Study Of Sucrose-fructosase

This thesis includes the researches on production, purification and properties of sucrose-fructosase, a fructooligosaccharide-producing enzyme from Aspergillus niger HNCO-8008. Fructooligosaccharides(FOS) have become of major interest in recent years because of their favorable functional properties such as low calorie and noncariogenic sweeteners, improvement of the intestinal bifidus bacteria, relief of constipation, decrease of total cholesterol and lipid in the serum, and promotion of animal growth.The main work and results are as follows :1 Selection of microorganisms and optimization of culture conditionsAn Aspergillus niger HNCO-8008 producing sucrose-fructosase was selected as the most suitable strain for FOS production from 8 strains. It was found that sucrose was the best carbon source for both cell growth and enzyme activity. The fermentation conditions were optimized by MRCO (Modified Random-Centroid Optimization Program), the results were sucrose 49.65%, K₂HPO₄ 1.089%, MgSO₄.7H₂O 0.134%, NaNO₃ 0.991%, Yeast extract 3.5%, initial pH8.3. The enzyme activity was 90.86u/ml after 48 hours incubation at 30 ℃.2 Purification of sucrose-fructosaseThe sucrose-fructosase from the cells and culture of HNCO-8008 was purified by (NH₄)₂SO₄ fractional precipitation, sephadex G-200 gel filtration and DEAE-Bio-Gel anion-exchange chromatographies. The molecular mass was estimated to be 59.5kDa by analytical gel filtration and 59kDa by SDS-PAGE(SDS-polyacrylamide gel electrophoresis). It appearred, therefore, to be composed of a single subunit. The enzyme was identified as a glycoprotein, the...