3.3. Optimization of culture conditions for improving enzyme activity
To improve enzyme activity, it is needed to determine the optimal culture conditions, including medium, growth temperature, and IPTG concentration, leading to regulation of expression level or condition to maximize the enzyme activity. Generally, LB broth was used for E. coli fermentation. However, this broth contains relatively low nutrient concentration. Since terrific broth (TB), super broth (SB), and 2× YT broth are commonly used as highly enriched media, these were investigated compared to LB broth to get enhanced enzyme activity and cell growth (Y. G. Hong et al., 2019; J. Kim et al., 2017). With the reinforced enzyme, the yhPA004 strain was cultured in the four different broths. After 0.1 mM IPTG induction, these were incubated at 25 °C. The whole-cell reaction was conducted as in the previous experiment. To measure the cell dry weight, aliquots of 5 mL were collected from the culture media and freeze-dried. LCD activity in whole cells harvested from TB broth was two-fold higher than from LB broth. The activity from SB broth and 2× YT broth was also higher than from LB, however, lower than from TB broth (Fig. 4A). Likewise, the cell dry weight from TB broth was highest at 2.4 g/L and 2.5-fold higher than that from LB (Fig. 4B). Based on these results, TB broth was determined to be the best induction media for the yhPA004 strain.
Overexpression of exogenous genes with plasmid system, especially when regulated by IPTG, is strongly affected by growth temperature and IPTG concentration (Baim, Labow, Levine, & Shenk, 1991; Hugouvieux-Cotte-Pattat, Dominguez, & Robert-Baudouy, 1992; Tobe et al., 1991). Thus, we investigated optimal induction conditions using TB broth. First, we investigated the induction temperature by incubating the cultures at 20 °C, 25 °C, 30 °C, or 37 °C after adding 0.1 mM IPTG. Whole-cell reactions were conducted using the same conditions as in the previous experiment. l-PA production increased as the induction temperature increased up to 30 °C, which showed the highest activity (Fig. 4C). Enzyme activity induced in 37°C was the lowest of all tested temperatures. Optimal IPTG concentration was also investigated. After IPTG was added (0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM, or 1 mM), expression was induced at 30 °C. We determined the best IPTG concentration for LCD expression was 0.1 mM, demonstrating that excessive or insufficient IPTG concentration decreased enzyme activity (Fig. 4D). Based on these experiments for efficient l-PA production, the yhPA004 strain was cultured in TB broth and induced with 0.1 mM IPTG at 30 °C.