded IC50 of 6.71 μg/ml, 1.76 μg/ml and IC25 of 0.27 μg/ml, respectively. Free radical scavenging and inhibition activities showed a concentration-dependent antioxidant activity with no significant differences with oligosaccharide standards (p < 0.01). However, vitamin C was significant in FRAP and NO assays. These results clearly demonstrated that an indigenous coprophilous fungus is a potential new reservoir of salient biotechnological enzymes that can be exploited for the production of prebiotics for subsequent biotechnological applications.