Theses and dissertations (Applied Sciences)
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Item Increasing catalytic activity of a fructosyltransferase using site-directed mutagenesis(2024-05) Wang Fanzhi; Permaul, Kugen; Singh, SurenFructooligosaccharides (FOS) are naturally occurring metabolites that have a wide application in the food industry. They are one of the most well-studied prebiotics and have been used as an alternative sweetener to sucrose, as the modern diet demands healthier and calorie-reduced foods. FOS is commercially produced either by hydrolysis of inulin into inulin-type FOS or by sucrose transfructosylation into levantype FOS. The levan-type FOS are short-chain FOS and are produced under the catalysis of fructosyltransferase (FTase) or fructofuranosidase (FFase). In this study, FOS production was studied using a fructosyltransferase, SucC, which was originally isolated from Aspergillus niger and was functionally expressed in Pichia pastoris. The tertiary structure of SucC was determined by bioinformatics analysis and catalytic sites were verified and validated by wet and dry experiments where the amino acid residues D64, D194 and E271 were proved to form the catalytic triad. Three mutants, C66S, G273V, L313H were constructed aiming to improve the enzyme performance. Only the C66S mutant showed improved enzymatic activity which was 61% increase in specific activity. The other mutants, G273V and L313H, led to a complete loss of enzyme activity. By simulating saturated mutagenesis, tertiary structure alignment, and molecular docking, it was predicted that the C66S mutation could increase the hydrophilic environment surrounding the active site without visible changes in its structure. Two more amino acid residues (E296, H310) in addition to D64, D122, R193, D194, E271 in mutant C66S were predicted to be interacting with sucrose, and the binding energy changed from -3.65 to -4.14 kcal/mol. Subsequently, mutant C66S was constructed by site-directed mutagenesis and expressed in Pichia pastoris GS115. The purified mutant C66S showed improved enzymatic activity with a 61.3% increase in its specific activity. Its Km value was decreased by 13.5% while the kcat value increased by 21.6%. Its transfructosylation efficiency significantly improved during the initial reaction stages of FOS production. These results clearly revealed that the increase of hydrophilicity surrounding the active site enhanced the transfructosylating activities. Therefore, modification of the hydrophilic micro-environment surrounding the active site could be an alternative way to artificially evolve an enzyme’s catalytic efficiency.Item The prebiotic effects of amadumbe (Colocasia Esculenta) and okra (Abelmoschus esculentus) mucilage(2023) Gajadhar, Sharmista; Amonsou, Eric Oscar; Mchunu, Nokuthula PeacePrebiotics have been shown to aid in the improvement and maintenance of human health through positive manipulation of gut microbiota. Diet-induced changes in gut microbial diversity has been recognized as a factor which contributes to the rising epidemics of chronic illnesses in both developed and developing countries. Traditional crops, amadumbe (Colocasia esculenta) and okra (Abelmoschus esculentus (L.) Moench) offer nutritional security to many communities in South Africa. These crops are rich in mucilage and are presumed prebiotics. Structural composition and functional properties of polysaccharides like mucilage are suggested to influence their fermentability by gut microbiota and potential health effects. The purpose of this study was to investigate the prebiotic effects of amadumbe and okra mucilages for potential application as dietary supplements. Mucilage was extracted from amadumbe and okra by cold water extraction. Purified mucilage was obtained by Sevag method, lipid removal and thereafter dialyzed. The composition and structure of crude and purified mucilage were analyzed using Fourier transform infrared spectroscopy (FT-IR), size exclusion chromatography (SEC) and high pressure liquid chromatography (HPLC). Functional properties including water and oil holding capacity, swelling and solubility were determined. The prebiotic potential of amadumbe and okra mucilage was carried out by in- vitro fermentation using human faecal sample. Glucose was the common monosaccharide present in both amadumbe and okra mucilage. Monosaccharides present in amadumbe mucilage were arabinose, mannose and xylose, while galactose, ribose and rhamnose were the main monosaccharides present in okra mucilage. The presence of β-glucan was found to be higher 0.20 g/100 g in amadumbe mucilage than in okra mucilage 0.07 g/100 g. The resistant starch content in amadumbe mucilage was higher 4 g/100 g than in okra mucilage 0.7 g/100 g. Asparagine, proline, glutamine, and threonine were the most common amino acids found in both amadumbe and okra mucilage samples. Purified amadumbe and okra mucilage displayed the same characteristic peaks as crude amadumbe and okra mucilage in the FT-IR spectrum but at a lower intensity suggesting that purification contributed to a more stable and uniform structure. The FT-IR spectrum indicated the presence of uronic acid and hydroxyl groups which confirm the existence of carbohydrate in both amadumbe and okra mucilage. The molecular weight of crude amadumbe and okra mucilages ranged between 219 and 224 kDa while molecular weight of purified amadumbe and okra mucilage ranged between 220 and 244 kDa. The purification process was seen to improve functional properties such as the water holding capacity, swelling and solubility of mucilages. In comparison to okra mucilage, crude and purified amadumbe mucilage showed low water holding capacity 5 and 9 g/100 g and high percentage solubility 61 and 73%. Amadumbe mucilage had a slightly higher oil holding capacity 11 g/100 g in comparison to okra mucilage 10 g/100 g. During in-vitro fermentation, inulin (positive control) rapidly decreased the pH of the fermentation medium from 7.0 to 6.5, in comparison to amadumbe (7.0 to 6.7) and okra (7.0 to 6.8) mucilage. At the end of fermentation inulin had maximum gas production of 233.19 mL, followed by amadumbe mucilage 158.98 mL and okra mucilage 113.98 mL. These results suggest inulin is more easily fermented by microbes compared to amadumbe and okra mucilage. Gut microbiota analysis at phylum level showed that amadumbe mucilage stimulated the proliferation of Actinobacteria and reduced the presence of Firmicutes in comparison to okra mucilage. At species level, okra mucilage promoted the growth of Bacteroidaceae bacteroidetes, Bacteroides ovatus and Bacteroides uniformis. These species are known to assist in protection of the gut and are capable of providing nutrients to other microbial species. This suggest that amadumbe and okra mucilages are fermented differently by gut microbiota possibly due to differences in their structure and composition. This study concluded that amadumbe and okra mucilages has potential to be utilized as an emerging prebiotic in food applications or as supplements.