| Fructooligosaccharides(FOS) is a new kind of functional oligosaccharide. As an important type of prebiotics, it has been widely used in food, nutraceutical and cosmetics industries, for its many physiological functions such as promoting the proliferation of Bifidobacterium, improving intestinal microflora, preventing tooth decay, reducing blood lipid levels and so on. Producing FOS by using microorganism or the corresponding enzymes has great prospects, since it can not only improve the efficiency but also lower down the production costs. According to the previous research of our group, Aspergillus niger 6640(A. niger 6640), which could produce high concentration of FOS and tolerate high temperature of 50-60 ?C, was successfully screened. In this study, the capability of producing FOS by A. niger 6640 with high salt stress when consuming molasses as carbon source were studied; the biotransformation conditions for the production of FOS by A. niger 6640 were optimized; two different processes were applied for producing crude enzyme of β-fructofuranosidase(β-Ffase) from A. niger 6640 and the properties of crude enzyme of β-Ffase were investigated. Then, β-Ffase with electrophoretic purity was isolated and purified from A. niger 6640 and the properties of β-Ffase were investigated.According to our results, A. niger 6640 had high salt tolerance and could yield 360.25 g/L FOS by consuming molasses when the concentration of KCl was 96.35 g / L. The optimal fermentation parameters for FOS production by A. niger 6640 were found to be: pH 7.6, temperature 33 ?C, time length 40 h and sucrose mass fraction 40 %. Then the crude enzyme of β-Ffase from A. niger 6640 was obtained by filtering, washing, drying and smashing. As for the drying process, our results showed that vacuum drying process was better than vacuum freeze-drying process for the preservation of enzyme activity. As for the production of FOS by crude enzyme of β-Ffase, Na2HPO4-citric acid buffer was more appropriate than Tris-HCl buffer. Specifically, the optimal conditions for the production of FOS by crude enzyme of β-Ffase in Na2HPO4-citric acid buffer were found to be: pH 6.5, time length 16 h and β-Ffase dosage 0.19 g/3mL.The crude enzyme of β-Ffase was yield by treating A. niger 6640 with snailase to broke the cell wall, because β-Ffase was an intracellular enzyme for 92.41% β-Ffase distributed inside the cell. The active β-Ffase with electrophoretic purity was obtained after separation processes such as the anion DEAE Sepharose Fast Flow chromatography and Phenyl Sepharose CL-4B column from crude enzyme solution of β-Ffase. The results showed that 4.068 mg of active enzyme protein with an activity of 16.95 U /mg can be obtained from 1L of the β-Ffase crude enzyme solution, and that the specific β-Ffase activity of the purified enzyme increases by 67.34 folds with a recovery of 12.12 %. In addition, SDS-PAGE electrophoresis results showed that the monomer molecular mass of β-Ffase is about 75 ku, and PCR electrophoresis results showed that the β-Ffase genomic DNA consisted of 1950 bp and the monomer molecular mass of β-Ffase was about 71.5 ku. Considering these two results, we inferred that the β-Ffase isolated from A. niger 6640 was monomeric structure.The optimal conditions for β-Ffase were found to be: pH 6.6-7.6, temperature 30-40 ?C, time length 24 h and the concentration of sucrose 60 %. The results also indicated that the enzyme had high stability below 40 ?C and abundant yield of FOS at high temperature of 50-60 ?C. 0.5m mol/L Mg2+ had a strongest stimulus effect on the activity of β-Ffase. In general, Mg2+、Ca2+、Co2+、Li+ could promote the activity of β-Ffase, while Cu2+ and Fe3+ could suppress the activity of β-Ffase. |
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