Promising Immobilization of Industrial-Class Phospholipase A1

applied sciences

Article Promising Immobilization of Industrial-Class Phospholipase A1 to Attain High-Yield Phospholipids Hydrolysis and Repeated Use with Optimal Water Content in Water-in-Oil Microemulsion Phase

Yusuke Hayakawa 1,2, Ryoichi Nakayama 2,*, Norikazu Namiki 2 and Masanao Imai 1

1 Course of Bioresource Utilization Sciences, Graduate School of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa 252-0880, Japan; [email protected] (Y.H.); [email protected] (M.I.) 2 Department of Environmental Chemistry and Chemical Engineering, School of Advanced Engineering, Kogakuin University, 2665-1 Nakano-machi, Hachioji, Tokyo 192-0015, Japan; [email protected] * Correspondence: [email protected]; Tel.: +81-42-628-4876

Abstract: In this study, we maximized the reactivity of phospholipids hydrolysis with immobilized

industrial-class phospholipase A1 (PLA1) at the desired water content in the water-in-oil (W/O) microemulsion phase. The optimal hydrophobic-hydrophilic condition of the reaction media in a hydrophobic enzyme reaction is critical to realize the maximum yields of enzyme activity of

phospholipase A1. It was attributed to enzymes disliking hydrophobic surroundings as a special molecular structure for reactivity. Immobilization of PLA1 was successfully achieved with the aid of a hydrophobic carrier (Accurel MP100) combination with the treatment using glutaraldehyde. The immobilized yield was over 90% based on simple adsorption. The hydrolysis reaction was kinetically Citation: Hayakawa, Y.; investigated through the effect of glutaraldehyde treatment of carrier and water content in the W/O Nakayama, R.; Namiki, N.; Imai, M. Promising Immobilization of microemulsion phase. The initial reaction rate increased linearly with an increasing glutaraldehyde concentration and then leveled off over a 6% glutaraldehyde concentration. The initial reaction rate, Industrial-Class Phospholipase A1 to Attain High-Yield Phospholipids which was predominantly driven by the water content in the organic phase, changed according to Hydrolysis and Repeated Use with a typical bell-shaped curve with respect to the molar ratio of water to phospholipid. It behaved Optimal Water Content in in a similar way with different glutaraldehyde concentrations. After 10 cycles of repeated use, the Water-in-Oil Microemulsion Phase. reactivity was well sustained at 40% of the initial reaction rate and the creation of the ﬁnal product. Appl. Sci. 2021, 11, 1456. https:// Accumulated yield after 10 times repetition was sufﬁcient for industrial applications. Immobilized doi.org/10.3390/app11041456 PLA1 has demonstrated potential as a biocatalyst for the production of phospholipid biochemicals.

Academic Editor: Adem Gharsallaoui Keywords: immobilization; phospholipase A1; hydrolysis; Accurel MP100; water content Received: 7 December 2020 Accepted: 29 January 2021 Published: 5 February 2021 1. Introduction Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in Phospholipids are major constituents of cell membranes and play a critical in the published maps and institutional afﬁl- metabolic activity of cells [1]. They have been beneﬁcial used as biocompatible amphiphilic iations. materials for foods, cosmetics and pharmaceutical components. Egg yolks and plant seeds are commonly known as major bioresources of phospholipids [2,3]. Phospholipase A1 (PLA1) is one subgroup of phospholipases with 1-acyl hydrolytic −1 activity. PLA1, particularly, hydrolyzes the ester bond at the sn position of a phospho-

Copyright: © 2021 by the authors. lipid [4,5], and therefore it is commonly employed to modify the fatty acid ester bond at −1 Licensee MDPI, Basel, Switzerland. the sn position. Fatty acid esters act as excellent biocompatible emulsiﬁers for the food This article is an open access article and pharmaceutical industries such as polyglycerol esters of fatty acids or sugar ester [6,7]. distributed under the terms and In the industrial applications for biotransformation, enzyme immobilization is a key tech- conditions of the Creative Commons nique that ensures the recycling of the biocatalyst [8–10]. However, the arising challenge Attribution (CC BY) license (https:// is to assess the industrial applications of hydrophobic reactions. Particularly, accurate creativecommons.org/licenses/by/ selection of lipase carrier which has good compatibility with hydrophobic solvent, requires 4.0/).

Appl. Sci. 2021, 11, 1456. https://doi.org/10.3390/app11041456 https://www.mdpi.com/journal/applsci Appl. Sci. 2021, 11, 1456 2 of 13

special attention. The enhanced reproducibility and repeatability allow for the continuous easy separation of the product and reduces the cost of the biochemical processing [11–13]. Hydrophobic enzyme reactions normally contain an antinomy. A fatty acid substrate needs a hydrophobic solvent [14–16]. However, enzymes dislike hydrophobic surroundings as they require a certain amount of water molecules to form a special molecular structure for reactivity. Therefore, the optimal hydrophobic-hydrophilic condition of the reaction media in a hydrophobic enzyme reaction is critical to realize the maximum yields of lipase-aided biochemical reactions. The optimal water content in water-in-oil (W/O) microemulsion systems has been the focus of previous investigations [17,18]. However, the relationship with the water content in the immobilized enzyme reaction system has not been reported in previous study in this field. Therefore, the investigation of the water content under the immobilized enzyme reaction should be considered and optimized. Stable reactivity by repeated use of immobilized lipases is a dominant factor to determine suitable carriers. Previously, hydrophilic gels (such as silica gel) and solid porous carriers have been employed for reactions with hydrophobic substrates. In the case of lipases, hydrolysis, synthesis, and esterification should be considered for industrial applications. Table1 summarizes previously studied hydrolysis, synthesis and esterification reactions using immobilized lipases and phospholipases.

