中国农业科学Issue(2)：334-342,9.DOI:10.3864/j.issn.0578-1752.2015.02.13
大蒜蔗糖﹕蔗糖1-果糖基转移酶（1-SST）的酶学特征
Characteristics of Sucrose：Sucrose 1-Fructosyltransferase in Garlic
摘要
Abstract
[Objective] Enzymologic characteristics of sucrose: sucrose 1-fructosyltransferase (1-SST) in garlic (Allium sativum L.) was investigated to provide a basis for the preservation, processing and further quality improvements of fresh garlic.[Method]1-SST was fractional precipitated by ammonium sulfate with different saturations of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100%, successively, in order to determine the fraction of the ammonium sulfate which 1-SST is in. 1-SST catalyses in fructan biosynthesis. After that, the 1-SST was further researched on the catalytic activity under different temperatures, pH, ionic strength and substrate concentrations. Here sucrose was used as substrate. The 1-SST enzymatic activity was revealed through production of 1-kestose which was analyzed by high performance liquid chromatography-evaporation light-scattering detection (HPLC-ELSD). Chromatographic separation of 1-kestose was achieved by the Prevail Carbohydrate ES column (250 mm×4.6 mm, 5 μm) at a flow rate of 1 mL·min-1 under 20 MP . The mobile phase consisted of a combination of B water and A acetonitrile. The gradient elution was applied as followings: 75% A : 25% B at 0 min, 65% A : 35% B at 15 min, 50% A : 50% B at 30 min, 75% A : 25% B at 35 min, 75% A : 25% B at 40 min. The column temperature was held at 40℃. ELSD impactor was off and drift tube temperature was kept at 90℃. The air was used as carrier gas for ELSD detection with a flow rate of 2.5 L·min-1.[Result]Fructose, glucose, sucrose, 1-kestose, nystose, 1F-fructofuranosylnystose were separated in 20 min. Thus the HPLC-ELSD method was proved a reliable and highly sensitive method for determining the 1-kestose. As early described, 1-kestose was produced when sucrose incubated with 1-SST and used to represent the enzymatic activity of 1-SST. Only 1-kestose was produced with 50% (w/w) sucrose solution as substrate. This phenomenon indicated that the 1-SST preserved very high substrate specificity under the above experiment conditions. Garlic 1-SST mainly existed in the fraction precipitated with 30%-40% ammonium sulfate solution while exhibited very weak enzymatic activity in the fractions precipitated with 0-30%, 40%-70% ammonium sulfate solution and no 1-SST was detected in the fraction precipitated with 70%-100% ammonium sulfate. Garlic 1-SST from the fraction precipitated with 30%-40% ammonium sulfate solution was further investigated. The optimal temperature was 35℃. The enzymatic activity was decreased dramatically when the temperature reached 40℃ which indicated this enzyme is heat labile. Therefore, high temperature should be avoided for studying the garlic 1-SST. Its optimum pH value was 5, which was similar to the 1-SST from Hordeum vulgare, Cichorium intybus L., Helianthus tuberosus Colombia and Lolium rigidum, Agave americana L. This result shows that the conformation of 1-SST activity site from different plants could be similar. The enzymatic activity of 1-SST was dependent on the sucrose concentration in the range of 100-600 mg·mL-1 which was increased with the substrate concentration below 500 mg·mL-1. However, the enzyme was saturated at the high concentration of substrate (over 500 mg·mL-1). This result fits into the Michaelis-Menten Equation. For Na+, Cl- ion, 1-SST enzymatic activity was reversed with the ion strength. Michaelis constant of 1-SST was 104 mmol·L-1 at 35℃.[Conclusion] In this study, The effect of different temperatures, pH, ionic strength and substrate concentration on the 1-SST enzymatic activity was investigated. This mechanism will help to understand the change of fructan, species and content of saccharides during processing and storage.关键词
果聚糖/蔗糖﹕蔗糖果糖基转移酶/酶学特征/大蒜/1-蔗果三糖Key words
fructan/sucrose:sucrose 1-Fructosyltransferase (1-SST)/enzymologic characteristics/Allium sativum L./1-kestose引用本文复制引用
文明,卜利伟,罗紫韵,王佳伟,董芬,黄雪松..大蒜蔗糖﹕蔗糖1-果糖基转移酶（1-SST）的酶学特征[J].中国农业科学,2015,(2):334-342,9.基金项目
国家自然科学基金 （）
