Sn2-monoacylglycerols (2-MAG) are very relevant in food chemistry. They are excellent emulsifiers and they may have excellent functional properties. Their synthesis via classical chemistry means that it is complex because the reactivity of the secondary hydroxyl of glycerol is much lower than the one of the primary hydroxyls. Because of the regioselectivity of certain lipase derivatives and the very mild reaction conditions, the different enzymatic routes may become very promising. In this mini-review, different enzymatic routes will be discussed: regioselective hydrolysis of oils, regioselectiveethanolysis of oils, glycerolysis of oils and glycerolysis of pure esters of fatty acids. “Pros” and “cons” of each strategy will be discussed. In addition, some problems related to the acyl migration of 2-MAG and to the relevance of biocatalyst engineering in activity, stability and regioselectivity of the immobilized lipase will be also commented.

Keywords: Food chemistry, Regioselectivity of lipases, Different lipase derivatives in different reaction media, Regioselectivity of lipases towards glycerol

INTRODUCTION

Sn2-monoacylglycerols (2-MAG) are non-ionic surfactants which have excellent properties as emulsifiers in food industry [1]. They are also useful in pharmaceutical and cosmetic industry [2]. Moreover, 2-MAG are intermediates in the synthesis of structured lipids, MLM. For example, these lipids may contain medium-size fatty acids (M) in the 1,3-positions of glycerol and polyunsaturated fatty acids (PUFA) in the position 2. These lipids have important benefits in the immune function [3] and they exhibit a better physiological adsorption than lipids containing PUFA in 1,3-positions or in random positions [4,5]. Structured lipids containing docosahexaenoic acid (DHA) in position 2 have an excellent bio-availability and they are related to brain development in early ages [6-8]. 2-monoolein is other MAG with also an excellent emulsifier and it has relevant antioxidant function [9-11]. The 2-MAG of arachidonic acid is also very useful for treatment of pain, anxiety and depression [12,13].

Chemical synthesis of 2-MAG is very complex because of the low reactivity of the secondary hydroxyl of glycerol. Chemical means that it produces undesirable by-products and they are not very suitable for food industry. Selective enzymatic synthesis under very mild conditions seems to be the most suitable strategy [14-19]. In this review, different enzymatic routes of 2-MAG catalyzed by selective lipases will be commented:

Regioselective Hydrolysis of Oils

It is considered the simplest protocol but it has been hardly reported because of relevant migration problems, from 2-MAG to 1-MAG, in aqueous medium at moderate temperatures and neutral pH values. Unmodified oils may be hydrolyzed, by a 1,3-regioselective lipase, to a 33% of 2-MAG and a 66% of fatty acids. Nieto et al. described the preparation of sn-2 eicosapentaenoyl glycerol (2-EPA) and sn-2 docosahexaenoyl glycerol by hydrolysis of fish oils catalyzed by a commercial derivative of a 1,3-regioselective lipase from Rhizomucormiehei(Lipozyme IM-20) [20]. At the beginning of hydrolysis a high amount of 2-MAG is produced but after long-term reactions, a high amount of 1-MAG is produced because of migration processes.

Regioselectiveethanolysis of Oils

It is the most widely reported route in literature. Unmodified oil and ethanol are the substrates and 1,3 regioselective lipase derivatives are the biocatalysts. Again, a 33% of a mixture of 2-MAG is obtained [3]. The by-products of the reaction are ethyl esters of fatty acids present in 1,3 position of the oil. The ideal reaction systems would be a mixture of oil and ethanol with no solvent (solvent-free systems). Nevertheless, lipase derivatives are unstable under these conditions and these processes have to be highly optimized. Ethanolysis in the presence of solvents, such as hexane, have to be carefully designed. The solvent, the biocatalyst, the excess of ethanol, the temperature, etc. may greatly modulate the activity, stability and selectivity of the biocatalyst and even the rate of migration of the product, 2-MAG. Piyatheerawong et al. [21] have observed that the polarity of the solvent strongly modulates the selectivity of some lipase derivatives (Novozym 435 andLipozyme 435).Next, the preparation of different MAGs by enzymatic ethanolysisis reported.