Table 1. Previous hydrophobic substrate reactions using immobilized lipases and phospholipases.

Enzyme Carrier Substrate Solvent Reaction Ref. Lipase from Candida rugosa Polypropylene Triolein Isooctane Hydrolysis [19] Lipase from Candida rugosa Zeolite Triglycerides Supercritical carbon dioxide Hydrolysis [20] Lipase B from Candida antractia Silica support rac-1-phenylethanol Ionic liquid/hexane Synthesis [21] Acrylic resin Lipase B from Candida antractia Sorbic acid Glycerol Esteriﬁcation [22] immobead 150

Phospholipase A2 Ion exchange support Lecithin Triton Hydrolysis [23]

Phospholipase A1 Microporous resin Phosphatidylcholine Supercritical carbon dioxide Synthesis [24]

The suitable immobilization of an enzyme allows for its long-term repeated use and higher purity of the product without any by-product separation. The physicochemical characteristics of the surface of the carrier and the morphological structure of the carrier particles determine the lifetime of the immobilized enzymes in the bioreactor. Immobilized enzymes are more tolerant to organic solvents, heat, and shearing forces and are much easier to recover than free lipases. Thus, these enzymes are more suitable for industrial applications. However, although a reaction component can be produced, there is a demand for higher reaction activity and reuse. Therefore, it is important to ﬁnd the optimized carrier of PLA1 for immobilization. Among the carriers used for immobilization of enzymes, Accurel has been widely used. This polymer has a highly hydrophobic microporous structure and has been used in this study. Moreover, in the preparation of immobilized enzymes, attention has also been paid to the low inactivation rate of enzymes and the long-term retention rate. However, since there are many types of enzymes, the optimum combination of carrier, enzyme, and reaction conditions should be altered individually. In this study, we adopted a composite method, in which an enzyme was adsorbed on a carrier and then crosslinked with glutaraldehyde (GA) treatment. Since the stabilization by GA treatment depending on the type of carrier and enzyme, the stability of immobilization due to the difference in GA concentration between Accurel MP 100 and Phospholipase A1 was investigated. The objectives of the study were to examine the effects of the optimized reaction conditions on hydrolysis between immobilized PLA1. Particularly, we used Accurel as a carrier to develop a higher immobilized enzyme. Moreover, we investigated phospholipid hydrolysis using an immobilized industrial-class PLA1 on Accurel MP100. The latter was made of a polypropylene-based hydrophobic granular support [25]. We solved this Appl. Sci. 2021, 11, 1456 3 of 13

problem of the water content under the immobilized enzyme reaction and obtained optimized water content. Moreover, we solved the problem about the impact of the enzyme immobilization process and the water content in the organic phase on the kinetic data to achieve maximization on reactivity and repeated productivity of immobilized PLA1 for a long period of time.

2. Materials and Methods 2.1. Materials

Accurel MP100 was provided by Evonik Industries AG (Essen, Germany). PLA1 from Aspergillus oryzae was donated by Mitsubishi-Kagaku Foods Corporation (Tokyo, Japan). It was commercially distributed for industrial enzymatic process use. Lecithin from soybeans was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). The purity of lecithin was assumed to be 60% phosphatidylcholine dipalmitoyl. The organic phase used 2,2,4-trimethylpentane (isooctane) as the main solvent and 1-butanol as the co-solvent. In this study, a volumetric ratio of 10% (v/v) of 1-butanol was employed during the organic phase. Phospholipid was adopted as a typical lipid substrate, and palmitic acid and lysolecithin assumed to be the main reaction products. Glutaraldehyde (GA) was employed as a cross-linker of PLA1 onto the outer and inner pore surface of the Accurel particle. Analytical grade GA was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan).

2.2. Hydrolysis of Phospholipids by Phospholipase A1 in the Water-in-Oil Microemulsion Phase

The enzymatic reactivity of PLA1 was determined by the hydrolysis of the phospholipids in W/O microemulsion. The PLA1 was solubilized into 0.2 M acetate buffer solution (pH 5.0). The enzyme buffer solution was prepared using 0.4 g of PLA1 powder and 10 mL of acetate buffer solution. The organic solution (50 mL) was prepared in 10% (v/v) 1-butaol/isooctane. The phospholipids were dissolved into the organic phase with a concentration of 6–18 mM, calculated based on phosphatidylcholine. The substrate organic solution was magnetically stirred in a thermostatic water bath (313 K). The enzymatic reaction was initiated by introducing PLA1 into the organic solution, which formed a W/O microemulsion. A supplemental amount of acetate buffer solution was then added to the W/O microemulsion to make the desired water content. The enzymatic reactivity was determined by measuring the increase in palmitic acid concentration according to the Lowry-Tinsley colorimetric method [26]. A mixture of cupric acetate aqueous solution (1 mL) and benzene (5 mL) was prepared in a vial. The pH of the cupric acetate aqueous solution was set at 6.0 using the additive pyridine. The stirring speed was 1300 min−1. A sample (1.0 mL) was taken from the reaction solution at the desired running time. The sample was introduced into the benzene phase of the benzene-cupric acetate mixture mentioned above. The solution was vigorously mixed for 1 min using a vortex-mixer (TTM-1, SIBATA, Tokyo, Japan). After centrifugation at 3000 min−1 for 3 min, the transparent upper benzene phase was added mixed for 1 min using a vortex mixer to dehydrate the benzene phase. After centrifugation at 3000 min−1 for 3 min, the optical density of the transparent upper benzene phase was measured by UV spectrophotometer (V-6301RM, JASCO Corporation, Tokyo, Japan) at 715 nm.

