## 2 Materials and Methods
### Reactivity of Immobilized Phospholipase A\({}_{1}\)
#### 3.3.2 Effect of Water Content on Production  
Figure 7 shows the effect of the amount of buffer solution into the W/O microemulsion phase on the reactivity of immobilized PLA\({}_{1}\). The productivity of the enzyme reaction was dependent on the supplemental aqueous volume in the W/O microemulsion phase. The palmitic acid concentration produced increased quickly with the reaction time and then dropped for over 40 min. When the Hb was 0.075 mL, the highest amount of palmitic acid was produced. Figure 8 shows the change of the initial reaction rate with the concentration of lecithin using a soybean dissolved in the organic phase. The initial reaction rate in this figure was a recalculated value which was a per unit mass of PLA\({}_{1}\). The reactivity of immobilized PLA\({}_{1}\) indicated by the initial reaction rate per unit mass of PLA\({}_{1}\) was well maintained, and the activity damage was fully suppressed. Industrial-class PLA\({}_{1}\) was successfully immobilized onto the Accurel particles with the aid of GA aqueous solution. Figure 8: Relationship between the substrate concentration and the initial reaction rate for non-immobilized phospholipase A\({}_{1}\) in W/O microemulsion (\(\blacksquare\)) at Hb = 0.3 mL, immobilized phospholipase A\({}_{1}\) (\(\bullet\)) at Hb = 0.075 mL. The concentration of glutaraldehyde for immobilization was 6%. Vi\({}^{\star}\): initial reaction rate of hydrolysis of phospholipid per unit mass of PLA\({}_{1}\). Figure 7: Time course of palmitic acid concentration (Cp) produced with immobilized phospholipase A\({}_{1}\) in various amounts of water. The volumetric amount of the supplemental acetate buffer solution, Hb was changed as follows; 0 (\(\bigstar\)), 0.05 (\(\bullet\)), 0.075 (\(\bigcirc\)), 0.1 (\(\blacktriangledown\)), and 0.2 (\(\blacksquare\)) mL. The C\({}_{\text{GA}}\) was set at 6%. The experiments were each conducted a minimum of three times. Figure 9 shows the relationship between the initial reaction rate and the molar ratio of water to lecithin, W. In this study, the W value was defined as the molar ratio of supplemental water moles to the phosphatidylcholine dipalmitoyl moles:  
\[\text{W}=\frac{[\text{phosphatidylcholine dipalmitoyl}]}{[\text{H}_{2}\text{O}]} \tag{1}\]  
The moles of water in the W/O microemulsion phase were calculated, assuming the supplemented aqueous volume with a density of 1.0 g/mL. The initial water content in the 1-butanol/isooctane mixed phase before the supplement of the aqueous phase was ignored. The remarkable decay of initial reaction rate for higher W values agreed with the change of hydrophobic character of the microwater pool. Previous studies of lipase reactivity in the W/O microemulsion phase have shown that water content is a dominant factor in regulating the reactivity of the lipase [17]. The size of the microemulsion is dependent on the water content. That correlates with the molar ratio of water to the amphiphilic component (W) [29]. Previously, the values of W have been often employed as a typical indicator of water content in the W/O microemulsion phase in other lipase [30; 31] and phospholipase reactions [32]. The initial reaction rate indicated a bell-shaped curve with increasing water content in the W/O microemulsion phase. The maximum reactivity of immobilized PLA\({}_{1}\) was obtained for W = 7. The maximum initial reaction rate in non-immobilized PLA\({}_{1}\) was W = 27. When the W value was low, the W/O microemulsion size was very small [33]. The water molecules were strongly associated with each other and demonstrated hydrophobic behavior [34; 35; 36]. In contrast, for the larger values of W, more water molecules recovered their hydrophilic character. The latter was discussed in literature from the viewpoint of the structural formation of water molecules [37]. These results suggest that the local surroundings of the immobilized PLA\({}_{1}\) in Accurel were more hydrophobic than that of the non-immobilized PLA\({}_{1}\) in the W/O microemulsion phase.