Regioselectiveglycerolysis of Oils

Glycerolysis is the alcoholysis of oils by using glycerol as acceptor alcohol. When using an excess of glycerol, a very high yield in monoacylglycerols (MAG) can be obtained [28,29]. Conversion of triglycerides in more than 90% of MAG have been reported [30,31]. When immobilized lipases have a regio-preference towards the 2-position of the glycerol as acceptor, a very high yield in 2-MAG may be obtained. A complex mixture of 2-MAG had to be separated into pure 2-MAG of a given fatty acid. Purification has to be very carefully designed in order to avoid acyl migrations.

Regioselectiveglycerolysis of Ethyl Esters of Pure Fatty Acids

The use of pure esters and a regioselective lipase (preference for the 2-position of glycerol as acceptor) may be useful to obtain a very high yield of pure 2-MAG. By using immobilized RML (immobilized by interfacial adsorption of C-18 supports) more than 95% of the 2-MAG of docosahexaenoic acid (2-DHA) was obtained. The process was carried out in a solvent-free system under very mild experimental conditions [32,33]. The obtained pure 2-MAG does not require complex purification.

Acyl migration of 2-MAG

Acyl migration is a relevant problem during synthesis and handling of 2-MAG. The synthesis of structured lipids (MLM or SLS) avoids this problem. In organic solvents, the hydrophobicity of the solvent and the excess of ethanol may modulate the migration [34-36]. Furthermore, the support used for lipase immobilization is another key factor. For example, hydrophobic supports do not promote acyl migration but polar alumina and cationic Amberlyst 15 greatly increase the migration rates [37].

In aqueous medium (for example oil hydrolysis) the temperature and pH can play a relevant role in acyl migration [20]. Moderately high temperatures and neutral or alkaline pH values may promote very intense migrations. Perhaps, the migration can be strongly reduced by working at low temperature (4°C) and acidic pH as it has been observed for the regioselective de-acetylation of per-acetylated sugars catalyzed by immobilized derivatives of lipases [38].

Biocatalyst Engineering

The immobilization of a given lipase on different supports strongly modulates its functional properties (activity, stability and regio-selectivity [39,40]. This is particularly the case of Thermomyceslanuginosus lipase (TLL). On one hand, when immobilized by interfacial adsorption on octadecyl supports it is not regioselective for ethanolysis of oils in solvent-free systems or in the presence of solvents [22]. In this way, a complete ethanolysis of oils can be performed. On the other hand, the same enzyme, also immobilized by interfacial adsorption but on divinylbenezene supports, exhibits a clear 1,3-regioselectivity and it promotes the synthesis of a 33% of 2-MAG. (Table 2). Additionally the pore sizes of supports seem to play a critical role in lipase activity in solvent-free systems containing very hydrophobic substrates (oils, fatty acid ethyl esters and so on) [33].

CONCLUSIONS

There are a number of enzymatic strategies to produce sn2-monoacylglycerols. All of them require a complex design of the biocatalyst and the reaction conditions. In addition to that acyl migration should be prevented. We propose an interesting strategy:

a- Complete ethanolysis of oil by non regioselective selective lipase derivatives (for example Tlladsorbed on C-18 supports). Production of more than 95% of ethyl esters.

b- The purification of the most interesting esters

c- Glycerolysis of a given pure ester with RML adsorbed on C18 supports (selective for the position 2 of glycerol as acceptor). Production of more than 95% of 2-MAG

d- Synthesis of structured lipids (MLM or SLS) in order to prevent acyl migration under physiological conditions.

1. Feltes MMC, de Oliveira D, Block JM, Ninow JL (2013) The Production, Benefits, and Applications of Monoacylglycerols and Diacylglycerols of Nutritional Interest. Food Bioprocess Technol 6: 17-35.