2.3. Scanning Electron Microscopy (SEM) Scanning electron microscopy (SEM) was employed to examine the surface of Accurel. The sample was coated with gold and viewed under a high vacuum. A surface of Accurel was conducted using a JSM-6701F scanning electron microscope (JEOL Ltd., Tokyo, Japan) at an acceleration voltage of 10.0 kV. Appl. Sci. 2021, 11, 1456 4 of 13

2.4. Phospholipase A1 Immobilization Method In total, 30 mL ethanol (guaranteed reagent, 99.5 w/w%) was introduced into a 150 mL capacity glass vial with an inner diameter of 45 mm. Accurel particles (1 g) were immersed into ethanol and then irradiated using an ultrasonic water bath (UT-206, SHARP Corporation, Osaka, Japan) for 60 min under 200 W. Accurel MP100 was pretreated by ethanol for modification of surface hydrophobicity [25]. PLA1 was simply adsorbed onto the Accurel MP100 carrier before the crosslinked immobilization. The desired amount of PLA1 was added to the acetate buffer solution (pH 5.0). The concentration of PLA1 was set at 10 g/L. The Accurel carrier was placed into the PLA1 solution and was then shaken to adsorb PLA1 for at least 24 h at 298 K. The unwashed wetted carrier was moderately dried in a desiccator for 24 h at room temperature (298 ± 2 K). The amount of adsorbed PLA1 on the Accurel carrier was evaluated from the mass balance based on decreasing concentration of PLA1 in the buffer solution. The dried Accurel carrier with the adsorbed PLA1 was then moderately shaken in a GA aqueous solution (CGA: 0–10%) for 60 min at room temperature to immobilize the PLA1 on the Accurel pore surfaces. The amount of PLA1 desorbed in the GA solution was then measured using the Bradford method [27,28], with the aid of a Protein quantification Kit-Rapid (Dojindo Molecular Technologies, Inc., Tokyo, Japan). The effective amount of immobilized PLA1 was estimated from the difference between the adsorbed and desorbed amounts of PLA1. The immobilized PLA1 carrier was briefly washed in distilled water and moderately dried in the desiccators for 2 days at room temperature.

2.5. Hydrolysis of Phospholipids Using Immobilized Phospholipase A1 on an Accurel Carrier The organic solvent was prepared with 10% (v/v) 1-butanol/isooctane. The initial concentration of the phospholipid was in the range of 6−18 mM, based on phosphatidylcholine. The substrate was stirred into the organic solution in a thermostatic water bath (313 K), and the aqueous buffer solution without PLA1 was added to the solution containing the substrate (phospholipid). It was stirred magnetically for 10 min to produce an optically transparent and mature W/O microemulsion phase. In this study, phospholipid acted not only as a substrate of hydrolysis but also as an amphiphilic component to prepare W/O microemulsion. The reaction was initiated when the immobilized PLA1 on the Accurel carrier was added to the W/O microemulsion phase. The concentration of palmitic acid in the sample after the initial reaction was measured using the same protocol as described in Section 2.2.

2.6. Statistical Analysis The experiments were performed in triplicates and the data deviation was expressed by error bars. To clear presentation on data for readers, the error bars were eliminated reluctantly in Figures 2, 5, 7, 9, and 11. In this study, the statistical analysis was examined that was results valid statistically signiﬁcant if p ≤ 0.05.

3. Results and Discussion 3.1. Progress of Hydrolysis Reaction in Water-in-Oil Microemulsion by Free Industrial-Class Phospholipase A1 Figure1 presents a schematic illustration of the hydrolysis of phospholipid by non- immobilized PLA1 containing W/O microemulsion. Figure2 shows the time course of the hydrolysis of phospholipid hydrolysis by PLA1 in the W/O microemulsion phase. The concentration of palmitic acid (Cp) was monitored as a typical product of the phospholipid hydrolysis, and the amounts of buffer solution containing PLA1 in the W/O microemulsion phase (Hb) were adjusted by the supplemental amount of buffer solution. In this study, the volume of the organic phase (butanol + isooctane) was set at 50 mL throughout the measurements. The reaction was initiated quickly with no reaction delay. The aqueous buffer solution was dispersed with Appl. Sci. 2021, 11, x FOR PEER REVIEW 5 of 15

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Appl. Sci. 2021, 11, 1456 3. Results and Discussion 5 of 13 3.1. Progress of Hydrolysis Reaction in Water-in-Oil Microemulsion by Free Industrial-Class Phospholipase A1 theFigure aid of W/O1 presents microemulsion. a schematic Whenillustration the Hb of wasthe hydrolysis 0.3 mL, the of concentration phospholipid of by palmitic non- immobilizedacid was superior PLA1 containing to other amounts W/O microemulsion. of water.

