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Preparation of Surfactant-Coated Lipase for the Esterification of Geraniol and Acetic Acid in Organic Solvents
Preparation of surfactant-coated lipase for the esterification of geraniol and acetic acid in organic solvents S. Y. Huang,* H. L. Chang,* and M. Goto† *Department of Chemical Engineering, National Taiwan University, Taipei, Taiwan, †Department of Chemical Science and Technology, Faculty of Engineering, Kyushu University, Hakozaki, Fukuoka, Japan Lipolytic esterification of geraniol and acetic acid in an organic solvent was investigated. A surfactant-coated lipase (SCL) was designed to enhance the esterification. The ester product, geranyl acetate, is an important component of flavor and fragrance compounds. Fatty acids such as acetic acid cause substantial deactivation of lipase, resulting in low esterification yields. SCL is expected to prevent direct contact of organic solvent and the substrates with lipase, thereby decreasing enzyme deactivation. The lipase from Candida cylindrecea interacted with the surfactant Span 85 in a buffer of pH 5.2 to form SCL for the esterification of geraniol and acetic acid. The SCL possessed low solubility in organic solvents which is beneficial for the recovery and repeated usage of enzyme. At the condition of [geraniol]/[acetic acid] 5 2, lipase/surfactant 5 0.5 and in isooctane at 35°C, the esterification yields after 24 and 36 h were 83.7 and 97%, respectively, which were much higher than those obtained by using reverse micelles and powdered lipase. The organic solvent had a dramatic effect on the esterification yield. The polarity of the product, substrate, and organic phase were used as the criteria for solvent selection. The SCL was easy to prepare, could be repeatedly used, and exhibited long-term stability. The esterification efficiency remained unchanged after nine days of use. © 1998 Elsevier Science Inc. Keywords: Surfactant-coated lipase; lipolytic esterification; acetic acid; geraniol; esterification yield
Introduction Enzymatic esterification, transesterification, and other lipolytic reactions in organic solvents have recently received much attention by bioindustries. Enzymes such as lipase often exhibit innovative catalytic functions in organic solvents including enhanced solubility of hydrophobic substrates, elimination of side reactions caused by water, facilitation of product recovery, and protection from microbial contamination. Modification of the surface of lipase to protect it from denaturation in an organic solvent has been achieved by some workers. Surfactant-coated lipase,1–3 surfactant-modified lipase,4 and lipid-coated lipase5,6 appear to be the most
Address reprint requests to Dr. S. Y. Huang, National Taiwan University, Department of Chemical Engineering, Taipei 10617, Taiwan The present address of H. L. Chang is Kao Yuan Junior College of Technology and Commerce Kaoshiung, Taiwan 82101 Received 15 October 1996; revised 23 September 1997; accepted 15 October 1997
Enzyme and Microbial Technology 22:552–557, 1998 © 1998 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
promising methods although some other methods for protecting lipases have been proposed, e.g., surface-modified lipase with activated polyethyleneglycol (PEG)7–9 and hosting lipases in reverse micelles.10 –12 Goto et al.1–3 studied the lipase-catalyzed esterification and interesterification by surfactant-coated lipase (SCL) in isooctane. With the nonionic surfactant, 2C18D 9GE, the esterification and interesterification reaction rates were improved 100-fold compared to unmodified lipase. The esterification yield after 72 h was as high as 94 –97%. Basheer et al.4 employed sorbitan esters to form a lipasesurfactant complex which could catalyze the interesterification of triglycerides and fatty acids in a microaqueous n-hexane system. Okahata et al.5,6 prepared a lipid-coated lipase (LCL) with synthetic dialkyl amphiphiles for the synthesis of triglycerides from monoglycerides and aliphatic acids in nonaqueous solvents. This LCL could also catalyze the enantioselective esterification of racemic alcohols in anhydrous organic solvents. The esterification yield from racemic 2-nonanol and dodecanoic acid was 63%. Fairly high enantioselectivity of racemic alcohol was ob-
