Enzymatic properties identification of pitcher fluid proteases from Nepenthes × miranda

Pitcher fluid of Nepenthes × miranda is usually discarded before transportation despite containing many functional materials, in which hydrolytic proteases are particularly attractive to us. In our study, we collected the pitcher fluid of Nepenthes × miranda and obtained pitcher fluid proteases with a proteolytic activity of 6.658 U/mL. Then the optimal pH and temperature were detected, and the activators, denaturants and inhibitors were screened respectively. Finally, Michaelis constants were calculated to quantify the affinity with different substrates. The results showed peak activity at pH 2.05 to 2.50 and temperatures between 50 °C and 55 °C, which indicated an advantage of exerting proteolytic activity at lower pH levels and higher temperature compared to nepenthesins from other Nepenthes species. Moreover, the pitcher fluid proteases could be activated by Mn 2+ significantly and tolerated glycerol of high content. We discover a way of the new protease resources, a rational use for biochemistry and a direction for the reuse of agricultural waste.