CRISPR-CAS9 MEDIATED EDITING OF THELACZGENE FOR OPTIMIZING LIPASE PRODUCTION AND CHARACTERIZATION IN ENGINEEREDESCHERICHIA COLIUSING POTATO PEEL AS A GLUCOSE SOURCE

Abstract

ABSTRACTImproper disposal of potato peels harms the environment, wasting nutrient-rich resources that could be used for beneficial enzyme production like lipase. This study aimed to edit thelacZ geneinEscherichiacoli using utilizing the CRISPR Cas9 technology. Both edited and uneditedE. coliwere used for submerged fermentation of potato peels to produce and characterize lipase. ThelacZgene was edited using the CRISPR Cas9 technology, and the efficiency was measured using multiplex PCR and gel electrophoresis. To measure lipolytic activity, olive oil screening was performed. The temperature and pH of the lipase were used to characterize it after partial purification and submerged fermentation at 10°C, 30°C, and 45°C. Blue colonies indicated thelacZ genewas unedited,lacZgene editing and repair was demonstrated by white colonies, and no colonies demonstrated the editing but not repair of thelacZgene. Bands at 1,100 bp indicated uneditedlacZ gene, while 650 bp showed editedlacZ gene. Increased cell mass was observed at 10°C. CRISPR Cas9 editedE. colishowed a clearer zone than the unedited in the lipase medium. The highest lipase activity from both edited and uneditedE. coliwas at 35°C, and the lowest at 65°C. Optimal pH was 7, with lowest activity at pH 4. The CRISPR Cas9 editedE. colidemonstrated significantly higher enzyme activities (p<0.05). This study concluded that CRISPR Cas9-mediatedlacZ geneediting inE. colienhances its ability to utilize potato peels, increasing lipase production.