Specific Activity Assay of Alkaline Pectinase Optimized by Kinetic Method

In this paper,the assay methods for protein and enzyme activity were optimized respectively. The rationality of the method optimization was investigated through the kinetic curve of the enzymolysis to confirm the accuracy of the endpoint determination of specific activity of alkaline pectinase. The results showed that optimal detection wavelength for pectinase protein was 550 nm,its linearity and intercept were significantly superior to the traditional method,and the effect of dilution factor on pectinase protein determination could be ignored. Calcium ion had an activating effect on this enzyme,polygalacturonic acid was more suitable as a substrate for enzyme activity measurement than pectin. The optimal substrate concentration and pH were 2 mg/mL and 9.5,respectively. The enzyme activity measured at a wavelength of 232 nm maintained a zero-order reaction state in the absorbance range within 0~2. The enzyme protein concentration was linearly related to the degradation rate and the intercept of the regression curve was closer to the origin. Therefore,this measurement condition could be used for the end point determination,and the enzymolysis time could be flexibly controlled,rather than being restricted to a fixed period of time.Taking 30 minutes as an example,the limit of detection(LOD)was 0.15 mU/mL due to the low sensitivity of the DNS method,the standard curve of galacturonic acid was not at the origin,resulting in measurement errors due to different dilutions of the enzyme solution. Therefore,the UV method was the best activity assay for alkaline pectinase.