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Immobilized MAS1 Lipase-catalyzed Synthesis of n-3 PUFA-rich Triacylglycerols in Deep Eutectic Solvents

and glycerolysis reactions prepare n-3 PUFA-rich TAG. Nevertheless, the synthesized n-3 PUFA-rich TAG content is low and the separation and pu-rification of the complex product are needed when enzymatic reactions are carried out in the solvent-free systems 8, 14, 15 ） . Although n-3 PUFA-rich TAG content is relatively high when enzymatic reactions are performed in the organic solvent systems, these organic solvents could cause food safety Abstract: n-3 polyunsaturated fatty acids (PUFA)-rich triacylglycerols (TAG) with many beneficial effects are still difficult to be synthesized efficiently and rapidly by current synthetic techniques. This study reports the fatty acid specificity of immobilized MAS1 lipase and its efficient synthesis of n-3 PUFA-rich TAG by esterification of glycerol with n-3 PUFA in natural deep eutectic solvents (NADES) systems. Immobilized MAS1 lipase showed the highest preference for capric acid [C10:0, the highest specificity constant (1/α)=1] whereas it discriminated strongly against docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) due to their lowest specificity constants (1/α=0.19 and 0.2). Moreover, the highest n-3 PUFA-rich TAG content (55.8%) with similar n-3 PUFA composition to the substrate was obtained in choline chloride/glycerol (CG) system. There was a 1.38-fold increase of TAG content in CG system compared with that in the solvent-free system. Interestingly, immobilized MAS1 lipase exhibited no regiospecificity in the solvent-free and various NADES systems. Besides, the potential reaction mechanism of immobilized MAS1 lipase-catalyzed esterification of glycerol with n-3 PUFA in NADES systems was described. It was found that the use of NADES as solvents could greatly enhance TAG content, and make it easy to separate the product. These results indicated that immobilized MAS1 lipase is a promising biocatalyst for the efficient synthesis of n-3 PUFA-rich TAG by esterification of glycerol with n-3 PUFA in NADES systems.

and environmental pollution 16,17 . Therefore, the exploration of green and more effective solvents for the synthesis of high content of n-3 PUFA-rich TAG and its easy separation is of great importance.
Recently, natural deep eutectic solvents NADES have received considerable attention as new generation of solvent due to their easier preparation, lower toxicity, and higher biodegradability when compared with conventional organic solvents 18,19 . Moreover, many studies have showed that NADES could facilitate the separation of products, enhance the yields, and protect the enzymes from inactivation 20,21 . Thus, NAEDS have been widely used as green media in enzymatic reactions for the synthesis of various epoxidized oil, glycolipids, and biodiesel 22 24 . However, there is little information about using NADES as solvents for the enzymatic synthesis of n-3 PUFA-rich TAG. Therefore, it would be an ideal choice to perform the enzymatic production of n-3 PUFA-rich TAG in some NADES systems.
Immobilized lipases are more desirable because they allow for easy recyclability, good resistant to environmental changes, good durability, high stability, activity and selectivity when compared with free lipases 25,26 . Moreover, studies have showed that the stability and recovery of immobilized lipase can be enhanced using NADES as reaction solvent 27 29 . Recently, an immobilized MAS1 lipase using XAD1180 resin as a carrier, which was from marine Streptomyces sp. strain W007 30 , was found to exhibit high catalytic activity during enzymatic esterification, glycerolysis, and transesterification reactions in the solvent-free system 31 33 . However, the fatty acid FA specificity of immobilized MAS1 lipase and its catalytic performances during enzymatic production of n-3 PUFA-rich TAG in NADES systems are generally unexplored. Therefore, the aim of this study was to assess the substrate specificity of immobilized MAS1 lipase and its catalytic performances for the production of n-3 PUFA-rich TAG during the esterification of n-3 PUFA with glycerol in some NADES systems. First, the fatty acid specificity of immobilized MAS1 lipase was evaluated in a multi-competitive esterification reaction. Subsequently, the abilities of immobilized MAS1 lipase to catalyze the esterification of n-3 PUFA with glycerol in the solvent-free and four types of NADES systems were compared and the analysis of n-3 PUFA compositions of the obtained n-3 PUFA-rich TAG in the different reaction systems were carried out in this study. Finally, the potential reaction mechanism of immobilized MAS1 lipase-catalyzed esterification of glycerol with n-3 PUFA in various NADES systems was discussed.