Figure 1. Schematic illustration of hydrolysis reaction in water-in-oil (W/O) microemulsion of non- immobilized phospholipase A1 (PLA1).

Figure 2 shows the time course of the hydrolysis of phospholipid hydrolysis by PLA1 in the W/O microemulsion phase. The concentration of palmitic acid (Cp) was monitored as a typical product of the phospholipid hydrolysis, and the amounts of buffer solution containing PLA1 in the W/O microemulsion phase (Hb) were adjusted by the supplemental amount of buffer solution. In this study, the volume of the organic phase (butanol + isooctane) was set at 50 mL throughout the measurements. The reaction was initiated quickly with no reaction delay. The aqueous buffer solution was dispersed with the aid of FigureFigure 1. 1.SchematicSchematic illustration illustration of hydrolysis of hydrolysis reaction reaction in water-in-oil in water-in-oil (W/O) (W/O) microemulsion microemulsion of non- of W/O microemulsion. When the Hb was 0.3 mL, the concentration of palmitic acid was immobilizednon-immobilized phospholipase phospholipase A1 (PLA A1).(PLA 1). superior to other amounts of water.

Figure 2 shows the time course of the hydrolysis of phospholipid hydrolysis by PLA1 in the W/O microemulsion phase. The concentration of palmitic acid (Cp) was monitored as a typical product of the phospholipid hydrolysis, and the amounts of buffer solution containing PLA1 in the W/O microemulsion phase (Hb) were adjusted by the supplemental amount of buffer solution. In this study, the volume of the organic phase (butanol + isooctane) was set at 50 mL throughout the measurements. The reaction was initiated quickly with no reaction delay. The aqueous buffer solution was dispersed with the aid of W/O microemulsion. When the Hb was 0.3 mL, the concentration of palmitic acid was superior to other amounts of water.

Figure 2. Time course ofof producedproduced palmiticpalmitic acid acid concentration concentration in in W/O W/O microemulsion. microemulsion. The The amounts amounts of buffer solution containing phospholipase A1 in the W/O microemulsion phase, Hb, of buffer solution containing phospholipase A1 in the W/O microemulsion phase, Hb, were 0.2 (•), were 0.2 (●), 0.225 (★), 0.25 (○), 0.3 (▼), 0.35 (▽), and 0.4 (■) mL. The experiments were each con- 0.225 (F), 0.25 ( ), 0.3 (H), 0.35 (5), and 0.4 () mL. The experiments were each conducted a ducted a minimum of three times. minimum of three# times.

3.2. Immobilized Phospholipase A1 on Accurel Particle

Figure3 depicts the adsorbed amount of PLA 1 on the pretreated Accurel MP100 particles. The adsorbed amount increased with the increasing concentration of GA, and the maximum adsorbed amount appeared when the GA concentration was 6%. Figure 2. Time course of produced palmitic acid concentration in W/O microemulsion. The amounts of buffer solution containing phospholipase A1 in the W/O microemulsion phase, Hb, were 0.2 (●), 0.225 (★), 0.25 (○), 0.3 (▼), 0.35 (▽), and 0.4 (■) mL. The experiments were each conducted a minimum of three times. Appl. Sci. 2021, 11, x FOR PEER REVIEW 6 of 15

3.2. Immobilized Phospholipase A1 on Accurel Particle

Appl. Sci. 2021, 11, 1456 Figure 3 depicts the adsorbed amount of PLA1 on the pretreated Accurel MP100 par- 6 of 13 ticles. The adsorbed amount increased with the increasing concentration of GA, and the maximum adsorbed amount appeared when the GA concentration was 6%.

Figure 3. Comparison of the adsorbed amount of phospholipase A1 per unit mass of Accurel Figure 3. Comparison of the adsorbed amount of phospholipase A1 per unit mass of Accurel MP100. MP100.

3.3. Reactivity of Immobilized Phospholipase A1 3.3. Reactivity of Immobilized Phospholipase A1 3.3.1. Effect of Glutaraldehyde as a Cross-Linker of Phospholipase A1 3.3.1. Effect of Glutaraldehyde as a Cross-Linker of Phospholipase A1 Figure4a illustrated the schematic image of substrate-based W/O microemulsion Figure 4a illustrated the schematic image of substrate-based W/O microemulsion as- associationsociation with with immobilized immobilized PLA1. The PLA W/O1. Themicroemulsion W/O microemulsion droplet readily dropletdiffused into readily diffused intothe inner the pore inner channeled pore channeled under the underlower wate ther lowercontent waterconditions. content The pore conditions. size at the The pore size atAccurel the Accurelsurface was surface found wasto be found1–5 μm to(SEM be images). 1–5 µm Figure (SEM 4b images). demonstrates Figure that4 sub-b demonstrates thatstrate substrate phospholipid phospholipid molecules were molecules located in were the outer located surrounding in the outer of the surrounding W/O micro- of the W/O Appl. Sci. 2021, 11, x FOR PEER REVIEWemulsion droplet. It is readily associated with immobilized PLA1. The hydrolysis reaction7 of 15 microemulsion droplet. It is readily associated with immobilized PLA1. The hydrolysis reactionwas quickly was initiated, quickly and initiated, a high reaction and a rate high was reaction realized. rate was realized. To achieve the highest yield in phospholipids hydrolysis using immobilized PLA1, the Accurel MP100 with glutaraldehyde treatment and the amount of water content in W/O microemulsion were the key factors. These are especially important for the industrial production of enzymes.

