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Ginger has a long history of cultivation, a the the important condiments and important of the traditional Chinese medicine. This experiment, as the raw material quality Laiwu big ginger extract of ginger protease, purification and activity detection technology, 38 kinds of ginger as raw material, the determination of the different varieties of ginger protease, curcumin, gingerol content. (1) ginger protease activity assay method the best enzymatic reaction temperature of 40 ℃, the reaction time of 5min tyrosine enzyme activity values ??calculated for the standard reference substance. By comparing UV spectrophotometry (Abs 280nm Act) and Lowery both French and enzymatic assays prove UV spectrophotometry (Abs 280nm law) has a simple, high stability, sensitivity, etc., this method measured ginger protease Michaelis constant optimum reaction conditions 46.18μg/mL. (2) of ginger protease extraction method by orthogonal test and variance analysis, the ginger extraction method program: buffer pH 7.5, ionic strength of 0.03 mol / L, the solid to liquid ratio of 1:2, the amount of ethanol 1:2.5 Click program ginger protease extraction, the obtained crude enzyme activity value crude enzyme by 843.7U the rate of 1.40%. Ethanol extraction powder ginger protease optimum extraction conditions: buffer pH6.5, ionic strength 0.10mol / L, beating solid to liquid ratio of 1:3 ethanol. Ginger protease extraction Under this program the measured enzyme values ??601.5U, crude enzyme was 1.39%. (3) was purified by column chromatography the ginger proteases using Sephadex G-50 Medium ginger proteases was purified as a filler, the preferred chromatography column 16 × 500mm, the elution conditions are: column bed volumes (CBV) to 100 ml, elution rate 1.5mL/min, the elution buffer as a phosphate buffer solution (pH 7.5 0.03mol / L), the sample volume was 3 ml (protein content 42.7μg/mL). This elution conditions purified by size exclusion chromatography (SEC) can be obtained by two distinct elution peak, wherein the peak I enzyme activity. Ginger enzyme activity as a reference to its 1.98 times, the specific activity of the crude enzyme, and and after SEC purified to a specific activity, purification factor investigation index reached 4.19 times, 2.11 times that of the crude enzyme. Cellulose DE-52 (elution rate 0.5mL/min) Sepharose the DE-52 (Fast Flow) (elution rate 1.0mL/min) two packing ion exchange chromatography (IEC) purification using phase elution method (total amount 50mL, variation gradient of starting buffer and limit buffer 5mL, 10 gradient) can be obtained six more significant protein peak, wherein the CelluloseDE-52 filler, peak III and peak Ⅳ enzyme activity; Sepharose DE-52 Fast Flow as a filler, the peak II and peak Ⅲ enzymatically. Ion exchange chromatography Results and the ginger proteases isoelectric point, i.e. ginger crude enzyme protein obtained after IEC purified two isoelectric points of different components, and are acidic proteins. (4) The ginger protease viability protection in buffer cysteine ??ginger protease activity can be effectively protected, 0.015mol / L cysteine ??to maintain enzyme activity at room temperature (25 ℃) best, save the 180min vitality of the enzyme sample value saved when 15min 85%. At low temperature (4 ℃), 0.020 mol / L cysteine ??enzyme activity remains the best 87% of the value of the first day, save the day 8 vitality. (5) 38 kinds of ginger resources survey protease different varieties of ginger, curcumin, ginger, capsaicin content have large differences in specific activity of ginger protease Objectives of the mission, Liaoning Dandong ginger, Jiangxi Huanglao door ginger, Shandong Laiwu ginger and so suitable for the extraction of ginger protease.
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