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Acidolysis Between Triolein and Short-Chain Fatty Acid by Lipase in Organic Solvents

Acidolysis Between Triolein and Short-Chain Fatty Acid by Lipase in Organic Solvents

Biosci. Biotechnol. Biochem., 69 (7), 1256–1261, 2005

Acidolysis between and Short-Chain by Lipase in Organic

Wakako TSUZUKI

National Food Research Institute, Kannondai 2-1-12, Tsukuba, Ibaraki 305-8642, Japan

Received December 1, 2004; Accepted April 14, 2005

Ten kinds of lipases were examined as biocatalysts for widely used for transesterification between triacylgly- the incorporation of short-chain fatty acids (acetic, cerol and fatty acids to produce LCSLs.6,7) In the case of propionic, and butyric acids) into triolein in order to Salatrim, biochemical production has also been devel- produce one kind of reduced-calorie structured . oped using Chirazyme L-2 as the catalyst by the Trans-esterification (acidolysis) was successfully done in esterification of with long-chain saturated fatty n- by several microbial lipases. Among them, acids.8) However, in Japan, triacetin, a substrate in the lipase from Aspergillus oryzae was used to investigate biochemical synthesis of Salatrim, is not permitted to be the effects of incubation time, substrate molar ratio, and used as a food stuff. water content on acidolysis. Finally, more than 80% of Instead of using triacetin, transesterification between triolein was incorporated by (molar ratio of long-chain triacylglycerols with short- or middle-chains triolein to butyric acid, 1:10) in the dried n-hexane at fatty acid, has been attemped.8–10) Biochemical produc- 52 C for 72 h. More than 90% of the products was tion of triaclyglycerols composed of long- and middle- monosubstituent, which was esterified with this short chains was facilitated and their mass production has chain fatty acid at the 1-position of the moiety been realized.11,12) However, study of enzymatic syn- of triolein. These results suggest that A. oryzae lipase thesis of triacylglycerols that contain mixtures of short- would be a powerful biocatalyst for the synthesis of low and long-chain fatty acids is hampered by the unfavor- caloric , such as triacylglycerol containing a mixture able substrate specificity of lipase toward short-chain of long- and short-chain aliphatic acids. fatty acids. Although the acidolysis ability of Rhizomu- cor miehei lipase was applied to esterify the mixture of Key words: lipase; acidolysis; triolein; short-chain fatty caproic and butyric acids into triolein in a previous acid study,13) the esterification rate of butyric acid alone was not determined. Salatrim synthesized chemically is Structured lipids (SLs) possess many unique nutri- mainly composed of acetic, propionic, butyric, and tional and metabolic characteristics, which depend on stearic acids. The biochemical synthesis of Salatrim is the molecular structures of acyl chains. Based on their also desirable to produce triacylglycerols similar to benefits to human nutrition, several reduced- or low- chemical synthesized ones. calorie have been designed and developed.1,2) The Previously, Tsujisaka et al. found lipases which could short and long acyl molecule (Salatrim) is a efficiently incorporate short-fatty acids into glycerol in SL whose caloric availability (4.5–6 kcal/g) is lower the aqueous phase.14) Referring to this study, some than other edible (9 kcal/g).3,4) It is composed of a lipases were examined to synthesize one kind of LCSL mixture of long-chain saturated fatty acids (predomi- similar to Salatrim in the organic in the present nantly stearic) and short chain fatty acids (acetic, study. In order to avoid the use of triacetin, triolein and propionic, and/or butyric) esterified to the glycerol several short-chain fatty acids were introduced as backbone,4) and its metabolic property is caused by the starting materials. Because the acidolysis products are chain length and positional distribution of acyl moieties to be food ingredients, n-hexane was mainly considered on the triacylglycerol.5) Salatrim is generally produced as the reaction solvent. The effects of various reactive by chemical interesterification among triacetin, tripro- parameters, including water content, molar ratio of tionin, tributyrin, and hydrogenated vegetable fats substrates, and reaction time on the yield of acidolysis containing a high quantity of .1) products were investigated in order to achieve an Recently, attempts at enzymatic synthesizing low- efficient LCSL synthesis. Finally, the validity of caloric structured lipids (LCSLs) have been made. In acydolysis by lipase between long chain triacylglycerol particular, the acidolysis activity of lipase has been and a short-chain fatty acid is discussed.