FigureFigure 4. ( 4.a) (Schematica) Schematic illustration illustration of pore ofstructure pore structureand W/O microemulsion and W/O microemulsion droplets association droplets association withwith immobilized immobilized phospholipase phospholipase A1 on the A outer1 on and the inner outer surfac andes innerof microspores surfaces in ofAccurel microspores in Accurel particles.particles. (b)( Phospholipid-basedb) Phospholipid-based W/O microemulsion W/O microemulsion droplets readily droplets diffused readily into diffused the micro- into the microspores spores of the PLA1 carrier. of the PLA1 carrier. Figure 5 shows the time course of concentration of palmitic acid using the immobilized PLA1 on Accurel with glutaraldehyde treatment. The enzyme reactions were quickly initiated regardless of the concentrations of GA. When the GA concentrations were 10% and 6%, the amounts of produced palmitic acid were the highest. The next highest production was for GA concentrations of 3%. There is no difference in the amount of production between 1.5% and 0% GA concentrations. Appl. Sci. 2021, 11, 1456 7 of 13

To achieve the highest yield in phospholipids hydrolysis using immobilized PLA1, the Accurel MP100 with glutaraldehyde treatment and the amount of water content in W/O microemulsion were the key factors. These are especially important for the industrial production of enzymes. Figure5 shows the time course of concentration of palmitic acid using the immobilized PLA1 on Accurel with glutaraldehyde treatment. The enzyme reactions were quickly initiated regardless of the concentrations of GA. When the GA concentrations were 10% and 6%, the amounts of produced palmitic acid were the highest. The next highest production

Appl. Sci. 2021, 11, x FOR PEER REVIEWwas for GA concentrations of 3%. There is no difference in the amount8 of 15 of production between 1.5% and 0% GA concentrations.

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Figure 5. Time course of palmitic acid production (Cp) using immobilized phospholipase A1 in Figure 5. Time course of palmitic acid production (Cp) using immobilized phospholipase A1 in various glutaraldehyde concentrations. The glutaraldehyde concentrations, CGA were 0% (○), 1.5% (various●), 3% (★), glutaraldehyde 6% (▼), and 10% (■ concentrations.). The experiments were The each glutaraldehyde conducted a minimum concentrations, of three CGA were 0% ( ), times.1.5% (•), 3% (F), 6% (H), and 10% (). The experiments were each conducted a minimum# of threeFigure times.5. Time course of palmitic acid production (Cp) using immobilized phospholipase A1 in variousDespite glutaraldehyde the small concentrations. difference in Thethe glutaraldehydeimmobilized amount concentrations, of PLA C1GA between were 0% GA(○), 1.5%concentrations(●), 3% (★), 6%of 0–10%,(▼), and the 10% amount (■). The ofexperiments produced were palmitic each conductedacid varied a minimum dramatically. of three There- Despite the small difference in the immobilized amount of PLA between GA concen- fore,times. the GA concentration not only effectively immobilized the PLA1 but also acted1 as a hydrophobictrations of modifier 0–10%, of the the amountcarrier surface. of produced palmitic acid varied dramatically. Therefore, Despite the small difference in the immobilized amount of PLA1 between GA con- theFigure GA concentration 6 shows the effect not of the only initial effectively hydrolysis reaction immobilized rate for immobilized the PLA1 butPLA1 also acted as a hy- ondrophobiccentrations the GA treatment of modiﬁer 0–10%, as the a ofcross-linker amount the carrier of ofproduced PLA surface.1. The palmitic initial acidreaction varied rate dramatically. increased with There- GA concentrationfore, the GA concentration and then dropped not only for effectivelyGA concentrations immobilized exceeding the PLA 6%.1 but According also acted to asthe a Figure6 shows the effect of the initial hydrolysis reaction rate for immobilized PLA1 results,hydrophobic a GA concentrationmodifier of the of carrier 6% was surface. optimal for effective immobilization of PLA1 and allowedon theFigure to GA attain 6 treatmentshows the therequired effect as a initiatedof cross-linker the initial reaction hydrolysis of rate PLA and reaction1 reaction. The rate initial yield. for immobilized reaction rate PLA1 increased with GA concentrationon the GA treatment and as thena cross-linker dropped of PLA for1.GA The initial concentrations reaction rate increased exceeding with 6%. GA According to the concentration and then dropped for GA concentrations exceeding 6%. According to the results, a GA concentration of 6% was optimal for effective immobilization of PLA1 and results, a GA concentration of 6% was optimal for effective immobilization of PLA1 and allowedallowed toto attain attain the required the required initiated initiated reaction rate reaction and reaction rate andyield. reaction yield.

Figure 6. Effect of immobilized phospholipase A1 with glutaraldehyde treatment on the initial reaction rate.

Figure 6. Effect of immobilized phospholipase A1 with glutaraldehyde treatment on the initial Figure 6. Effect of immobilized phospholipase A1 with glutaraldehyde treatment on the initial reaction rate. reaction rate.

