关键词: 甜菊, β-葡萄糖苷酶, 提取, 对硝基苯酚-β-D葡萄糖苷, 酶活性

Abstract:

The paper aims to study the role of stevia β-glucosidase in catabolism of steviol glycosides, and to lay a theoretical foundation for breeding of increasing steviol glycosides content by inhibiting its degradation in future. Stevia rebaudiana was used as test material in this study, and we firstly optimized the methods of extraction and determination of enzyme activity of β- glucosidase from stevia leaves, and then preliminarily analyzed the enzyme properties further. The results indicated that: the enzyme solution, which was extracted by 50 mmol/L phosphate buffer with pH 7.0, and added equivalent PVP during grinding, then salted out with 20%-40% saturation ammonium sulfate by the method of ammonium sulfate grade precipitation, had the highest enzyme activity; using pNPG as the substrate to determine enzyme activity, the best method was reacted at 37℃ for 60 min; and the results of enzyme property analysis showed that: stevia β-glucosidase possessed sound thermal stability and pH stability, its optimum temperature was 30℃, the optimum pH value was 7.0; but in 4℃, the enzyme activity gradually reduced, and was only 26.4% up to 26 d; among 9 common metal ions, Mn ²⁺ and Fe ²⁺ had significant activation effect on the enzyme, while Cu ²⁺ and Hg ²⁺ had obvious inhibition effect.

Key words: Stevia rebaudiana, β-glucosidase, extraction, 4-Nitrophenyl β-D-glucopyranoside, enzyme activity