To whom correspondence should be addressed. Tel: +81-29-838-8039; Fax: +81-29-838-7996; E-mail: [email protected]ffrc.go.jp Biosynthesis of Long- and Short-Chain Triacylglycerols by Lipase 1257 Materials and Methods synthesis, 10 commercially available lipases were examined for their acidolysis ability between triolein Materials. Short-chain fatty acids, (C2:0), and short-chain fatty acids (C2:0,C3:0, and C4:0) in dried (C3:0), butyric acid (C4:0), and triolein n-hexane. As shown in Fig. 1, lipase from porcine were obtained from Sigma Chemical (St. Louis, MO). pancreas did not act on any substrates in this solvent. In Lipases from Aspergillus oryzae, Rhizomucor miehei, the case of lipase from C. cylindracea, of and Candida cylindracea were purchased from Fluka triolein proceeded in preference to acidolysis. The eight Chemie (Buchs, Switzerland). And lipases from Pseu- other lipases mainly catalyzed acidolysis between domonas cepacia, Rhizopus arrhizus, Pseudomonas triolein and a fatty acid. The progress rates of acidolysis fluorescens, Phycomyces nitens, and Porcine pancreas by lipases from A. oryzae, P. cepacia, R. niveus, R. ar- (Type VI-S) were obtained from Sigma Chemical. rhizus, and M. miehei depended on the length of a fatty Lipase from Rhizopus niveus was purchased from acid chain used. The monosubstituents of triacylglycerol Nagase Biochemicals (Fukuchiyama, Japan), and lipase with C4:0,C3:0, and C2:0 increased in this order. from Rhizopus delemer was from Seikagaku Kogyo However, no differences were found among the incor- (Tokyo). All solvents used in this study were of poration rates of these short-chain fatty acids into analytical grade and were obtained from Wako Pure triolein by lipases from R. delemar or P. fluorescence. Chemical Industries (Osaka, Japan). Among the lipases examined, A. oryzae lipase showed high acidolysis activity and a strict substrate specificity Acidolysis reaction. Commercially available lipases in acidolysis reaction. Hence, this enzyme was selected were used as biocatalysts for acidolysis of triolein with a as a catalyst for the subsequent experiments. short fatty acid. A typical acidolysis reaction was carried out with 0.1 mmole triolein (88.6 mg) and 0.1–1.6 Water content mmole of C2:0,C3:0,orC4:0, using 1 mg lipase in 1 ml The influence of water in the reaction solvent on of n-hexane, which was dried with Molecular Sieves 4A acidolysis was investigated using lipase from A. oryzae. in advance. The enzyme reaction was performed in a For a routine enzymatic reaction, n-hexane was dried glass vial with vigorous stirring at 37 C. with molecular sieves 4A in advance. But no molecular sieves were added to the enzymatic reaction medium. Extraction and analytical methods. After enzymatic Under this condition, the water content of the reaction reaction, the reaction mixture was cooled and filtered medium was about 0.01% and acidolysis occurred prior through a sodium sulfate column to remove any to hydrolysis, as shown in Fig. 1 and Fig. 2, line 2. moisture and enzyme particles. The products of acid- When water was added to the dried n-hexane to 0.1% of olysis and hydrolysis products were traced by a HPLC its concentration, only hydrolysis was promoted and method developed by Lin et al.15) HPLC was carried out acidolysis was completely restrained (Fig. 2, line 1). on a liquid chromatograph consisting of a chromatog- These results suggest that the balance between the raphy manager (Toso HPLC system, Toso, Tokyo), acidolysis and hydrolysis of the lipase activity was operated by a computer (PX-8010), an in-line degasser strongly dependent on the water content in the reaction (SD-8012), a pump (CCPM), an autosampler (AS-8020), medium. When the water content was above 0.05%, the and a UV detector (UV-8020) detecting at 205 nm. incorporation rate of fatty acid into triolein decreased Molecular species of the products by lipase were extremely, due to the predominance of the hydrolysis of separated using a C18 column (25 Â 0:46 cm, 5 mm, triolein. When molecular sieves 4A was added to the C18M, Shodex, Tokyo) with a linear gradient starting at dried n-hexane, the water content fell below 0.001% and 100% methanol to 100% iso-propanol in 40 min. The no enzymatic progress was detected (Fig. 2, line 3). molecular structure of the acidolysis product was Previous reports have suggested that a physical dis- identified by LC-MS. Each reaction mixture was sub- ruption of the enzyme-bound water resulted in enzyme jected to thin layer chromatography (TLC) to check the deactivation.16) Further addition of molecular sieves into enzymatic progress. Lipids were separated by TLC on dried n-hexane probably induces a lack of essential Silica gel 60 plates purchased from Merck (Darmstadt, water for the catalytic activities of the enzyme. Germany) in the solvent system, /methanol/ acetic acid (8:3:0.1, v/v). After development, lipids The effect of temperature were visualized by spraying the plate with 5% phos- Adequate temperature control is important for repro- phomolybdic acid in . The water content was ducible assay of the enzymatic catalyzed reaction. measured with a Novasina water activity measurement Acidolysis by A. oryzae lipase was conducted at various instrument. reaction temperatures to investigate the effect of reaction temperature on the preponderance of competing Results and Discussion reactions (hydrolysis and acidolysis). As shown in Fig. 3, the acidolysis rate increased proportionally to Lipase screening the reaction temperature from 25 Cto52C. In To find an appropriate catalyst for aimed LCSLs contrast, hydrolysis one decreased with increase in the 1258 W. TSUZUKI