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3.3.2. Effect of Water Content on Production

3.3.2. EffectFigure of7 Water shows Content the effect on Production of the amount of buffer solution into the W/O microemulsion phaseFigure on the7 shows reactivity the effect of of immobilized the amount of PLAbuffer1 .solution The productivity into the W/O microemul- of the enzyme reaction was dependentsion phase on the on reactivity the supplemental of immobilized aqueous PLA1. The volume productivity in the of the W/O enzyme microemulsion reaction phase. The palmiticwas dependent acid on concentration the supplemental produced aqueous volume increased in the quicklyW/O microemulsion with the reactionphase. time and then droppedThe palmitic for acid over concentration 40 min. When produced the in Hbcreased was quickly 0.075 mL,with the reaction highest time amount and of palmitic acid then dropped for over 40 min. When the Hb was 0.075 mL, the highest amount of palmitic wasacid was produced. produced.

Figure 7.7. TimeTime course course of palmitic of palmitic acid concentration acid concentration (Cp) produced (Cp) with produced immobilized with phospho- immobilized phospholipase lipase A1 in various amounts of water. The volumetric amount of the supplemental acetate buffer Asolution,1 in various Hb was amountschanged as of follows; water. 0 ( The★), 0.05 volumetric (●), 0.075 (○ amount), 0.1 (▼), ofand the 0.2 supplemental(■) mL. The CGA acetate buffer solution, Hbwas wasset at changed6%. The experiments as follows; were 0 (eachF), conducted 0.05 (•), 0.075a minimum ( ), 0.1of three (H), times. and 0.2 () mL. The CGA was set at 6%. The experiments were each conducted a minimum# of three times. 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 Figure8 shows the change of the initial reaction rate with the concentration of lecithin a recalculated value which was a per unit mass of PLA1. The reactivity of immobilized usingPLA1 indicated a soybean by the dissolved initial reaction in the rate organic per unit mass phase. of PLA The1 was initial well reaction maintained, rate and in this ﬁgure was a recalculatedthe activity damage value was which fully su wasppressed. a per Industrial-class unit mass of PLA1 1was. The successfully reactivity immo- of immobilized PLA1 bilized onto the Accurel particles with the aid of GA aqueous solution. indicated by the initial reaction rate per unit mass of PLA1 was well maintained, and the activity damage was fully suppressed. Industrial-class PLA was successfully immobilized Appl. Sci. 2021, 11, x FOR PEER REVIEW 1 10 of 15 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- Figure 8. Relationship between the substrate concentration and the initial reaction rate for non- immobilized phospholipase A1 in W/O microemulsion (■) at Hb = 0.3 mL, immobilized phospho- immobilizedlipase A1 (●) at Hb phospholipase = 0.075 mL. The Aconcentration1 in W/O of microemulsion glutaraldehyde for ( immobilization) at Hb = 0.3 was mL, 6%. immobilized phospholi- Vi*: initial reaction rate of hydrolysis of phospholipid per unit mass of PLA1. pase A1 (•) at Hb = 0.075 mL. The concentration of glutaraldehyde for immobilization was 6%. Vi*: initialFigure reaction 9 shows rate the of relationship hydrolysis ofbetween phospholipid the initial perreaction unit rate mass and of the PLA molar1. 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: [phosphatidylcholine dipalmitoyl] W= (1) [HO]

Figure 9. Effect of the water content on the initial reaction rate (Vi) in immobilized phospholipase A1. W/O microemulsion system with non-immobilized phospholipase A1 was chosen as a reference (★). The glutaraldehyde concentration, CGA was 1.5% (●), 3% (○), 6% (▼), and 10% (■). The experiments were each conducted a minimum of three times.

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Appl. Sci. 2021, 11, 1456 Figure 8. Relationship between the substrate concentration and the initial reaction rate for non-9 of 13 immobilized phospholipase A1 in W/O microemulsion (■) at Hb = 0.3 mL, immobilized phospholipase A1 (●) at Hb = 0.075 mL. The concentration of glutaraldehyde for immobilization was 6%. Vi*: initial reaction rate of hydrolysis of phospholipid per unit mass of PLA1. Figure9 shows the relationship between the initial reaction rate and the molar ratio of waterFigure to 9 lecithin, shows the W. relationship In this study, between the W the value initial was reaction deﬁned rate as and the the molar molar ratio ratio of supplementalof water to lecithin, water W. moles In this to the study, phosphatidylcholine the W value was dipalmitoyl defined as the moles: molar ratio of supplemental water moles to the phosphatidylcholine dipalmitoyl moles: [phosphatidylcholine dipalmitoyl] W = (1) [phosphatidylcholine[ ] dipalmitoyl] W= H2O (1) [HO]

Figure 9. Effect ofof thethe waterwater contentcontenton on the the initial initial reaction reaction rate rate (Vi) (Vi) in in immobilized immobilized phospholipase phospholipase A1 . A1. W/O microemulsion system with non-immobilized phospholipase A1 was chosen as a refer- W/O microemulsion system with non-immobilized phospholipase A1 was chosen as a reference (F). ence (★). The glutaraldehyde concentration, CGA was 1.5% (●), 3% (○), 6% (▼), and 10% (■). The The glutaraldehyde concentration, CGA was 1.5% (•), 3% ( ), 6% (H), and 10% (). The experiments experiments were each conducted a minimum of three times. were each conducted a minimum of three times. #