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Preparation of surfactant-coated lipase: S. Y. Huang et al. tained. Inada et al.7–9 modified lipase with activated polyethyleneglycol. The modified lipase was dissolved in benzene and catalyzed various lipolytic reactions. The recovery of the modified enzyme was straightforward and enzyme activity was stable after repeated use. The preparation of the modified lipase, however, was fairly complicated. Han et al.10 investigated the lipase-catalyzed hydrolysis of olive oil in an [sodium bis(2-ethyl-1-hexyl)sulfosuccinate] AOTisooctane reverse micellar system. Hayes et al.12 also investigated esterification by lipases in reverse micelles. Factors affecting the activity of lipases were elucidated.10,12 The most serious problem of reverse micelles with lipases was rapid loss of enzyme activity.12 The esters obtained from some short-chain fatty acids (acetic, propionic, and butyric acids) and the naturally formed alcohols, e.g., geraniol, are important components of flavor and fragrance compounds.13 Langrand et al.13 reported that the esterification of propionic/butyric acids and geraniol/isoamylic alcohols could be facilitated with a commercially available lipase; however, the acetic acid esters were more difficult to produce. Acetic acid preferentially reacts with the serine residue at the active site of lipase to form an enzyme-acetic acid complex during the first step of geraniol/acetic acid esterification.14 During esterification, acetic acid is thought to strip the water on the surface of lipase due to its strong hydrophilicity. This results in the deactivation of lipase. It also causes a dead-end inhibition14; therefore, the yield of the ester is low.13 The aim of this work was to develop a lipase protection system to minimize the direct contact of organic solvents and substrates with lipase and to facilitate the lipolytic esterification of acetic acid and gerainol. A SCL was designed and employed for lipolytic esterification. An SCL system and the reaction conditions were found that greatly improved the esterification yield. The SCL exhibited long-term stability and reusability. The protection of enzymes with surfactant is considered to be widely applicable especially when organic solvents are employed in the reaction.
Materials and methods Enzymes and reagents Four kinds of lipase (EC 3.1.1.3) were used in this study. Lipase Cc from Candida cylindracea, lipase Pp from porcine pancreas, and lipase Wg from wheat germ were purchased from Sigma Chemical Co. (St. Louis, MO); lipase An from Aspergillus niger was purchased from Aldrich Chemical Co. (Milwaukee, WI).
Surfactants Span 85 and Tween 85 were purchased from Sigma. n-octanoyln-methyl-d-glucamine (OMG) was purchased from Aldrich. Cetyltrimethyl ammonium bromide (CTAB) was purchased from Tokyo Kasei Co. (Tokyo, Japan). The surfactants, 1C18D 9GE, 2C12GE, 2C16GE, and 2C18D 9GEC2QAC2PA were synthesized in the Goto laboratory.2 Bis (2-ethylhexyl)sulfosuccinate sodium salt (AOT) was purchased from Fluka AG Chemische Fabrik (Buchs, Switzerland).
Substrates Geraniol (98% purity) was purchased from Sigma. Acetic acid was from J. T. Baker Inc. Phillipsburg, (NJ).
Organic solvents The organic solvents used in this work (isooctane, n-hexane, n-heptane, toluene, benzene, chloroform, decane, n-butanol, acetonitrile, and dimethylsufoxide) were all analytical grade. Other chemicals used were reagent grade.
Preparation of surfactant-coated lipase The method of preparation was similar to that presented by Goto et al.3 Lipase (250 mg) was solubilized in 125 ml of 100 mm phosphate buffer pH 5.2 and centrifuged at 1,250 g for 5 min at 4°C to remove insoluble residue. The supernatant was mixed with an aqueous dispersion of 125 mg Span 85 (125 ml) and sonicated in an ultrasonic bath at 80 W for 20 min. After incubating for 24 hours at 4°C, the precipitates were collected by centrifugation at 4°C (8,000 g for 5 min) and lyophilized. The yield and protein content of the SCL were about 27% and 10%, respectively. The yield of SCL is defined as the weight of SCL obtained divided by the weights of original lipase and surfactant. Unless otherwise stated, all the SCL employed in this study were prepared by mixing 1 mg ml21 lipase Cc and 0.5 mg ml21 Span 85 at pH 5.2.
