Extraction, Separation And Activity Of Fructan Metabolism Enzyme From Garlic

Objective: This article introduce that the activity of the metabolic enzymes of the garlic is studied and the isolation, purification and characterization of the enzyme are studied.Methods:(1) 1-SST was precipitates with ammonium sulfate precipitation to determine the position of 1-SST in different ammonium sulfate saturation. 1-SST which has the highest activity of ammonium sulfate precipitate and sucrose were taken to reaction for studying its enzymological characteristics.(2) TLC rapidly detectcs garlic fructan metabolism enzyme product of low fructan by changing the expansion type and flow agent phase ratio.(3) The crude enzyme of Garlic was separated by DEAE Sepharose FF of anion exchange chromatography and Superdex 200 gel filtration chromatography method, meanwhile using TLC method for rapid detection of garlic fructan metabolism enzyme.(4) Garlic total protein which were extracted by TCA/ acetone method, modified TCA/acetone method, phenol methanol/ammonium acetate precipitation, TCA/ acetone/redistilled phenol method were separated by two-dimensional electrophoresis.Results and Discusssion:(1) Garlic 1-SST mainly existed in the fraction precipitated with 30%-40% ammonium sulfate solution. The optimal temperature was 35℃. The optimal reaction p H is 5.0. The quality of the optimal concentration of reactant sucrose was 500 mg·m L-1. The lower Na+, Cl- ion, the stronger 1-SST of activity. Michaelis constant of 1-SST was 104 mmol·L-1 and Vmax was 2500μg·m L-1·h-1. In this study, the effect of different temperatures, p H, 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.(2) A new TLC method for n-butanol: isopropyl alcohol: acetic acid: water =35:25:20:10(unfold 3 times) is suitable for rapid detection of low fructan. This provides appropriate the method for rapid qualitative detection of garlic low fructan during garlic fructan metabolism enzyme separation and purification process.(3) We also found that there may be 6-SFT, 1-FFT or 6G-FFT in garlic through the subdivision of the crude enzyme of garlic solution and TLC method. The results indicate that it may also exists other kinds of protein except for 1-FEH, 1-SST in garlic.We further understand the fructan metabolism enzyme for providing the basis for garlic.(4) TCA/acetone method/redistilled phenol method which is a good method for further research on proteomics is more suitable for the extraction of total protein of the garlic by comparing several kinds of garlic protein extraction method.