Promotion of the lipase-catalyzed hydrolysis of conjugated linoleic acid l-menthyl ester by addition of an organic solvent
© Kobayashi et al.; licensee Springer. 2012
Received: 16 November 2012
Accepted: 11 December 2012
Published: 14 December 2012
Conjugated linoleic acid l-menthyl ester was hydrolyzed in water by the lipase from Candida rugosa with the addition of an organic solvent. The degree of hydrolysis (yield) greatly improved when a tertiary alcohol, such as t-butyl alcohol, was added. However, the addition of a less polar solvent, such as hexane, decreased the degree of hydrolysis, and some water-miscible solvents, such as acetone, caused inactivation of the lipase. With the addition of t-butyl alcohol, the reaction mixture formed a one- or two-phase system, and the mixing ratio of substrates and t-butyl alcohol determined the number of phases. Although the degree of hydrolysis at 10 d was higher in the one-phase system, the initial reaction rate was generally lower. Meanwhile, the reaction was much faster in the two-phase system while maintaining a moderate degree of hydrolysis.
Commercially available conjugated linoleic acid (CLA) contains two structural isomers (9-cis,11-trans- and 10t,12c-CLA). It has been reported that each isomer has different physiological activities, such as an anti-cancer activity (Soel et al.2007), decreasing the body fat content (Park et al.1999), and suppressing the development of hypertension (Nagao et al.2003a). To develop nutraceuticals containing the two CLA isomers of arbitrary amounts, a fractionation process of the CLA isomers is needed.
Some fractionation processes have been developed using the lipase-catalyzed selective esterification (Haas et al.1999; Kobayashi et al.2006; Nagao et al.2003b). In these processes, lipases, such as from Candida rugosa, one of the most effective lipases for the esterification of CLA, were used, and some alcohols were used for the hydroxyl donors. l-Menthol is one these alcohols and very effective for the large-scale fractionation by selective esterification with CLA (Kobayashi et al.2006). In this process, the alkali hydrolysis of CLA menthyl ester was needed to recover CLA as a free fatty acid. However, the hydrolysis requires heating under a strong alkaline condition, which may cause decomposition or isomerization of CLA. The lipase-catalyzed hydrolysis of CLA menthyl ester is considered to avoid these drawbacks. However, a degree of hydrolysis (yield) for CLA menthyl ester would be low in an oil/water two-phase system because the degree of the reverse reaction (esterification) is very high (ca. 80%) at equilibrium (Kobayashi et al.2007).
In general, the hydrolytic reaction system by lipases consists of two phases, i.e., water and oil phases in a neat reaction mixture. The water content in the oil phase is fixed in this case. However, addition of an organic solvent would promote the partition of water from the water phase to the oil phase, which results in changing the water content in the oil phase. To promote the hydrolysis, this change would be effective because the water content in the oil phase plays an important role in determining the degree of reaction in the two-phase system (Kobayashi et al.2007). In this study, some organic solvents were added to the reaction system at various mixing ratios of the substrates and the solvent in order to evaluate the performance of the lipase-catalyzed hydrolysis of CLA menthyl ester.
Materials and methods
CLA in a free fatty acid form was a gift from The Nisshin OilliO Group, Ltd. (Tokyo, Japan), the fatty acid contents of which were 33% 9c,11t-CLA, 34% 10t,12c-CLA, 4.9% other CLAs, 16% oleic acid, 6.4% palmitic acid, 2.7% stearic acid, and 2.2% other fatty acids. Lipase from Candida rugosa (Lipase-OF) was from Meito Sangyo (Aichi, Japan).
Preparation of CLA menthyl ester
Lipase from C. rugosa (1.8×104 U/g-powdery enzyme) was used as an aqueous solution (1.8×104 U/mL) in which 1 U lipase was defined as the amount of lipase which liberated 1 μmol free fatty acid/min during the hydrolysis of olive oil. Esterification of CLA was performed by stirring 100 g (0.36 mol) CLA, 55.8 g (0.36 mol) l-menthol, and 8.67 mL lipase solution at 500 rpm and 30°C for 7 d under a nitrogen atmosphere. After the reaction, the reaction mixture was separated into the oil and water phases by centrifugation. CLA menthyl ester and unreacted l-menthol were extracted with hexane after adding a 0.1 M sodium hydroxide aqueous solution to the oil phase to remove any unreacted CLA (free fatty acid). The mixture of CLA menthyl ester and l-menthol was distilled under vacuum at 150°C and 500 Pa to obtain CLA menthyl ester as a distillation residue.
Hydrolysis of CLA menthyl ester in the presence of an organic solvent
CLA menthyl ester, organic solvent, and water at specific amounts were weighed into a screw-capped vial. In a typical procedure, 2 g CLA menthyl ester, 2 g t-butyl alcohol and 1 g water were used. To the substrate mixture was added the powdery lipase from C. rugosa at 8.9×103 U/g-ester. The mixture was then stirred at 500 rpm and 30°C for 10 d under a nitrogen atmosphere. The organic solvents used in this reaction were t-butyl alcohol, t-amyl alcohol, diacetone alcohol, diethyl ether, diisopropyl ether, hexane, cyclohexane, acetone, 2-butanone, cyclohexanone, and acetonitrile. The reaction without any organic solvent was also tested as the control. At appropriate intervals, the reaction mixture (ca. 250 mg) was sampled and analyzed to estimate the initial reaction rate, water content in the oil phase, and degree of hydrolysis.