Fig. 1. The Catalytic Modes of 10 Lipases in Dried n-Hexane Using Triolein (TO) and Acetic Acid (Ac), Propionic Acid (Pro), and Buthyric Acid (Bu). The products of hydrolysis and acidolysis by each lipase were shown by ( ) and ( ) respectively. The un-reactive triolein ( ) is also shown. The data represent the mean of three sets. Replicate experiments indicated a similar trend.

Fig. 3. The Yield of Enzymatic Products from Triolein and Butyric Aicd in Dried n-Hexane by A. oryzae Lipase after 24 h Incubation at Various Temperatures. Fig. 2. Thin Layer Chromatography of the Enzymatic Products from The yield of acidolysis product (1-butyl-2,3-dioleoyl-glycerol) Triolein and Butyric Acid Catalyzed by A. oryzae Lipase after 24 h ( ), the hydrolysis one (2,3-diolein) ( ) and un-reactive triolein Incubation in Dried n-Hexane with 0.1% Water (1), without Water ( ) are plotted. The data represent the mean Æ SD of three (2) and with Molecular Sieves (3). independent reaction sets from separate duplicate experiments. Biosynthesis of Long- and Short-Chain Triacylglycerols by Lipase 1259 reaction temperatures. Above 55 C, both activities Reactive volume (hydrolysis and acidolysis) decreased and the mass of To investigate the effect of a reaction volume on un-reactive triolein increased (Fig. 3). This means that acidolysis by A. oryzae lipase, reactions were performed degradation of lipase molecule is induced by high in 0, 0.05, 0.1, 0.2, 0.5, 1, 2, and 4 ml of dried n-hexane temperature. These results suggest that 52 C is most (88.6 mg of triolein; molar ratio of butyric acid to suitable reaction temperature for the acidolysis reaction triolein, 10:1, 1 mg of lipase). Very little acidolysis by lipase. product was detected in less than 0.1 ml of n-hexane (data not shown). Because the relative concentration of Molar ratio of substrates butyric acid was increased on a small reaction scale, the The effect of substrate molar ratio on acidolysis was resultant low pH exerted an inhibitory effect on lipase studied by varying the mole ratio of triolein to C2:0,C3:0, activity. A reaction scale of 1 ml was selected as the and C4:0 from 1:1 to 1:16 respectively. All yields were optimal volume for acidolysis reaction. Although a calculated based on the amount of the product and the solvent-free process is desirable for foodstuffs, n-hexane un-reacted triolein. As shown in Fig. 4, the yield of as the reaction medium was essential for the acidolysis acidolysis product between triolein and C2:0,C3:0,or reactions. C4:0 achieved a maximum at molar ratios of each fatty acid and triolein of 3:1, 6:1, and 10:1 respectively Reaction media (Fig. 4). When the molar ratio of acetic acid to triolein The organic solvents listed in Table 1 were examined became more than 6:1, no acidolysis could be detected, as