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 PLA1 was obtained for W = 7. The maximum initial reaction rate in non-immobilized PLA1 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 [34–36]. 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 PLA1 in Accurel were more hydrophobic than that of the non-immobilized PLA1 in the W/O microemulsion phase. In a previous study, the maximum reactivity of immobilized lipase, W = 10, was achieved using AOT [38]. Appl. Sci. 2021, 11, 1456 10 of 13

In general, the size of the W/O microemulsion droplet was dependent upon the molar ratio of water and amphiphiles. According to the AOT system used previously, the characteristic radius (RC) of the W/O microemulsion was linearly proportional to the molar ratio of water and amphiphile AOT. The correlation formula was previously reported as RC (Å) = 1.5 W [33]. The molecular size of lecithin was roughly assumed to be 10 times that of the AOT molecule. The outer diameter of the W/O microemulsion was estimated as 420 Å (42 nm) for W = 7.

3.4. Repeated Use of Immobilized Phospholipase A1 for High Yield Production Figure 10 shows the concentration of palmitic acid after 60 min and the initial reaction rate in the repeated use of immobilized PLA1. The initial reaction rate and the production of palmitic acid until repeated ﬁve times were almost constant and then remained at 40% of the initial value after its sixth repetition. The results promise practical applications of immobilized industrial-class PLA1 onto Accurel particles for the prospective production of phospholipid chemicals. Repeated used was conducted 10 times in this study. Reactivity Appl. Sci. 2021, 11, x FOR PEER REVIEW 12 of 15 of immobilized PLA1 was regrettably decayed according to repeated number. Effective reactivation of immobilized PLA1 was main limitation was this application. Appl.Appl. Sci.Sci. 20212021,, 1111,, xx FORFOR PEERPEER REVIEWREVIEW 1212 ofof 1515

FigureFigure 10.10. ComparisonComparison ofof thetheFigure repeatedrepeated 10.FigureComparison initialinitial 10. reactionreaction Comparison of the raterate repeated andand ofreactionreaction initialthe repeated yields reactionyields ofof rateinitial hydrolysishydrolysis and reaction reaction withwith rate yields and of hydrolysisreaction yields with immobilized of hydrolysis phospholipase with immobilizedimmobilized phospholipasephospholipaseA AA111(Hb: (Hb:(Hb: 0.075 0.0750.075immobilized mL, mL,mL, C CCGAGAGA:: 6%).phospholipase 6%).6%). : : InitialInitial Initial A reactionreaction reaction1 (Hb: 0.075 rate,rate, Vi. Vi. mL, CGA : : Reac- Reaction:Reac- 6%). product : Initial (palmitic reaction acid) rate, concentration, Vi. : Reac- Cp. tiontion productproduct (palmitic(palmitic acacid)id) concentration,concentration,tion product Cp.Cp. (palmitic acid) concentration, Cp. Figure 11 indicates the accumulated yield of palmitic acid after 60 min using immobi- Figure 11 indicates the accumulated yield of lizedpalmitic PLA acidthroughout after 60 min 10 cycle’s using repetition.immo- Accumulated production was 3.5-fold superior Figure 11 indicates the accumulatedFigure yield 11 of indicatespalmitic acid1 the after accumulated 60 min using yield immo- of palmitic acid after 60 min using immo- bilizedbilized PLAPLA11 throughoutthroughout 1010 cycle’scycle’s repetition.repetition. AccumulatedAccumulatedto that of a production singleproduction use of waswas non-immobilized 3.5-fold3.5-fold su-su- PLA1. Immobilization of industrial-class PLA1 bilized PLA1 throughout 10 cycle’s repetition. Accumulated production was 3.5-fold su- periorperior toto thatthat ofof aa singlesingle useuse ofof non-immobilizednon-immobilized PLAPLAwas11. successfully. ImmobilizationImmobilization realized ofof industrial-classindustrial-class as a promising biocatalyst for production of the phospholipid perior to that of a single use of non-immobilized PLA1. Immobilization of industrial-class PLAPLA11 waswas successfullysuccessfully realizedrealized asas aa promisingpromising biocatalystbiocatalystbiochemicals. forfor productionproduction ofof thethe phospho-phospho- lipidlipid biochemicals.biochemicals. PLA1 was successfully realized as a promising biocatalyst for production of the phospholipid biochemicals.

