THE DEVELOPMENT OF ACE2-LIKE ENZYME FROM YOGHURT-ISOLATED KLUYVEROMYCES MARXIANUS

• Main Author: Rafi Ibrahim, Sulthan
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/75097
• Institution: Institut Teknologi Bandung

Angiotensin-converting enzyme 2 (ACE2) is an important enzyme found on the cell membrane of organs that has a profound function in the physiological esprocess of the human body. However, the ACE2 protein can also be used by the SARS-CoV-2 virus as an entry point for infection. Furthermore, this infection will downregulate the function of ACE2 itself and eventually lead to the appearance of COVID-19 symptoms such as decreased lung function, myocarditis, and hypertension. Therefore, one mechanism that can be utilized to treat COVID-19 is to reduce the binding of SARS-CoV-2 with the human ACE2 receptor by utilizing 'ACE2-like' enzymes. It is known from previous studies that ACE2-like enzyme can be isolated from microorganisms present in fermented food products. Therefore, this study will isolate microorganisms from fermented foods and develop an expression system for the obtained ACE2-like enzyme. This research begins with the selection of microorganisms in yogurt using MRS agar selective medium. Then, the growing colonies were extracted and tested for ACE2-like activity and continued with whole genome sequencing using Nanopore. The WGS results were assembled and annotated to find candidate genes encoding ACE2-like enzyme and then in silico analysis was carried out on the candidate genes. Gene candidates with the best in silico test results will be designed expression plasmids, cloned, and expressed in recombinant Escherichia coli BL21 (DE3) bacteria. The expression of recombinant ACE2-like protein was confirmed by SDS-PAGE method and ACE2-like activity test. From the results, 16 different colonies were successfully grown on selective medium, and four of them had the highest ACE2-like enzyme activity. Of the four colonies, one colony was successfully analyzed and identified its whole genome, namely Kluyveromyces marxianus. From the results of genome annotation, this species has four candidate genes encoding ACE2-like enzyme. From the results of in silico analysis, it was concluded that the KEX1 gene was the best ACE2-like enzyme candidate coding gene. The KEX1 gene was then cloned and expressed with 0.25mM IPTG induction and confirmed by SDS-PAGE. SDS-PAGE results confirmed the successful expression of KEX1. However, further research is needed to optimize the expression and characterize the resulting recombinant KEX1 protein.