Materials
Lipase MAS1 PDB ID: 5H6B was produced by the method described earlier 34 . DHA/EPA-rich EE were sourced from Sinomega Biotech Engineering Co., Ltd. Zhejiang, China . Standards of 37-component fatty acid methyl esters FAME mix C 4 -C 24 , monooleoylglycerol, dioleoylglycerol 15 of 1,2-dioleoylglycerol and 85 of 1,3-dioleoylglycerol , trioleoylglycerol were purchased from Sigma-Aldrich. Xylitol 98 , choline chloride 98 , betaine 98 , anhydrous , glycerol 99 , and urea 99 were all sourced from Aladdin Chemistry Co. Ltd. Shanghai, China . Formic acid, n-hexane and 2-propanol of chromatographic grade were obtained from Kermel Chemical Reagent Co., Ltd. Tianjin, China . All other chemicals were of analytical grade unless otherwise stated. In this study, the composition of n-3 PUFA was the sum of the composition of EPA, docosapentaenoic acid DPA and DHA.

Preparation of immobilized MAS1 lipase
Lipase MAS1 was immobilized using XAD1180 resin hydrophobic support as a carrier according to the method described previously 31 . First, the supernatant of crude lipase MAS1 75 mg/g resin was mixed with an equal volume of 0.02 mol/L sodium phosphate buffer pH 8.0 . Subsequently, the mixed solutions were transferred to a conical flask containing XAD1180 resin and then the flask was placed in a shaking water-bath with a speed of 200 rpm at a temperature of 30 for 8 h. After that, the resulting immobilized MAS1 lipase was recovered by filtration of the supernatant and then washed with 0.02 mol/L sodium phosphate buffer pH 8.0 several times until no protein was detected in the eluate. Finally, the obtained immobilized MAS1 lipase was dried under vacuum at 40 for 8 h and stored in sealed vials at 4 until use.
2.3 Determination of the esteri cation activity of immobilized MAS1 lipase The esterification activity of immobilized MAS1 lipase was determined by the method described previously 35 . First, the conical flask containing 20 mM lauric acid, 20 mM 1-propanol, and 3 water w/w, with respect to total reactants was incubated in a thermostatic water bath oscillator at a temperature of 60 for 10 min. Then, immobilized MAS1 lipase 15 mg was added to the flask and mixed with the reaction mixture for 10 min. After that, reaction samples 30 μL was withdrawn and mixed with n-heptane 970 μL . Finally, gas chromatography GC equipped with a column OV351 60 m 0.32 mm 0.10 μm was used to analyze the propyl ester according to the previous report 36 . According to the above mentioned method, the esterification activity of immobilized MAS1 lipase was 1500 U/g.
The FA specificity of immobilized MAS1 lipase was determined as follows: First, the conical flask containing 11.5 mmol equimolar FAs, 23 mmol glycerol, and 60 μL phosphate buffer 0.1 mol/L, pH 7.0 was incubated at a temperature of 60 for 10 min. Then, immobilized MAS1 lipase 50 mg was added to start the reaction. After 10 min of reaction, the reaction was stopped and the reaction mixture was centrifuged at 10,000 g for 10 min. After that, 0.1 g the upper oil solution was added to a 100-mL conical flask containing 25 mL isopropanol and 100 μL 1 phenolphthalein for the acid value determination according to the previous report 37 . Meanwhile, the remaining reaction mixture containing glycerides and FAs was mixed with 20 mL 30 KOH/ethanol solution. After that, n-hexane 10 mL was added to extract the produced glycerides. Subsequently, the upper phase was collected by a separatory funnel and then was heated under reduced pressure at a temperature of 30 to remove n-hexane. The obtained glyceride mixtures were methylated to fatty acid methyl esters FAME according to the method of Wang et al. 38 . Finally, the FA composition of the obtained glyceride mixtures was analyzed using an Agilent 7890A GC equipped with a capillary column CP-Sil 88 60 m 0.25 mm 0.2 μm as described previously 39 . The conversion degree C n for each FA was calculated as follows: where AV 0 and AV t were acid values of the samples at time zero and time t, respectively. F n was the fatty acid composition of the glyceride mixtures determined by GC.

