CONCENTRATION AND INDUCTION TIMING OPTIMIZATION OF LACTOSE AS AN INDUCER IN HETEROLOGOUS ECTOINE PRODUCTION IN ESCHERICHIA COLI BL21(DE3)

• Main Author: Caroline, Evelyn
• Format: Final Project
• Language: Indonesia
• Subjects: Ilmu hayati ; Biologi
• Online Access: https://digilib.itb.ac.id/gdl/view/84423
• Institution: Institut Teknologi Bandung

Ectoine is an osmoprotectant capable of protecting cells from various environment stimuli such as salinity, extreme temperature, and drought which makes it beneficial for various industrial applications. Ectoine is mainly produced by halophilic bacteria, though also recently discovered in Archaea and Eukarya groups. Ectoine production using halophilic bacteria has a few setbacks, including the need of high-salinity medium causing corrosion on bioreactors, and complex extraction and purification processes. Previous study by Deep Sea ITB team on Virgibacillus salarius had discovered ectoine gene cluster which had been successfully inserted into E. coli BL21(DE3) for heterologous production. Optimizations of various parameters for industrial scale production are mandatory, including the concentration and induction timing of inducer. IPTG, which was used in the previous study, has negative impact on cell viability, relatively expensive especially on industrial scale, and thus could be substituted with other inducers such as the natural inducer lactose. Optimization of concentration and induction timing would affect the expression level of recombinant products and increase production efficiency, hence this research attempted to optimize these two factors in using lactose as an inducer in ectoine production. Lactose concentration optimization was done at 5gr/L, 10gr/L, and 20gr/L while optimization on induction time was done in the middle of exponential phase, early stationary phase, and stationary phase on 2nd, 4th, and 6th hour respectively. Induction by 5gr/L lactose on the 6th hour was discovered to yield the highest result on 1.560 ± 0.013 gr/L. This value was relatively higher compared to the previous study where induction was done with IPTG (0,37 ± 0,018 gr/L). A two-way ANOVA analysis was conducted to examine the effect of lactose concentration and induction time on ectoine production. There was no statistically significant difference in induction time, but there was statistically significant difference on lactose concentration and interaction between effects of lactose concentration and induction time (P-value=0.001 and 0.024 respectively). Further analysis by Tukey HSD confirmed a significant difference between lactose concentrations. This result signifies the potential of lactose as inducer in industrial-scale production.