2. Wang X, Wang X, Jin Q, Wang T (2013) Improved synthesis of monopalmitin on a large scale by two enzymatic methods. J Am Oil ChemSoc 90: 1455-1463.
3. Muñío MM, Robles A, Esteban L, González PA, Molina E (2009) Synthesis of structured lipids by two enzymatic steps: Ethanolysis of fish oils and esterification of 2-monoacylglycerols. Process Biochem 44: 723-730.
4. Christensen MS, Høy CE, Becker CC, Redgrave TG (1995) Intestinal absorption and lymphatic transport of eicosapentaenoic (EPA), docosahexaenoic (DHA), and decanoic acids: dependence on intramolecular triacylglycerol structure. Am J ClinNutr 61: 56-61.
5. Christensen MS, Høy CE, Becker CC, Redgrave TG (1994) Lymphatic absorption of n-3 polyunsaturated fatty acids from marine oils with different intramolecular fatty acid distributions BiochemBiophysActa 1215: 198-204.
6. Pfeffer J, Freund A, Bel-Rhlid R, Hansen CE, Reuss M, et al. (2007) Highly efficient enzymatic synthesis of 2-monoacylglycerides and structured lipids and their production on a technical scale. Lipids 42: 947-953.
7. Nettleton JA (1993) Are n−3 fatty acids essential nutrients for fetal and infant development?. J Am Dietetic Ass 93: 58-64.
8. Rodríguez A, Esteban L, Martín L, Jiménez MJ, Hita E, et al. (2012) Synthesis of 2-monoacylglycerols and structured triacylglycerols rich in polyunsaturated fatty acids by enzyme catalyzed reactions. Enzyme Microbial Technol 51: 148-155.
9. Cho KH, Hong JH, Lee KT (2010) Monoacylglycerol (MAG)-oleic acid has stronger antioxidant, anti-atherosclerotic, and protein glycation inhibitory activities than MAG-palmitic acid. J Med Food 13: 99-107.
10. Cho KH, Lee JH, Kim JM, Park SH. et al. (2009) Blood lipid-lowering and antioxidant effects of a structured lipid containing monoacylglyceride enriched with monounsaturated fatty acids in C57BL/6 mice. J Med Food 12: 452-460.
11. Zhang Y, Wang X, Zou S, Xie D, Jin Q, et al. (2018) Synthesis of 2-docosahexaenoylglycerol by enzymatic ethanolysis. BioresourceTechnol 251: 334-340.
12. Long JZ, Li W, Booker L, Burston JJ, et al. (2008) Selective blockade of 2-arachidonoylglycerol hydrolysis produces cannabinoid behavioral effects. Nat ChemBiol 5: 37-44.
13. Di Marzo V (2008) Targeting the endocannabinoid system: To enhance or reduce? Nat. Rev. Drug Discov 7: 438-455.
14. Wang X, Liang L, Yu Z, Rui L, Jin Q, et al. (2014) Scalable synthesis of highly pure 2-monoolein by enzymatic ethanolysis. Eur J Lipid SciTechnol 116: 627-634.
15. Muñío MM, Esteban L, Robles A, Hita E, et al. (2008) Synthesis of 2-monoacylglycerols rich in polyunsaturated fatty acids by ethanolysis of fish oil catalyzed by 1,3 specific lipases. Process Biochem 43: 1033-1039.
16. Compton D L, Eller FJ, Laszlo JA, Evans KO (2012) Purification of 2-monoacylglycerols using liquid CO2 extraction. J Am Oil ChemSoc 89: 1529-1536.