Calculation of the speci city constant
A series of competitive factors α values , each of which is proportional to the specificity constant 1/α , K cat /K m where K m is measure of the specificity/affinity of the enzyme towards the substrate and K cat is catalytic constant for a particular substrate, was employed to evaluate the FA specificity of immobilized MAS1 lipase toward competing FAs in the esterification reactions. The competitive factor is determined using Eq. 2 proposed by Rangheard et al. 40 : where A 0 and A t are the concentrations of the substrate A at time zero and time t, respectively; B 0 and B t are the concentrations of the substrate B at time zero and time t, respectively.
In this study, an equation for the competitive factor α , which is based on Eq. 2 , can also be written as: where C ref and C n are the conversion degree Eq. 1 of the reference FA and the FA of interest, respectively. Among eleven FAs, a fatty acid with the highest conversion degree was taken as the reference FA and its competitive factor was equal to 1. Since the specificity constant for each FA was defined as the reciprocal of the competitive factors α , the specificity constant of the reference FA was 1 and the specificity constant for other FAs was calculated accordingly.

Preparation of different NADES
The detailed compositions of various NADES are given in Table 1. Various NADES were prepared at a temperature of 80 using vacuum evaporation. After that, homogeneous and colorless liquids were obtained. Finally, the obtained NADES were stored in sealed vials and placed in a desiccator at room temperature until use.

Esterification of Glycerol with n-3 PUFA by immobi-
lized MAS1 lipase Free n-3 PUFA were produced from DHA/EPA-rich EE by the previous method 41 . The catalytic performances of immobilized MAS1 lipase in NADES systems were evaluated in the production of n-3 PUFA-rich TAG by esterification of glycerol with n-3 PUFA. The esterification reactions were carried out in a conical flask containing 10 g substrates glycerol to n-3 PUFA molar ratio of 5:1 with an equal mass of various NADES, and enzyme loading of 200 U/g substrate under N 2 at a temperature of 65 with a shaking speed of 200 rpm for 12 h. The control group without any NADES was also performed under the same conditions. Samples were withdrawn periodically and prepared for high-performance liquid chromatography HPLC and gas chromatography GC analysis.

Analysis of n-3 PUFA composition of TAG by GC
Before the analysis, separation of TAG from the reaction mixtures was performed on a thin layer chromatography plate using a mixture of n-hexane, ethyl ether, and acetic acid 80:20:1, v/v/v as the developing solvent according to the method of Qin et al. 42 . Then, the substrate n-3 PUFA and the scraped TAG bands were separately methylated to FAME using the method described earlier 38 . Finally, the n-3 PUFA composition of the substrate and TAG in the final product was analyzed using an Agilent 7890A GC equipped with a capillary column CP-Sil 88 60 m 0.25 mm 0.2 μm as described previously 39 .
2.9 Analysis of the composition of the reaction mixture by HPLC The detailed composition of the reaction mixture was analyzed using a normal-phase HPLC equipped with a refractive index detector and a Phenomenex Luna column 250 mm 4.6 mm i.d., 5 μm particle size, Phenomenex Corporation as described previously 33 . The mobile phase consisted of n-hexane, isopropanol with formic acid 21:1:0.003, v/v/v and its flow rate was 1 mL/min. Peaks in HPLC were identified by comparison of their retention times with those known standards. Retention times were 3.10 min TAG , 3.76 min FA , 4.63 min 1,3-diacylglycerols DAG , 5.86 min 1,2 2,3 -DAG , 29.51 min 1 3monoacylglycerols MAG , 37.93 min 2-MAG . Waters 2695 integration software was employed to analyze the data and calculate peak-area percentages. In this study, TAG content, DAG content and MAG content were defined as the weight percentage of TAG, DAG and MAG in the reaction mixture, respectively. The percentage of each acylglycerol species was obtained from peak areas in HPLC. Esterification degree was defined as the percentage of initial FA consumed in the reaction mixture as calculated from peak areas.

Statistical analysis
All experiments were performed in triplicate and the results were presented as the means standard deviations SD . In addition, an ANOVA procedure was employed to determine significant differences among the measured values through significant differences test and variance analysis of Statistical Program for Social Sciences for Windows 13.0.