Esterification SCL that was insoluble in organic solvent (mainly isooctane) was employed as a biocatalyst to perform the esterification of geraniol by acetic acid: CH3CHAC~CH3!~CH2!2CHAC~CH3! CH2OH 1 CH3COOH i CH3CHAC~CH3!~CH2!2CHA C~CH3!CH2COOCH3 1 H2O
(1)
An appropriate amount of SCL or unmodified enzyme was added into a screw-cap vial containing freshly prepared geraniol and acetic acid dissolved in an organic solvent. The organic solvent was dewatered overnight with molecular sieve 3. Esterification was conducted in a reciprocating waterbath at 200 rpm. Samples (0.4 ml) were periodically removed to determine the concentration of geranyl acetate. The esterification yield is defined as the concentration of geranyl acetate formed divided by the initial concentration of the acetic acid. Unless otherwise stated, all the esterification experiments were performed with 1.5 mg protein ml21 isooctane, 30 mm geraniol, and 15 mm acetic acid at 35°C.
Selection of organic solvent Log P was used as an index of solvent polarity where P is the distribution coefficient of an organic solvent between octanol and water, i.e., P5
@Solvent#octanol @Solvent#water
(2)
Ten organic solvents15,16 with log P values ranging betwen 21.3 and 5.6 were tested for esterification. The solvent which produced the highest conversion was selected. The strategy employed to select the optimal solvent was to maximize |log Pcph 2 log Ps| and to minimize |log Pcph 2 log Pp| where log Pcph, log Ps, and log Pp represent the log P of the continuous organic phase, substrate, and product, respectively.
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Lipolytic esterification in reverse micelles Isooctane containing 50 mm or 100 mm AOT [sodium bis (2-ethyl-1-hexyl)sulfosuccinate] were prepared. Geraniol (30 mm) and 15 mm acetic acid were added. Lipase dissolved in a trace amount of pH 5.2 buffer was added into the organic solvent containing surfactant. Various reverse micellar solutions with Wo (molar ratio of water and surfactant) ranging between 0;40 were prepared by vigorously shaking the liquid. Esterification was performed at 35°C. All experiments were performed in duplicate or triplicate.
Analysis of protein content of SCL A modified Lowry method was employed to determine the protein content in SCL.17,18 The SCL was vigorously shaken with 1 ml deionized water until the solution was homogeneous, and then reagent 1 and reagent 2 (see Appendix) were added with vigorous shaking. After the reaction was completed, suspended matter was removed by centrifugation and the optical density of the supernatant was measured at 750 nm. With the aid of a calibration curve obtained with lipase Cc, the concentration of protein was determined. The data obtained by the modified Lowry method was consistent with the results obtained using an elementary analyzer (Perkin-Elmer 2400-CHN, Perkin-Elmer, Norwalk, CT).
Determination of esterification yield Gas chromatography was used to determine the concentration of geranyl acetate and calculate the reaction yield. A DB-5 fusedsilica capillary column, 30 m 3 2.5 mm I.D., J&W Scientific (Folson, CA) was used with methanol employed as an internal standard. The temperatures at injection and FID was 250 and 260°C, respectively.
Results and discussion
Figure 1 Comparison of geranyl acetate formation by surfactant-coated lipase, reverse micellar-entrapped lipase, and unmodified lipase (lipase from C. cylindracea)
observed after 24 h. The esterification yield tended to decrease at longer times. This likely was due to enhancement of ester hydrolysis by the small amount of water in the microemulsions. The SCL showed higher esterification yields than the other two enzyme systems. The yield increased almost linearly until 24 h. The yield obtained employing SCL with a lipase/surfactant ratio of 2 was 97% at 36 h (Figure 1) whereas a lipase/surfactant ratio of 4 produced 100% conversion after 36 h (data not shown). These results show that lipase coated with Span 85 was protected from denaturation in organic solvents and exhibited higher catalytic performance compared to the reverse micellar-entrapped lipase and the unmodified lipase.
