Expression of recombinant protease gene from Bacillus subtilis 50a in Escherichia coli BL21 (DE3) harbouring pGEX

• Main Author: Nik Roshdi, Nik Wardah Sofiah
• Format: Undergraduate Final Project Report
• Published: 2020
• Online Access: http://discol.umk.edu.my/id/eprint/4286/

As the Bacillus subtilis, the wild type protease producer can only produce a small amount of protease enzyme, hence the other approach need to be taken to produce more enzyme like protease. The main purpose of this study is to express the recombinant thermostable protease in E.coli BL21 (DE3) and to compare the enzyme activity by new host and existing host which is E. coli BL21 (DE3) harbouring recombinant pGEX /protease 50a and E. coli BL21 (DE3) harbouring recombinant pET / protease 50a respectively. The method used in this study consists of preparation of stock and inoculum, the production and harvesting of thermostable protease, the assay of protease activity and also the purification by heat treatment. However, the bacteria was screened qualitatively on skim milk and LB agar plate and both of the E. coli BL21 (DE3) pGEX4T-1/50a and E. coli BL21 (DE3) pLySs pET22b(+)/50a showed positives result with the form of single colonies on the LB-SMA plates. Furthermore, it can be seen that there are a form of clearing zone around the single colonies on LB-SMA plate of the E.coli BL21 (DE3) harbouring recombinant pGEX4T-1/50a but only single colonies on the plate of E. coli BL21 (DE3) pLysS harbouring recombinant pET22b(+)/50a. Theresult of enzyme activity obtained is not as expected. There is a little bit of contradiction from the expected result where the activity of enzyme for both of the new host and existing host should be much higher than the result obtained.