Extraction And Purification Of Ginger Protease And Comparison Of Different Ginger Varieties

In China, there's a long history of planting, eating ginger and having it as herbal medicine. This article focuses on the extraction, purification and proteolytic activity measurement of ginger protease by the material of Laiwu Dajiang and the concentration of ginger protease, curcumin and gingerol with 38 kinds of Chinese ginger varieties.(1) Proteolytic assay of ginger proteaseThe optical catalyst factors are: temperature 40℃, catalyst time 5min and take tyrosine standard solution to measure the units of proteolytic activity. With comparison of two methods of Lowery Protein Assay and Abs 280nm Value, the best measurement of protease activity is the later which features simplicity, sensitivity and stability. The Km value is 46.18μg/mL measured by the methods of Abs 280nm Values.(2) Extraction of ginger proteaseThe optimal ginger protease extraction factors by ginger juice are: buffer pH value 7.5, ion strength 0.03mol/L, the ratio of ginger mass and buffer volume 1:2, and the final crude ginger protease activity is 843.7U, with 280.3mg ginger protease extracted for 20g fresh ginger. On the other hand, the optimal factors of ginger powder precipitated by anhynous ethonal are: ginger mass and anhydrous ethanol ratio 1:3, buffer pH 6.5 and ion strength 0.10mol/L, and the final crude ginger protease activity is 601.5U, with 277.1mg ginger protease extracted from 20g fresh ginger.(3) Ginger protease purification by column chromatographyAs employing Sephadex G-50 Medium as the gel to purifying ginger protease, the best elution factors are: column 16*500mm with a CBV of 100mL, elution speed 1.5mL/min, elution buffer phosphate buffer (pH 7.5, 0.03mol/L) and the applying amount 3mL (protein concentration 42.7μg/mL). The SEC elution figure shows two separately protein peaks among which the peak I performs protease activity. Taking the ginger juice's specific protease activity as benchmark, the purified protease's specific activity is its 4.19 folds, and 2.11 folds with the benchmark of crude ginger protease.6 clear protein peaks are shown in the elution figures with gel staff of Cellulose DE-52 (elution speed 0.5mL/min) and Sepharose DE-52 Fast Flow (elution speed 1.0mL/min) with gradient elution (totally 50mL with 10 gradients, and the step is 5mL while the volume of initial buffer decreasing and limited buffer increasing). In those peaks, peakⅢand peakⅣshow protease activities in Cellulose DE-52 elution while peakⅡand peakⅢshow proteases activities in Sepharose DE-52 Fast Flow elution. Because proteins'separation in IEC is according to their isoelectric point, the two IEC purified ginger protease must be two different kind of component and they are acidic proteins.(4) Preservation of ginger protease activityL-Cysteine is employed to preserve the ginger protease activity in this experiment and under the room temperature (25℃), the concentration of 0.015mol/L shows best effect after 180min standing which is 85% compared with the value measured at 15min. As preserving under low temperature (4℃), the best concentration is 0.020mol/L which can preserve 87% proteolytic activity after 8-day store.(5) Investigation of 38 kinds of Chinese gingersThere are lots of ginger varieties in China, and in this article, the 38 kinds ginger's protease concentration are tested and the following varieties are suit for protease extraction: Dandong ginger of Liaoning, Huangmen ginger of Jiangxi and Laiwu ginger of Shandong.