Effect of lipase source
Effect of surfactant source
Preparation of SCL was conducted using Span 85 surfactant and four lipases, Cc, Pp, An, and Wg. The yield of SCL ranged from 21–28%. The SCL obtained with Wg lipase was dark green whereas the SCLs containing the other lipases were white. All SCLs contained about 10% protein. The four SCLs were insoluble or sparingly soluble in the ten organic solvents examined, similar to the results reported by Basheeer et al.4 Although the SCLs were insoluble in the organic solvents, the lipase was protected and worked effectively as a biocatalyst. The esterification of geraniol by acetic acid induced by the prepared SCLs, reverse micellar entrapped lipase, and unmodified lipase were compared. The conversions catalyzed by An, Cc, and Pp lipases in SCLs were 3.28-, 72.3-, and 6.24-fold greater, respectively, than the yield obtained with unmodified lipase. The lipase from Wg did not catalyze the esterification shown in Eq. (1) and was not studied further. SCL containing Cc produced the highest conversion (83.67% in 24 h) among the lipases tested in this work. The esterification kinetics of unmodified lipase, reverse micelles, and SCL with a lipase/surfactant ratio of 2/1 were compared (Figure 1). The esterification yields were identical during the first hour; however, the conversion leveled off to 10% after 5 h with the unmodified lipase. In the reverse system, a maximum conversion of 23% was
Various commercial surfactants were employed to produce SCLs containing Cc lipase. The surfactants used in this experiment were: (1) nonionic: Span 85, Tween 85, OMG, (2) cationic: CTAB, and (3) anionic: AOT. Table 1 shows the yields and protein content of SCLs as well as the
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Table 1 Effect of different type of surfactants on the preparation of SCL and esterification yield Preparation
Surfactant Span 85 Tween 85 OMG CTAB AOT 1C18D9GE 2C12GE 2C16GE 2C18D9GEC2QAG2PA
SCL yield (%)
Protein content
Esterification yield after 24 h (%)
27.4 15.6 0 21.12 0 49.5 35.3 44.8 39.7
10.4 8.8 – 10.3 – 11.1 10.6 9.8 10.0
83.7 0.5 – 9.7 – 30.6 42.5 28.2 23.8
N-octanoyl-N-methyl-d-glucamine (OMG); cetyltrimethylammonium bromide (CTAB); Bis (2-ethylhexyl) sulfosuccinate sodium salt (AOT)
Preparation of surfactant-coated lipase: S. Y. Huang et al. Table 2 Effect of pH on preparation of surfactant-coated lipase and the esterification yield
Table 3 Effect of lipase/surfactant ratio on protein content and esterification conversion of the surfactant-coated lipase
Preparation pH 3.0 4.0 5.2 6.0 7.2 7.6 8.0 9.0 10.0
Preparation
SCL yield (%)
Protein content (%)
Esterification yield in 24 h (%)
22.9 22.6 27.4 29.3 33.9 36.5 35.1 34.1 26.5
9.8 11.2 10.4 10.3 10.5 11.0 11.5 11.3 7.1
13.3 56.1 83.7 19.3 31.8 26.7 21.1 31.9 15.1
esterification yields. OMG and AOT were unable to coat the surface of lipase. The surfactants synthesized by one of the authors,2 1C18D 9GE, 2C12GE, 2C16GE, and 2C18D9GEC2QAC2PA in contrast, were able to form SCLs. The yields of the SCLs prepared with the synthesized surfactants were higher than those prepared with commercially available surfactants whereas the protein contents of the SCLs were similar. Esterification yields depended on the surfactants used to coat the lipase. Span 85 produced the highest esterification yield of 96.8% after 36 h (Figure 1) and 83.7% after 24 h. The SCLs prepared with the synthesized nonionic surfactants produced 30 ; 40% esterification yields. Tween 85 exhibited the poorest SCL in yield and esterification yield. The cationic surfactant, CTAB and the amphoteric surfactant, 2C18D 9GEC2QAG2PA produced relatively low yields of SCL. Charged surfactants interact through attractive and repulsive forces with the electric charges present on the surface of enzymes. This might alter the stereoconfiguration of the enzymes and result in the deterioration of the catalytic activity. It is suggested that the interaction between the nonionic surfactant and the lipase via a weak interaction like van der Waal forces favor maintainance of the stereoconfiguration of lipase for catalytic performance, thus producing the highest yield.
Lipase/surfactant ratio (w/w) 4/1 2/1 1/1 0.5/1
SCL yield (%)
Protein content (%)
Esterification conversion in 24 h (%)
14.2 27.4 32.1 41.2
11.5 10.4 9.4 7.0
89.6 83.7 78.4 74.4
formed to determine the factors affecting the esterification efficiency with the SCL constituted with Span 85 and Cc lipase at pH 5.2.
Effect of lipase/surfactant ratio The weight ratio of lipase/surfactant produced a remarkable effect on the yield of SCL, protein recovery, and esterification yield (Table 3). The yield of SCL was only 14.2% for a lipase/surfactant ratio of 4 whereas at a ratio at 0.5, the yield increased to 41.2%. The yield of SCL increased as the lipase/surfactant ratio was decreased. Protein content, in contrast, increased with the lipase/ surfactant ratio. This might be attributed to the formation of a thicker surfactant layer on the lipase at higher surfactant loadings. Larger particles increase the yield of SCL.