The water content in the oil phase of the reaction mixture was measured by Karl-Fischer titration using a CA-07 moisture meter (Mitsubishi-Kagaku, Tokyo). The measurement was performed at least five times using an ca. 40 mg oil phase in each measurement, and the median value was adopted as the water content.
The degree of hydrolysis was determined by gas chromatography (GC). Prior to applying a sample to the GC, the reaction mixture was diluted 10 times with hexane. The diluted sample was centrifuged at 9000×g for 2 min to separate it into the hexane and water phases. After removing the water phase, solvents in the hexane phase were removed under vacuum. Free fatty acids in the residual oil underwent methylation as follows (Kobayashi et al.2011): Ten milligrams of the oil was dissolved in 1 mL toluene/methanol (3:2, v/v). The solution was mixed well with a 25 μL (trimethylsilyl)-diazomethane diethyl ether solution (2 M), and the mixture was stored for 10 min at room temperature. After methylation, the mixture was applied to a 6890N GC (Agilent Technologies, CA, USA) connected to a DB-23 column (0.25 mm I.D. × 30 m; Agilent). The column temperature was raised as follows: 150 to 200°C, 10°C/min; 200 to 210°C, 2°C/min; 210 to 230°C, 15°C/min; kept at 230°C for 10 min.
Results and discussion
Effect of the type of organic solvent on the hydrolysis
Effect of the type of an organic solvent on the degree of hydrolysis at 1 d
Degree of hydrolysis (%)
These results indicate that lipase from C. rugosa is not completely inactivated in a tertiary alcohol. On the other hand, other water miscible organic solvents, such as acetone and acetonitrile, readily inactivate the lipase. These facts contrast the fact that lipase from Candida antarctica (fraction B) shows a high catalytic activity not only in t-butyl alcohol, but also in acetone and acetonitrile (Kobayashi et al.2010; Zhu et al.2012). Based on these results, t-butyl alcohol was used for the following studies.
State of the reaction mixture
Because t-butyl alcohol is a water-miscible solvent and also miscible with CLA menthyl ester, the addition of t-butyl alcohol greatly changes the state of the reaction mixture which consists of two phases, i.e., aqueous and oil phases. When a small amount of t-butyl alcohol is added to the mixture, water and CLA menthyl ester are partitioned into the oil and aqueous phases, respectively. Meanwhile, the mixture will become one phase when more t-butyl alcohol is added. Therefore, the initial ratios of t-butyl alcohol, CLA menthyl ester and water were changed, and effects of the ratio on the hydrolysis were discussed from the viewpoint of the state of the reaction mixture, the initial reaction rate, and the degree of hydrolysis.
Hydrolysis in one-phase system
In the one-phase system, the initial reaction rates were ca. 1–4 fold higher than that of the control in most cases (Figure2). However, the significant addition of t-butyl alcohol tended to decrease the initial rate. Meanwhile, the degree of hydrolysis at 10 d greatly improved to 42–68% in all cases compared to that of the control as shown in Figure3, maybe due to the high concentration of water in the reaction mixture. However, the addition of a large amount of t-butyl alcohol lowers the substrate concentration. Therefore, performing the reaction under the one-phase state is not practical.
There are some possibilities for the low reaction rate at the higher ratio of t-butyl alcohol: 1) Low substrate concentration is directly related to the low reaction rate. 2) Water-miscible organic solvents cause unfolding of the protein (Klyosov et al.1975) or elimination of water molecules from an enzyme molecule that are essential to maintain its catalytic activity (Krishna et al.2001). Therefore, lipase may be gradually inactivated by the significant addition of t-butyl alcohol. 3) A lipase molecule is generally adsorbed on the interface between the oil/water phases, and the interfacial activation produces the catalytic activity (Grochulski et al.1993). In the one-phase system, however, there is no interface, and the behavior of the interfacial activation would be different from that in the typical two-phase system.
Hydrolysis in two-phase system
The initial reaction rates in the two-phase system were much improved for all cases compared to that of the control (Figure2). Especially when the ratio of t-butyl alcohol was 9–50 wt%, the reaction rates tended to greatly increase, and the maximum reaction rate reached ca. 10.8 times that of the control. These results would support the fact that the addition of a small amount of t-butyl alcohol activates the lipase from C. rugosa, and that the formation of the interface between the two phases is very important for the lipase to sufficiently exhibit its catalytic activity even in the presence of t-butyl alcohol.
In conclusion, the addition of an organic solvent greatly affected the degree of hydrolysis of CLA menthyl ester by the lipase from C. rugosa. When tertiary alcohols, such as t-butyl alcohol, were used, the degree of hydrolysis improved. The mixing ratio of substrates and t-butyl alcohol affected the number of phases, and the state of the phase much influenced the initial reaction rate and degree of hydrolysis. The initial reaction rate was generally lower in the one-phase system. In the two-phase system, the reaction was much faster, and a higher degree of hydrolysis could be achieved when the reaction mixture formed an emulsion. Therefore, to efficiently perform the hydrolysis, it is favorable to adopt the two-phase system in an emulsified form.
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