| Glucansucrase enzymes from Glycoside-Hydrolase family 70 are able to synthesize α-glucan polymers from sucrose in which the growing glucan chain is used as acceptor, and oligosaccharides in which oligosaccharides are used as acceptor. Oligosaccharides catalytically synthesized by glucansucrases enzymes can be a potential source of new prebiotics. The oligosaccharides are neither hydrolyzed by mammalian digestive enzymes nor absorbed from the gastrointestinal tract by the host animal. Consequently, they can only be fermented in the gastrointestinal tract, which ―beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improves host healththeir. They are increasingly demanded within the food and health industries. This study focused on glucansucrase enzyme from Lactobacillus reuteri SK24.003, including purification, characterisation and application of the enzyme.Firstly, after culturing of L. reuteri SK24.003, glucansucrase enzyme from L. reuteri SK24.003 was isolated and purified. Enzyme was effectively extracted by treatment with 8 mol/L urea and the crude extract was purified by freeze-drying, DEAE-FF ion exchange chromatography and Sepharose CL-6B gel chromatography. The final specific activity of enzyme was 1.27 U/mg protein form with 8.64-fold purification. The relative molecular mass of purified enzyme was 1.66×105 as evaluated by both gel chromatography and SDS-PAGE.Secondly, the characterisation of purified glucansucrase were investigated. The activity of glucansucrase enzyme showed the optimum activity at temperature 30-35 oC and pH 5.0-5.5, respectively. The double-charge ions including Mg2+, Mn2+, Ni2+, Co2+, Ca2+, Fe2+ and Zn2+ activated the enzyme activities and Ca2+ ion highly stimulated the activity by approximately 4 times. The enzyme activity induced by Cu2+, Al3+, Fe3+ and EDTA. The purified enzyme hydrolyzed sucrose only. The Km and Vmax of purified enzyme for sucrose were calculated to be 3.71 mmol/L and 0.81 μmol/(min·mg), respectively.Thirdly, to gain insight into the products specificity of the glucansucrase enzyme and the mechanism of oligosaccharides formation, incubations with sucrose and maltose were study. The effects of synthesis conditions on the formation of transfer products were evaluated, including ratio of donor/acceptor, concentration of substrates and enzyme concentration. The decrease of maltose which lower than sucrose resulted in more forming of DP3 while less forming of DP4 and DP5. The optimum enzyme concentration for synthesize oligosaccharides was 6 U/g sucrose. The oligosaccharides products were isolated via size-exclusion chromatography and analyzed. Oligosaccharides products were composed of isomaltose,panose, maltotriose, maltotetraose, tetrasaccharide with an α-1,4/α-1,6 alternating and pentasaccharide products with an α-1,4/α-1,6 alternating. A five-step-six-parameter model of glucansucrase from L. reuteri SK24.003 with sucrose and maltose as substrate was developed using 1stOpt software. Simulated results from developed model matched exceeding well with experimental results.Finally, in vitro fermentation of glucansucrase-derived oligosaccharides was carried out with human fecal cultures, which fructooligosaccharide95, galactooligosaccharide57 and long chain inulin were used as reference carbohydrates. Changes in bacterial population, pH and SCFA were evaluated in 0、5、10、24 and 48 h. A significant decrease in pH and increase in OD600 were observed for fecal microbiota exposed to oligosaccharides which was no significant different with galactooligosaccharide57. Short-chain fatty acid production of oligosaccharides were higher than other reference carbohydrates. Butyric acid production of oligosaccharides was no significant different from galactooligosaccharide57 and fructooligosaccharide95. The oligosaccharides are a potential source of new prebiotic. It’s more accessible and can be utilized to produce more SCFAs than reference carbohydrates. |
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