N-ACYL HOMOSERINE LACTONASE ENZYME PRODUCTION OPTIMIZATION FROM BACILLUS LICHENIFORMIS DAHB1 PLASMID IN ESCHERICHIA COLI BL21 (DE3) IN FED-BATCH CONTINUOUS FEEDING SYSTEM WITH VARIATION OF GLYCEROL CONCENTRATION

• Main Author: Ramadhan Aryoaji, Kenan
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/83720
• Institution: Institut Teknologi Bandung

Vibrio parahaemolyticus is a bacterium implicated in Acute Hepatopancreatic Necrosis Disease (AHPND) in whiteleg shrimp (Litopenaeus vannamei). AHPND occurs when the quorum sensing mechanisms of V. parahaemolyticus are activated, primarily regulated by the autoinducer acyl homoserine lactone (AHL). This process can be inhibited by the enzyme N-acyl homoserine lactonase (AHL-lactonase), which degrades the ester bonds in AHL. This enzyme is produced within the periplasmic regions of E. coli BL21 (DE3) harboring the plasmid pET-26b(+)-N20-aiiA-6xHis. Previous studies employing a batch system have successfully secreted and optimized the enzyme using a two-stage temperature method, adjusting from 27°C to 37°C. The current study employs a fed-batch fermentation process using the same temperature approach but varies the glycerol concentration in the feed media (300, 400, and 600 g/L) with a carbon to nitrogen (C:N) ratio of 5:1 to enhance cell productivity and enzyme yield. The objective was to identify the optimal glycerol concentration affecting cell growth, pH stability, and enzyme production. Results indicate that glycerol is not the limiting factor; instead, oxygen availability is the constraint, as evidenced by significant pH drops signaling a shift to anaerobic metabolism and acetic acid production as a byproduct. The highest protein yield observed was at a glycerol concentration of 600 g/L, achieving 6,944 mg/mL