3.4. Effect of agitation, buffer, and metal ions in the whole-cell
reaction
In previous studies, optimal conditions for LCD activity, such as pH,
temperature, and substrate concentration (l-lysine), were
reported, and exogenous addition of iron (II) sulfate,
NAD+, and detergent such as Triton X-100 improved
productivity (Ying et al., 2015). Herein,
we tested whether additional conditions including agitation, buffer
concentration, and metal ions affected productivity
(Y. G. Hong et al., 2019;
Moon et al., 2019). To test the effect of
agitation, the whole-cell reaction for l-PA production was
carried out for 48 h, and agitating the reaction was compared to the
settling method. Performing the reaction with agitation increased
l-PA production by two-fold compared to using the settling
method (Fig. 5A). The two-fold difference in conversion could be
observed from early in the reaction. Therefore, adding agitation
improves l-PA production in the whole-cell system.
In the whole-cell reaction, pH 7.5 is optimal for LCD enzyme activity,
and l-PA increases the acidity of the mixture. Phosphate buffer
(pH 7) was used to prevent the decrease in pH owing to the production of
l-PA, which is important to produce high concentration
l-PA. The whole-cell reaction was conducted with different
buffer concentrations compared to the control sample without buffer.
Addition of buffer up to 150 mM increased l-PA production, and
increasing the buffer concentration over 200 mM did not affect
production (Fig. 5B). Therefore, we chose 150 mM phosphate buffer for
subsequent reactions.
To investigate the effect of metal ions, different metal ions were
tested in the whole-cell reaction. Only FeCl2 enhanced
LCD enzyme activity, while other metals decreased activity (Fig. 5C). In
the first test, iron chloride was used, and we wanted to investigate
whether the form of the iron salt had an effect. Both the chloride and
sulfate forms can improve the enzyme activity. The chloride form showed
1.3-fold higher production, and the sulfate form showed 1.45-fold of the
control (Fig. 5D). When the optimal iron concentration was tested, the
highest conversion was observed with 20 mM iron sulfate (data not
shown). According to previous literature on LCD, NAD+increases enzyme activity as a cofactor
(Gatto et al., 2006;
Ying et al., 2017;
Ying et al., 2015). However, in our
system, NAD+ addition did not improve whole-cell
conversion compared to control (Supplementary Fig. 2).