Purification of recombinant proteases from Escherichia coli BL21 (DE3) pLysS

Enzymes are biological catalyst in biochemical reaction It provides an alternative route to achieve the activation energy required and speed up the reaction. Proteolytic enzymes help break down and digest protein and can be found in animal, plants, and microorganism. Microorganism is known to be the excellent source of protease. Purification of protease is a time consuming and complicated process. So the research for another method where it will reduce time and cost of the process should be done. The protease was purified by ultrafiltration and heat treatment methods and the yield of purification was measured. Protease which being extracted by using the E.coli BL21 pLysS is purified by using ultrafiltration and heat treatment method at 70°C for 1, 2, and 3 hours. The E.coli was streaked on Luria Bertani agar supplemented with skim milk and was incubated at 37°C for overnight. The enzyme of 10kDa membrane size which was purified for 3 hours has the lowest protein content and second highest protease content. The protein content of crude enzyme was higher than purified enzyme. Protein profiling, SDS PAGE was carried out to determine whether the sample is fully purified by observing the bands in order to further confirm the purity level of the protease enzyme with different incubation time heat treatment. It is observed that fully purified enzyme has lesser band existed compared to the crude enzyme which act as control. This enzyme can withstand high temperature from 70°C to 80°C. The research was successfully purified the recombinant protease by using different treatment and get high yield of purification.