PRODUCTION OF RECOMBINANT CANDIDATE PROTEASE FROM BACILLUS VELEZENSIS, A SYMBIONT OF FRESHWATER SPONGE EUNAPIUS CARTERI AND TEST OF ITS ACTIVITY AND INHIBITORY ON THE GROWTH OF PATHOGENIC BACTERIA.

• Main Author: Dwi Cahyanti, Naomi
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/70762
• Institution: Institut Teknologi Bandung

In previous research, a coding gene of protease candidate from fresh water sponge (Eunapius carteri) symbiont, Bacillus velezensis, has been successfully isolated using degenerated primer. Sequencing analysis result of the gene was identical to the S8 serine peptidase of B. velezensis in NCBI genebank. The protease candidate gene was cloned into pET16b and produced recombinantly in Escherichia coli with IPTG induction. The purpose of this research is to obtain the optimum conditions for overproduction of the recombinant candidate protease and to verify its protease activity as well as its inhibitory effect on growth of pathogenic bacteria. Overproduction of the protease in E. coli BL21(DE3), E. coli BL21-Gold(DE3), E. coli BL21-Codonplus(DE3)-RIPL at 37 ?C and 20 ?C showed that protease was produced dominantly in insoluble form. Overproduction using the GroES/EL-TF chaperone system in E. coli BL21(DE3) host at 20 ?C induced by tetracyclin 10 ng/ml and IPTG 0.01 mM showed an increase in the solubility of Full protease 13.53% ± 2.04 and Half protease 36.39% ± 8.96. Intracellular soluble crude protein from overproduction using the GroES/EL-TF chaperon system was tested for its protease activity in caseinolytic media with proteinase K as a positive control. Based on this test, it was found that the recombinant proteins have protease activity with the formation of a clear zone around the disc. Inhibition activity test on the growth of pathogenic bacteria showed that recombinant proteases have more potential to inhibit Methicilin Resistant Staphylococcus aureus (MRSA) than ESBL E. coli. The protease activity is assumed by a catalytic triad mechanism according to the active site contained in S8 serine peptidase in B. velezensis.