ISOLATION AND EXPRESSION OF THE ACE2-LIKE ENZYME GENE FROM INDONESIAN TRADITIONAL FERMENTED FOOD BACTERIA ISOLATES AS AN ALTERNATIVE TREATMENT FOR COVID-19

• Main Author: Nur Laily, Izza
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/67142
• Institution: Institut Teknologi Bandung

ACE2 and S1 SARS-CoV-2 interaction lead to accumulate Ang-II in COVID-19 patients. On the other hand, it is known that M32-carboxypeptidase as an ACE2- like enzyme derived from food-fermenting bacteria Paenibacillus sp. B38 could reduce plasma Ang-II levels in mice. Therefore, this study aims to isolate the ACE2-like enzyme gene from Indonesian traditional fermented food bacteria and examine their inhibitory activity against ACE2-S1 SARS-CoV-2 interaction. Indonesian fermented foods were collected from different traditional markets of Bandung City, West Java, Indonesia. The bacteria were isolated and grown in selective MRS media. Furthermore, those samples were screened for hydrolysis activity against Nma-His-Pro-Lys(Dnp). Bacterial isolates exhibit ACE2-like enzyme then identified by the 16s rRNA sequencing method. Subsequently, the recombinant ACE2-like enzyme expressed in E. coli BL21(DE3) was confirmed by sequencing, SDS-PAGE, and substrate hydrolysis activity. Inhibitory activity of the recombinant protein against ACE2 and S1 SARS-CoV-2 binding was determined by in vitro and in silico analysis. As many as three out of 38 isolates derived from seven fermented-food exhibited ACE2-like enzyme activity. The 16s RNA results confirmed that those isolates were Bacillus cereus, Lactobacillus delbrueckii, and Lactobacillus fermentum. However, only B. cereus encodes M32- carboxypeptidase gene and it was defined as BceCP. The recombinant BceCP was successfully cloned to E. coli BL21(DE3) although it was a truncated protein. Truncated BceCP show lower substrate hydrolysis activity compared to the native full-length BceCP. Nevertheless, its inhibitory activity against ACE2 and SARS- CoV-2 binding interaction remains either in vitro or in silico analysis. Thus, full- length BceCP is believed to serve as an ACE2 "receptor mimic" that blocks the entry virus to human cells and reduces Ang-II levels in COVID-19 patients. .