DEVELOPMENT OF RECOMBINANT ACE2-LIKE ENZYME PROTEIN FROM BACILLUS CEREUS AS A THERAPEUTIC AGENT FOR COVID-19 PATIENTS

• Main Author: Sendra Maulida, Alya
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/76446
• Institution: Institut Teknologi Bandung

SARS-CoV-2 infection can cause downregulation of ACE2, resulting in higher levels of pro-inflammatory Angiotensin II in the body. To overcome this, a therapy with recombinant human ACE2 protein (rhACE2) is needed to restore the body's homeostasis. However, the use of rhACE2 is expensive, so an alternative is ACE2-like enzyme that has the same therapeutic effect. Based on previous research, a carboxypeptidase protein (CAP) belonging to Bacillus cereus isolated from Indonesian fermented food, namely cassava tape, was identified and has the potential as an ACE2-like enzyme. However, in that study, this carboxypeptidase enzyme has not been further characterized. Therefore, this study will characterize, express, and test the activity of CAP protein from Bacillus cereus that has been isolated from previous studies. The work phase began with the synthetic construction of a plasmid containing the BceCP gene encoding CAP from Bacillus cereus (pET21b(+)-BceCP). Prediction of the structure and psychochemical characteristics of the protein was carried out using the Prabi and Expasy websites. The plasmid was transformed into E. coli BL21 (DE3) cells. Protein expression was induced with IPTG at concentrations of 0, 0.25, 0.5, and 1 mM. The protein concentration was measured using software called ImageJ then the data were statistically tested with SPSS. The recombinant BceCP protein (rBceCP) produced was tested for hydrolysis activity and compared with the positive control in the form of recombinant carboxypeptidase protein derived from Enterobacter sp. (rEntCP). The rBceCP protein was also examined for its inhibitory ability towards the binding between ACE2 and SARS-CoV-2 S1 protein. The results of in silico analysis show that the protein structure is a homodimer dominated by ?-helix (53.66%) and has a molecular weight of 58.99 kDa. The protein is stable, hydrophilic, and has an isoelectric point at pH 4.93. Transformation of the plasmid into E. coli BL21(DE3) cells produced 82 transformant colonies with a transformation efficiency of 1.64 x 104 cfu/?g. The results of rBceCP expression showed no significant difference among the three variations of IPTG concentration (p-value >0.05). Based on the results of protein hydrolysis activity analysis, it was found that the trend of rBceCP protein activity was similar to rEntCP, but the purified protein sample had lower activity than the crude sample. The results of the inhibition test showed that the rBceCP protein sample had a high relative activity of inhibition with the highest relative activity of 60.94%. The relative activity of inhibition in crude and purified samples did not show significant differences (p-value> 0.05). Based on the results obtained, it can be concluded that the CAP protein from Bacillus cereus has the potential as an ACE2-like enzyme and can be further developed as a candidate therapeutic agent for COVID-19 patients. It is hoped that the results of this study can help Indonesia to reduce mortality and increase the recovery rate of COVID-19 patients.