17. Esteban L, Muñío MM, Robles A, Hita E, et al. (2009) Synthesis of 2-monoacylglycerols (2-MAG) by enzymatic alcoholysis of fish oils using different reactor types. BiochemEng J 44: 271-279.
18. Irimescu R, Furihata K, Hata K, Iwasaki Y, Yamane T (2001) Utilization of reaction medium-dependent regiospecificity of Candida antarctica lipase (Novozym 435) for the synthesis of 1,3-dicapryloyl-2-docosahexaenoyl (or eicosapentaenoyl) glycerol. J Am Oil Chemists' Soc 78: 285-289.
19. Kim BH, Akoh CC (2015) Recent Research Trends on the Enzymatic Synthesis of Structured Lipids. J Food Sci 80: C1713-C1724.
20. Nieto S, Gutiérrez J, Sanhueza J, Valenzuela A (1999) Preparation of sn-2 long-chainpolyunsaturatedmonoacylglycerolsfromfishoilbyhydrolysiswith a stereo-specificlipasefrommucormiehei. Grasas y Aceites. 50: 111-113.
21. Piyatheerawong W, Iwasaki Y, Xu X, Yamane T (2004) Dependency of water concentration on ethanolysis of trioleoylglycerol by lipases. Journal of Molecular Catalysis B. Enzymatic 28: 19-24.
22. Abreu Silveira E, Moreno-Perez S, Basso A, Serban S, Pestana Mamede R, et al. (2017) Modulation of the regioselectivity of Thermomyceslanuginosus lipase via biocatalyst engineering for the Ethanolysis of oil in fully anhydrous medium. BMC Biotechnol 17: 88.
23. Lee HJ, Haq M, Saravana PS, Cho YN, Chun BS (2017) Omega-3 fatty acids concentrate production by enzyme-catalyzed ethanolysis of supercritical CO2extracted oyster oil. Biotechnology and Bio Eng 22: 518-528.
24. Keskin H, KoçakYanik D, Mucuk HN, Göğüş F, Fadiloğlu S (2016) Valorization of Olive Pomace Oil with Enzymatic Synthesis of 2-Monoacylglycerol. J Food Sci 81: C841-C848.
25. Wang X, Li M, Wang T, Jin Q, Wang X (2014) An improved method for the synthesis of 2-arachidonoylglycerol. Process Biochem 49: 1415-1421.
26. Compton DL, Laszlo JA, Appell M, Vermillion KE, Evans KO (2014) Synthesis, purification, and Acyl migration kinetics of 2-monoricinoleoylglycerol. J Am Oil Chemists' Soc 91: 271-279.
27. Irimescu R, Iwasaki Y, Hou CT (2002) Study of TAG ethanolysis to 2-MAG by immobilized Candida antarctica lipase and synthesis of symmetrically structured TAG. J Am Oil ChemSoc 79: 879-883.
28. Pawongrat R, Xu X, H-KittikunA (2007) Synthesis of monoacylglycerol rich in polyunsaturated fatty acids from tuna oil with immobilized lipase AK. Food Chem 104: 251-258.
29. Solaesa ÁG, Sanz MT, Falkeborg M, Beltrán S, Guo Z (2016) Production and concentration of monoacylglycerols rich in omega-3 polyunsaturated fatty acids by enzymatic glycerolysis and molecular distillation. Food Chem 190: 960-967.
30. YangT, RebsdorfM, EngelrudU, Xu X (2005) Enzymatic production of monoacylglycerols containing polyunsaturated fatty acids through an efficient glycerolysis system. J Agric Food Chem 53: 1475-1481.

31. Kaewthong W, H-Kittikun A (2004) Glycerolysis of palm olein by immobilized lipase PS in organic solvents. Enzyme Microbial Technol 35: 218-222.
32. Moreno-Perez S, Luna P, Señorans J, Rocha-Martin J, Guisan JM, et al. (2017) Enzymatic transesterification in a solvent-free system: synthesis of sn-2 docosahexaenoylmonoacylglycerol. Biocatalysis Biotransformation.
33. Moreno-Perez S, Turati DFM, Borges JP, Luna P, Señorans FJ, et al. (2017) Critical role of different immobilized biocatalysts of a given lipase in the selective ethanolysis of sardine oil. J Agric Food Chem 65: 117-122.
34. Compton DL, Laszlo JA, Appell M, Vermillion KE, Evans KO (2012) Influence of fatty acid desaturation on spontaneous acyl migration in 2-monoacylglycerols. J Am Oil Chemists' Soc 89: 2259-2267.
35. Watanabe Y, Sato S, Sera S, Sato C, Yoshinaga K, et al. (2014) Enzymatic analysis of positional distribution of fatty acids in solid fat by 1,3-selective Transesterification with Candida antarctica lipase B. J Am Oil Chemists' Soc 91: 1323-1330.
36. RincónCervera MÁ, VenegasVenegas E, Ramos Bueno R, Rodríguez García I, Guil-Guerrero JL (2013) Acyl migration evaluation in monoacylglycerols from Echiumplantagineum seed oil and Marinol J BiosciBioeng 115: 518-522.
37. Compton DL, Laszlo JA, Evans KO (2013) Influence of solid supports on acyl migration in 2-monoacylglycerols: Purification of 2-mag via flash chromatography. J Am Oil Chemists' Soc 90: 1397-1403.
38. Mateo C, Palomo JM, Fernandez-Lorente G, Guisan JM, Fernandez-Lafuente R (2007) Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme Microbial Technol 40: 1451-1463.
39. Fernandez-Lorente G, Cabrera Z, Godoy C, Fernandez-Lafuente R, Palomo JM, Guisan JM (2008) Interfacially activated lipases against hydrophobic supports: Effect of the support nature on the biocatalytic properties. ProcessBiochem 43: 1061-1067.
40. Fernandez-Lorente G, Palomo JM, Cocca J, Mateo C, Moro P, et al. (2003) Regio-selective deprotection of peracetylated sugars via lipase hydrolysis. Tetrahedron 59: 5705-5711.