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).