solvents for the acidolysis between triolein and short- as shown in Fig. 4. In the previous study, Nishio and chain fatty acids. Each solvent was dried before use with Kamimura have also found some inactivation of P. fragi the same procedure as n-hexane. All acidolysis reactions 22.39B lipase by short chain fatty acid.17) Because the were conducted in the absence of molecular sieve. As shorter chain fatty acids showed stronger acidic activity, shown in Table 1, the final yields of the acidolysis the increase in acetic acid concentration can be product depended on the solvent used. It was found that attributed to acidification of the micro-aqueous environ- non-polar solvents, such as n-hexane, n-octane, iso- ment of the lipase. It finally induced the loss of lipase octane, and benzene, gave higher yields than polar activity in this experiment. These molar ratios which solvents, such as , acetonitrile, and so on. When gave the maximum yields were used for the remainder lipase was placed in organic solvents, their stereo- of the study. selectivities, stabilities, and mode of altered.18) These enzymatic properties changed by a surrounding solvent influenced the acidolysis process. Furthermore, the acidolysis progress was subjected to the effects of several physiological factors, such as the of the enzyme, substrates, and products in an organic solvent.

Time course Finally, the time course of the acidolysis catalyzed by lipase from A. oryzae was studied using triolein and three fatty acids (C2:0,C3:0, and C4:0). As shown in

Table 1. Yield of Acidolysis Product between Triolein and Butyric Acid by A. oryzae Lipase

37 C 52 C Solvents (%) (%) n-Hexane 72 86 n-Octane 69 82 iso-Octane 70 82 Benzene 59 65 Toluene 2 Tetrahydrofuran 4 Chloroform 5 0 Fig. 4. The Effect of Molar Ratio between Triolein and a Short- Acetonitrile 0 Chain Fatty Acid on the Yield of Acidolysis Product. Acetone 0 For the acidolysis reaction, the molar ratio of acetic acid ( ), n-Propyl 0 propionic acid ( ), and butyric acid ( ) to triolein was changed Ethanol 0 from 1:1 to 16:1. The data represent the mean of three sets. Replicate Methanol 0 experiments indicated a similar trend. 1260 W. TSUZUKI

Fig. 5. Typical Time Course of Incorporation of a Short-Chain Fatty Acid into Triolein Catalyzed by A. oryzae Lipase. The yields of incorporation products of acetic acid into triolein ( ) and into 1-acetyl-2,3-dioleoyl glycerol ( ) were plotted in relation to the incubation periods. In case of propionic acid and butylic acid, the yields of the acidolysis products, 1-propionyl-2,3-dioleolyl glycerol ( ), 1,3- dipropionyl-2-oleoyl glycerol ( ), 1-butyl-2,3-dioleoyl glycerol ( ), and 1,3-dibutyl-2-oleoyl glycerol ( ) were plotted. The data represent the mean of four sets. Replicate experiments indicated a similar trend.