FigureFigure 11.11. AccumulatedAccumulated yieldyield ofof palmiticpalmitic acidacid usingusing immobilizedimmobilized phospholipasephospholipase AA11 inin W/OW/O micro-micro- emulsionemulsion system.system. ItIt waswas comparedcompared wiwithth non-immobilizednon-immobilized phospholipasephospholipase AA11 (non-immobilized(non-immobilized phospholipasephospholipase AA11).). Here,Here, HbHb waswas 0.0750.075 mL,mL, andand CCGAGA waswas 6%,6%, respectively.respectively. TheThe experimentsexperiments werewere eacheach conductedconducted aa minimumminimum ofof threethree times.times.Figure Cp*:Cp*: 11. concconcAccumulatedentrationentration ofof yield palmiticpalmitic of palmitic acidacid fromfrom acid hydrolysishydrolysis using immobilized ofof phospholipase A1 in W/O micro- phospholipidphospholipid perper unitunit massmass ofof PLAPLA11.. emulsion system. It was compared with non-immobilized phospholipase A1 (non-immobilized phospholipase A1). Here, Hb was 0.075 mL, and CGA was 6%, respectively. The experiments were 4.4. ConclusionsConclusions each conducted a minimum of three times. Cp*: concentration of palmitic acid from hydrolysis of phospholipid per unit mass of PLA1. MaximizationMaximization ofof immobilizedimmobilized industrial-classindustrial-class phospholipasephospholipase AA11 waswas successfullysuccessfully re-re- alizedalized byby regulationregulation ofof waterwater contentcontent ofof W/W/OO microemulsionmicroemulsion phasephase asas aa reactionreaction media.media. Industrial-classIndustrial-class PLAPLA11 waswas successfullysuccessfully4. Conclusions immobilizedimmobilized ontoonto aa polypropylenepolypropylene porousporous particleparticle (Accurel(Accurel MP100)MP100) withwith thethe aidaid ofof glutarglutaraldehyde.aldehyde.Maximization TheThe yieldyield of immobilized ofof immobilizedimmobilized industrial-class PLAPLA11 waswas overover phospholipase A1 was successfully realized by regulation of water content of W/O microemulsion phase as a reaction media. Industrial-class PLA1 was successfully immobilized onto a polypropylene porous particle (Accurel MP100) with the aid of glutaraldehyde. The yield of immobilized PLA1 was over Appl. Sci. 2021, 11, x FOR PEER REVIEW 12 of 15

Figure 10. Comparison of the repeated initial reaction rate and reaction yields of hydrolysis with immobilized phospholipase A1 (Hb: 0.075 mL, CGA: 6%). : Initial reaction rate, Vi. : Reac- tion product (palmitic acid) concentration, Cp.

Figure 11 indicates the accumulated yield of palmitic acid after 60 min using immobilized PLA1 throughout 10 cycle’s repetition. Accumulated production was 3.5-fold su- Appl. Sci. 2021, 11, 1456 perior to that of a single use of non-immobilized PLA1. Immobilization of industrial-class11 of 13 PLA1 was successfully realized as a promising biocatalyst for production of the phospholipid biochemicals.

Figure 11. 11. AccumulatedAccumulated yield yield of of palmitic palmitic acid acid using using immobilized immobilized phospholipase phospholipase A1 Ain1 W/Oin W/O micro- mi- emulsion system. It was compared with non-immobilized phospholipase A1 (non-immobilized croemulsion system. It was compared with non-immobilized phospholipase A1 (non-immobilized phospholipase A1). Here, Hb was 0.075 mL, and CGA was 6%, respectively. The experiments were phospholipase A1). Here, Hb was 0.075 mL, and CGA was 6%, respectively. The experiments were each conducted a minimum of three times. Cp*: concentration of palmitic acid from hydrolysis of each conducted a minimum of three times. Cp*: concentration of palmitic acid from hydrolysis of phospholipid per unit mass of PLA1. phospholipid per unit mass of PLA1. 4. Conclusions Conclusions

Maximization of of immobilized immobilized industrial-class industrial-class phospholipase phospholipase A1A was1 was successfully successfully realizedrealized by by regulation regulation of of water water content content of of W/ W/OO microemulsion microemulsion phase phase as as a a reaction reaction media. media. Industrial-classIndustrial-class PLA 1 waswas successfully successfully immobilized immobilized onto a polypropylene polypropylene porous particle (Accurel MP100) MP100) with with the the aid aid of of glutar glutaraldehyde.aldehyde. The The yield yield of immobilized of immobilized PLA PLA1 was1 overwas over 90% based on simple adsorption. A hydrolysis reaction with the immobilized PLA1 was quickly initiated without any time lag. The minimization of activity loss of the PLA1 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 PLA1 increased with an increasing glutaraldehyde concentration. A glutaraldehyde concentration of 6% provided a sufﬁciently stable immobilization of PLA1 and the initial reaction rate. The initial reaction rate could be compared with that of the non-immobilized PLA1 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 PLA1. 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 PLA1 under the appropriate regulation of the water content in the W/O microemulsion phase. Industrial-class immobilized PLA1 on Accurel MP100 has been suggested as a promising biocatalyst to product phospholipid biochemicals.

Author Contributions: 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. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Appl. Sci. 2021, 11, 1456 12 of 13

Acknowledgments: Phospholipase A1 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.

Nomenclature Am the adsorbed amount of PLA1 on Accurel MP100 [g-PLA1/g-Accurel] CGA concentration of glutaraldehyde to immobilize PLA1 on Accurel MP100 [wt%] Cp concentration of palmitic acid from hydrolysis of phospholipid [mM] concentration of palmitic acid from hydrolysis of phospholipid per unit mass of Cp* PLA1 [mM/g] Cs concentration of soybean phospholipid [mM] the supplemental amount of buffer solution in the W/O microemulsion phase Hb [mL] (volume of the organic phase was set at 50 mL throughout this study) RC characteristic radius of the W/O microemulsion [Å] t reaction time [min] Vi initial reaction rate of hydrolysis of phospholipid [µM/s] initial reaction rate of hydrolysis of phospholipid per unit mass of Vi* PLA1 [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®OT”. It is a typical anionic AOT amphiphilic reagent. The main component of AOT is sodium dioctyl sulfosuccinate GA glutaraldehyde PLA1 phospholipase A1 SEM scanning electron microscopy W/O water-in-oil

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