The FA speci city of immobilized MAS1 lipase
The specificity constant 1/α was used as an indicator to evaluate the FA specificity of immobilized MAS1 lipase. The larger the specificity constant of a fatty acid, the greater the selectivity of immobilized MAS1 lipase for this fatty acid. The specificity constants 1/α of immobilized MAS1 lipase for eleven fatty acids ranging from C8:0 to C22:6 in the esterification are shown in Fig. 1. Immobilized MAS1 lipase exhibited the highest preference for C10:0 followed by C18:2. However, immobilized MAS1 lipase discriminated strongly against EPA and DHA due to their lowest specificity constants 1/α 0.2 and 0.19, respectively . The results indicated that the specificity of immobilized MAS1 lipase toward EPA and DHA was very similar. Therefore, the scientists and manufactures could utilize the specificity of immobilized MAS1 lipase for different fatty acids to design experiments for the modification of fats and oils.

n-3 PUFA-rich TAG content improvement
Among the FAs tested, EPA and DHA were poorer substrates for immobilized MAS1 lipase, as shown in Fig. 1. It was reported that the selectivity of lipase-catalyzed esterification reactions could be affected by NADES 43 . Moreover, it was demonstrated that n-3 PUFA-rich TAG could prevent cardiovascular disease and cancer, inhibit inflammation, and reduce the risk of atherosclerosis 44 47 . Therefore, the catalytic performances of immobilized MAS1 lipase-catalyzed esterification of n-3 PUFA with glycerol for the production of n-3 PUFA-rich TAG in the solvent-free and different NADES systems were investigated in this study. The effects of different NADES on esterification degree are shown in Fig. 2. It was observed that the esterification degree in the solvent-free system for the first 3 h was quicker than those in various NADES system. After that, the esterification degree in the solvent-free system was slower than or equal to those in NADES system. After 12 h,  and CX as reaction solvents resulted in a lower performance whereas the use of BG and CG as the reaction media showed a similar influence on esterification degree.
The effects of different NADES on TAG, MAG, and DAG contents during the esterification of n-3 PUFA with glycerol catalyzed by immobilized MAS1 lipase are presented in Fig.  3. Figure 3A shows the effects of different NADES on TAG content. It was found that TAG content separately reached 23.44 , 41.77 , 34.62 , 37.72 , and 55.8 after 12 h when the esterification reactions catalyzed by immobilized MAS1 lipase were carried out in the solvent-free, BU, BG, CX, and CG systems, respectively. Moreover, it was observed that in addition to TAG content in BG system, the accumulation rates of TAG in the other NADES systems were quicker than that in the solvent-free system. Although the accumulation rate of TAG in BG system for the first 5 h was slower than that in the solvent-free system, a sudden shift to a higher accumulation rate could be seen for subsequent reactions relative to the reaction performed in the solvent-free system. Therefore, it could be concluded that TAG content was significantly improved in various NADES system when compared with the solvent-free system. In particular, when the esterification reactions were performed in CG system, the maximum TAG content was obtained. However, lower MAG and DAG contents were obtained in various NADES systems than those in the solventfree system Figs. 3B and 3C . Besides, it was observed that MAG content showed different trend with DAG    Fig. 3C . Nevertheless, MAG content in various NADES systems separately increased firstly and then decreased gradually or kept increasing slowly as the reaction proceeded whereas MAG content in the solventfree system increased dramatically with time Fig. 3B . After 12 h, MAG content separately reached 21.71 , 7.59 , 13.04 , 6.25 , and 4.35 when the esterification reactions catalyzed by immobilized MAS1 lipase were carried out in the solvent-free, BU, BG, CX, and CG systems, respectively. This was probably because water produced during the esterification process was quickly absorbed by NADES, thus shifting the equilibrium in the positive direction. The results showed that the conversion from glycerol to MAG, from MAG to DAG, and from DAG to TAG could be promoted using NADES as reaction solvents during the esterification of n-3 PUFA with glycerol catalyzed by immobilized MAS1 lipase. Therefore, the use of various NADES as reaction solvents was favorable to synthesize n-3 PUFA-rich TAG.