Fig. 5, incorporation of a fatty acid into the 1-position of case, it is important to accommodate the water content the glycerol moiety of triolein reached a maximum after in the reactive solvent. 72 h incubation under both temperature conditions Recently, several approaches have focused on the use (37 C and 52 C). The highest yields produced among of lipase in an organic solvent to modify triacylglycer- triolein and C2:0,C3:0, and C4:0 were 72%, 64%, and ols. This method is attractive compared with the 53% respectively, in incubation at 37 C. On the other chemical process because of the reduction in the number hand, these values increased to 86%, 71%, and 60% side products.1) A previous study suggested that the respectively when the acidolysis reactions were con- lipases from Aspergillus and several species can specif- ducted at 52 C. These findings indicated that the ically acylate a short-chain fatty acid into glycerol in the efficiency of incorporation of a short-chain fatty acid aqueous system.14) This diversity of lipase specificity into triolein depends on the chain length of the fatty acid depending on the origin encouraged us to screen the used. More than 72 h of incubation with A. oryzae lipase lipase as a catalyst for constructing triacylglycerol with induced a moderate increase in the second substitution short- and long-chain fatty acids. The emphasis in this by each fatty acid at the 3-position of the glycerol study was placed on the finding of a lipase which could moiety of triacylglycerol, viz., 1,3-diacetyl (dipropionyl, dominantly incorporate short-chain fatty acids into dibutyl)-2-oleoyl-glycerol, especially when the acid- triacylglycerols. The results indicate that the biochem- olysis was conducted at 52 C. During this period, the ical production of a reduced-calorie oil similar to yield of the monosubstitution product, 1-acetyl (pro- Salatrim is possible using lipase without using triacetin. pionyl, butyl)-1,2 dioleoyl-glycerol, decreased slightly. In the previous study, propionic acid (C6:0) and This suggests that the incorporation rates of these fatty butyric acid (C4:0) were simultaneously incorporated acids into triolein were significantly different from those into triacylglycerol with a high yield by a lipase from into 1-acetyl (or propionyl, butyl)-2,3-dioleoyl-glycerol. R. miehei.13) In that case, half of the acidolysis products The distinction of physical properties between the two was disubstituted triacylglycerols and the other half was types of triacylglycerols is probably responsible for the monosubstituted ones. On the other hand, the product by substrate specificity of A. oryzae lipase. the lipase from A. oryzae accounts for monosubstituted triacylglycerols (more than 90% of the products for 72 h It is well known that non-aqueous enzymatic reactions at 52 C, for example), and disubstituted ones were critically depend on the water level in the reaction produced partly. This study is also distinguished from systems. Several previous reports found that water has a the previous one in relation to the main components of profound influence on the rate of competitive reactions the acidolysis product. by enzymes.5,6) The results of this study also indicate Because the substrate specificity of lipase is different that the water content in the reaction system is to be depending on its origin, other lipases should be attributed to the mode of lipase catalysis between considered as catalysts for the biosynthesis of LCSLs, hydrolysis and acidolysis. To minimize the unfavorable which are composed of specific fatty acids at specific enzyme-catalyzed side reaction, viz., hydrolysis in this positions of triacylglycerols. This is important for Biosynthesis of Long- and Short-Chain Triacylglycerols by Lipase 1261 considering a digestion in vivo, because fatty acids reaction yield and rate of synthesis of structured at the sn-2 position of triacylglycerols are easily triacylglycerol containing eicosapentaentaenoic acid absorbed.19) Further study for an efficient acidolysis of under vacuum with water activity control. Lipids, 34, triacylglycerols with specific fatty acids is in progress 989–995 (1999). in our laboratory using other lipases. Biosynthesis of 9) Jennings, B. H., and Akoh, C. C., Lipase catalyzed modification of fish oil to incorporate . Food LSCLs can be beneficial not only in certain food Chem., 72, 273–278 (2001). materials but also in other nutritional applications. 10) Akoh, C. C., Structured lipid-enzymatic approach. 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