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.