Abstract: A method for rapid determination of protease activity based on the substrate of un-denatured hide powder (collagen fibers) labeled with a low temperature reactive dyestuff was developed. The preparation method of the substrate was optimized, the properties of substrate were investigated, the conditions of detection were optimized, and the activities of several proteases were measured. The results show that the structure of the dyed hide powder does not change significantly, and it is suitable for the determination of protease activity in a wide pH range. The performances of proteases towards different substrates are different. Compared with casein and heat-denaturated staining powder, the results of the new method have more practical guiding significance for the selection of proteases and the optimization of using conditions in the process of leather making.

Key words: protease, enzyme activity, Un-denatured dyeing substrate