## 4 Conclusions  
Maximization of immobilized industrial-class phospholipase A\({}_{1}\) was successfully realized by regulation of water content of W/O microemulsion phase as a reaction media. Industrial-class PLA\({}_{1}\) was successfully immobilized onto a polypropylene porous particle (Accurel MP100) with the aid of glutaraldehyde. The yield of immobilized PLA\({}_{1}\) was over 90% based on simple adsorption. A hydrolysis reaction with the immobilized PLA\({}_{1}\) was quickly initiated without any time lag. The minimization of activity loss of the PLA\({}_{1}\) throughout the immobilization process was effectively accomplished using appropriate concentrations of glutaraldehyde with moderate shaking under room temperature. The initial reaction rate of the immobilized PLA\({}_{1}\) increased with an increasing glutaraldehyde concentration. A glutaraldehyde concentration of 6% provided a sufficiently stable immobilization of PLA\({}_{1}\) and the initial reaction rate. The initial reaction rate could be compared with that of the non-immobilized PLA\({}_{1}\) in the W/O microemulsion system. It was strongly dependent on the water content, W (molar ratio of water and amphiphilic molecules (phospholipids)). In this study, the maximum reaction rate of 7 was achieved. The background of the maximum reactivity of immobilized PLA\({}_{1}\). It was considered in terms of easy diffusion of substrate involved with the size of the W/O microemulsion and the inlet opening of pores on the surface of the Accurel particles. The productivity was examined after 10 repeated uses. Accumulated production was 3.5 times superior to that of a single use of non-immobilized PLA\({}_{1}\) under the appropriate regulation of the water content in the W/O microemulsion phase. Industrial-class immobilized PLA\({}_{1}\) on Accurel MP100 has been suggested as a promising biocatalyst to product phospholipid biochemicals.  
Conceptualization, R.N.; methodology, R.N.; formal analysis, Y.H.; investigation, Y.H.; writing--original draft preparation, Y.H.; writing--review and editing, Y.H., R.N. and M.I.; visualization, R.N., Y.H. and M.I.; supervision, N.N. All authors have read and agree to the published version of the manuscript.  
This  
**Acknowledgments:** Phospholipase A\({}_{1}\) was donated from Mitsubishi-Kagaku Foods Corporation (Tokyo, Japan). Accurel MP100 was provided by Evonik Industries AG (Essen, Germany). We sincerely appreciate their kind cooperation.  
**Conflicts of Interest:** The authors declare no conflict of interest.  
\begin{tabular}{p{142.3 pt} p{142.3 pt}}
**Nomenclature** \\ Am & the adsorbed amount of PLA\({}_{1}\) on Accurel MP100 [g-PLA\({}_{1}\)/g-Accurel] \\ C\({}_{GA}\) & concentration of glutaraldehydehyde to immobilize PLA\({}_{1}\) on Accurel MP100 [wt\%] \\ Cp & concentration of palmitic acid from hydrolysis of phospholipid [mM] \\ Cp\({}^{\star}\) & concentration of palmitic acid from hydrolysis of phospholipid per unit mass of PLA\({}_{1}\) [mM/g] \\ Cs & concentration of soybean phospholipid [mM] \\ Hb & the supplemental amount of buffer solution in the W/O microemulsion phase [mL] (volume of the organic phase was set at 50 mL throughout this study) \\ R\({}_{\rm C}\) & characteristic radius of the W/O microemulsion [Å] \\ t & reaction time [min] \\ Vi & initial reaction rate of hydrolysis of phospholipid [mM/s] \\ Vi\({}^{\star}\) & initial reaction rate of hydrolysis of phospholipid per unit mass of PLA\({}_{1}\) [mM/s/g] \\  & molar ratio of water to phospholipid (as an amphiphilic component) \\ W & [mol-water of aqueous buffer solution/mol-phosphatidylcholine as an amphiphilic substrate (phospholipid)] \\
**Abbreviations** & abbreviation of commercial name of \"Aerosol\(\otimes\)OT\". It is a typical anionic AOT & amphiphilic reagent. The main component of AOT is sodium dioctyl sulfosuccinate \\ GA & glutaraldehydehyde \\ PLA\({}_{1}\) & phospholipase A\({}_{1}\) \\ SEM & scanning electron microscopy \\ W/O & water-in-oil \\ \end{tabular}