Study On The Isolation And Identification, Degeneration Characteristics And Structure Properties For ?-glucosidase From Hevea Brasiliensis Seed

?-glucosidase can hydrolyze non-reducing?-D-glycosidic linkages at the end of saccharides and glycoside compounds, with releasing aglycone and one glucose. P-glucosidase plays an important role in the biological bodies, such as the cyanogenic effect, the metabolism of glycolipid and exogenous glucoside, the degradation of cellulose, etc. This paper studies the methods of purifying the p-glucosidase in the rubber seeds and using mass spectrometry to identify the purified results; it tests P-glucosidase variations in enzyme activity and conformation under the influence of denaturant; it simulates the spatial structure of ?-glucosidase and analyzes the molecular docking of enzyme and substrate. The main study results are as follows:Rubber seed protein is classified by Osborn into albumin, globulin, prolamin and glutelin, while ?-glucosidase mainly exists in albumin. The preliminarily purified enzyme solution was get through acetone precipitation, reconstitution, centrifugation and isoelectric precipitation, and then was purified by Sephadex G-200chromatography and non-denaturing electrophoresis and the purification result was identified through mass spectromete. The specific activity of P-glucosidase is increased from the original10.9U/mg to175.96U/mg. It can be found that the p-glucosidase exists in the form of polymer under the natural circumstances through the measurement of its molecular weight by means of SDS-PAGE electrophoresis and non-denaturing electrophoresis.Under the influence of a certain concentration of urea or guanidine hydrochloride, the spatial structure of P-glucosidase will change and the denaturation and inactivation will occur. The enzyme activity of ?-glucosidase shows a trend of decline with the concentration increase of the denaturant, and the increase of temperature will accelerate the pace of the decline, ?-glucosidase can be activated at a low concentration of the urea within one hour, and the enzyme activity improves significantly. It has been found through the laser particle size analysis that after P-glucosidase is denatured by the urea, the molecular particle size of the enzyme solution decreases, which infers that the polymer structure of ?glucosidase is depolymerized. By analyzing the fluorescence spectra, it shows that the location of the fluorescence peak moves with the increase of urea's concentration in the enzyme solution, which indicates that the tryptophan residues continuously is exposed and the structure of protein is spread.The result shows that the sequence consistent rate of ?-glucosidase between rubber seed and clover is53%by the amino acid sequence comparison. The three-dimensional structure of P-glucosidase in rubber seed was constructed by using the homology modeling method and the rationality of spatial structure model was proved with various kinds of evaluation methods, ?-glucosidase has a typical (P/a)s barrel structure that is in the GH1family. The proton donor is Glul95in the4th ?-folded and the nucleophilic reagents is Glu410in the7th ?-folded. The molecular docking of the different kinds of substrates and ?-glucosidase was investigated through the Autodock4.2software. The results show that, the binding ability of flavonoids and isoflavonoids with ?-glucosidase is the strongest, with the range of-8.6to-7.92kcal/mol, the binding ability between trehalose and maltose with beta glucosidase is relatively week. The binding region of the substrate and enzyme is in the active pocket, the glycosyl of the substrate is located on the bottom of the pocket. The catalytic region of ?-glucosidase has a series of polar amino acid residues and the aromatic amino acid residues, which are conduced to bind the substrates and related with the substrate specificity of ?-glucosidase.