No change in the selectivity and regiospecificity
The effects of different NADES on 1,3-DAG/1,2-DAG ratio are displayed in Fig. 4. 1,3-DAG/1,2-DAG ratio firstly increased and then decreased gradually with time when the esterification reactions were catalyzed by immobilized MAS1 lipase in the solvent-free and various NADES systems. The 1,3-DAG/1,2-DAG ratio reached the maximum at 3 h and separately decreased to 2.04, 2.19, 2.21, 2.24, and 2.06 after 12 h of reaction when the esterification reactions were carried out in the solvent-free, BU, BG, CX, and CG systems, respectively. It could be concluded that immobilized MAS1 lipase showed similar selectivity between 1,3-DAG and 1,2-DAG no matter whether the esterification reactions were carried out in the solvent-free system or in various NADES systems. Besides, it could be seen from Fig. 3A that TAG content in the solvent-free and NADES systems increased as the reaction proceeded. However, the secondary hydroxyl group of 1,3-DAG was difficult to react with n-3 PUFA when compared with the primary hydroxyl group of 1,2-DAG. It could be concluded that TAG was formed mainly through the acylation of 1,2-DAG. Moreover, fatty acids could be transferred not only from sn-1 or sn-3 position to sn-2 position, but also from sn-2 position to sn-1 or sn-3 position. Thus, the decreased 1,3-DAG/1,2-DAG ratio at 12 h indicated that a weak acyl transfer occurred during the reaction process. The results indicated that immobilized MAS1 lipase had no regiospecificity in the solvent-free and various NADES systems. 3.2.3 The n-3 PUFA compositions of the prepared n-3 PU-FA-rich TAG The n-3 PUFA compositions of the substrate and n-3 PU-FA-rich TAG in the solvent-free and various NADES systems after 12 h of reaction were analyzed and the results are presented in Table 2. It was found that the substrate mainly consisted of 38.78 EPA, 6.42 DPA, and  3.3 The potential reaction mechanism of immobilized MAS1 lipase-catalyzed esterification of n-3 PUFA with glycerol in NADES systems According to the above results, it could be conferred that the potential reaction mechanism of immobilized MAS1 lipase-catalyzed esterification of n-3 PUFA with glycerol in NADES systems Fig. 5 could proceed as follows: Glycerol was firstly converted to MAG, followed by the conversion from MAG to DAG, and finally DAG was converted to TAG. At each step, one mole of water was produced and then was quickly captured by NADES during the reaction Fig. 5A . Thus, the reaction equilibrium was shifted toward the positive direction, resulting in an increase in TAG content. Although the reaction was a heterogeneous system at beginning and then turned to be a pseudo-homogeneous system after stirring, the produced MAG, DAG, and TAG were all not actually soluble in NADES. Thus, the side-reactions were significantly minimized. Moreover, the produced TAG became a single phase at upper layer after the reaction was stopped and the mixture was centrifuged. However, the mixture of NADES, glycerol, and water was remained at the lower layer and immobilized MAS1 lipase suspended at the medium layer Fig. 5B . Therefore, the separation of products and immobilized lipase from the reaction mixture was simple and effective.
Overall, n-3 PUFA-rich TAG content obtained by immobilized MAS1 lipase was significantly improved in various NADES systems when compared with the solvent-free system. The maximal n-3 PUFA-rich TAG content 55.8 with similar n-3 PUFA composition to the substrate was obtained at 12 h in CG system. There was a 1.38-fold increase of TAG content in CG system compared with that in the solvent-free system in this study, which was higher than that 1.2-fold at 48 h obtained by Novozym 435 in the literature 48 . It could be concluded that the catalytic efficiency of immobilized MAS1 lipase was better than that of Novozym 435. Therefore, the results indicated that immobilized MAS1 lipase is a promising and efficient biocatalyst for the synthesis of n-3 PUFA-rich TAG by esterification of glycerol with n-3 PUFA in NADES systems.

Conclusions
In this study, immobilized MAS1 lipase exhibited the highest preference for C10:0 whereas it discriminated strongly against EPA and DHA. However, n-3 PUFA-rich TAG content was efficiently improved by immobilized MAS1 lipase-catalyzed esterification of glycerol with n-3 PUFA in NADES systems when compared with the solventfree system and the maximal n-3 PUFA-rich TAG content reached 55.8 at 12 h in CG system in this study. It was also found that immobilized MAS1 lipase had no regiospecificity and exhibited similar selectivity between 1,3-DAG and 1,2-DAG in the solvent-free and various NADES systems. Besides, n-3 PUFA-rich TAG in the solvent-free and various NADES systems showed similar n-3 PUFA compositions to the substrate. Finally, the potential reaction mechanism of immobilized MAS1 lipase-catalyzed esterification of n-3 PUFA with glycerol in NADES systems was described. It was observed that the use of NADES as reaction solvents not only facilitated product separations, but also shifted the reaction equilibrium in the positive di- Fig. 5 Reaction mechanism A and TAG preparation process B of immobilized MAS1 lipase-catalyzed esterification of n-3 PUFA with glycerol in NADES systems. rection due to their absorption of the produced water during the reaction process. These results indicated that immobilized MAS1 lipase is a promising and efficient biocatalyst for the synthesis of n-3 PUFA-rich TAG by esterification of glycerol with n-3 PUFA in NADES systems.