Effect of reaction temperature The effect of reaction temperature on SCL-catalyzed esterification resembled the behavior of the native enzyme. Geranyl ester synthesis was performed at various temperatures in dried isooctane in the presence of SCL. The esterification yield was 71.6% after 24 h at 25°C. Yield increased at higher temperatures until a maximum yield was observed at 35°C. Beyond this temperature the conversion declined; the conversion dropped to 77.2% at 45°C; hence, the esterification temperature adopted in this work was 35°C.
Effect of aqueous pH on the preparation of SCL
Effect of organic solvent
pH values between 3.0 –10.0 were tested for preparation of SCL and esterification yield. As shown in Table 2, a wide distribution in SCL yield (23–36.5%) at various pH values was found. This is thought to be attributed to the different net charges possessed by the lipase (pI of lipase is 4.2). Protein content ranged from 10 –11% except for a lower protein content found at pH 10.0. A large variation in esterification yield was found at different pH values. The highest esterification yield after 24 h (83.7%) was achieved at a pH of 5.2. The esterification yield of pH 4.0 decreased to 56.1% whereas the yield was below 30% at other pH values. These results show that the pH of the microaqueous layer covering the surface of lipase inside the SCL plays a crucial role in maintaining lipase activity. A series of experiments were therefore per-
Ten organic solvents possessing different polarities were selected for the SCL-catalyzed esterification of geraniol. Figure 2 shows the esterification proceeded when the log P value of the solvents was greater than 2. n-Hexane (log P 5 3.5) and isooctane (log P 5 4.51) produced the highest yields of 82.2% and 83.7%, respectively. nHeptane, with a log P value of 4.0, also facilitated a high conversion (70.65%). These solvents are hydrophobic and do not penetrate the water layer surrounding the surface of lipase. This favored the maintenance of the stereoconfiguration of the enzyme and resulted in a high esterification yield. Esterification yield in decane was relatively low (20%) even though it is hydrophobic (log P 5 5.6). This result can be explained by the optimization policy for selecting a solvent,14,15 i.e., maximizing |log Pcph 2 log Ps| and Enzyme Microb. Technol., 1998, vol. 22, May 15
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Figure 2 Selection of organic solvent with the aid of log P values for enhancing esterification with surfactant-coated lipase. Solvents (log P): decane (5.6); isooctane (4.51); n-heptane (4.0); n-hexane (3.5); toluene (2.5); chloroform (2.0); benzene (2.0); n-butanol (0.8); acetonitrile (20.33); dimethylsulfoxide [DMSO] (21.3)
minimizing |log Pcph 2 log Pp|. The substrates were geraniol and acetic acid whereas the product was geranyl acetate. The log P values of these compounds were estimated by the method of the Rekker et al.16 as 3.17, 20.23, and 4.09, respectively. Acetic acid was a bottleneck to the esterification, indicating that maximizing |log Pcph 2 log Pacetic acid| was the first requisite. The three organic solvents mentioned above and decane all fulfill this requisite; however, the value of |log Pcph 2 log Pp| for decane and geranyl acetate is high (1.51); therefore, decane is not a suitable solvent for this reaction. The log P values of the three solvents selected above are around 4; this validates the experimental results.
Effect of concentration ratio of geraniol and acetic acid The effect of the substrate concentration on the esterification yield was investigated. The esterification was conducted in dewatered isooctane containing 1.5 mg protein ml21 at 35°C for 24 h. Figure 3A shows the effect of acetic acid concentration on the ester formation at a geraniol concentration of 30 mm. A maximum yield of geranyl acetate (13 mm, esterification yield of 86.7%) was achieved at an acetic acid concentration of 15 mm. Higher acetic acid concentrations inhibited the esterification reaction and decreased the yield of geranyl acetate; at 60 mm acetic acid, the concentration of geranyl acetate decreased to less than 3 mm. Figure 3B shows the effect of geraniol concentration on ester formation at an acetic acid concentration of 15 mm. The formation of geranyl acetate was relatively insensitive to geraniol concentrations between 15–75 mm, although the product formation slightly declined at 75 mm geraniol. Based on the esterification yield (amount of product formed) and economic considerations (cost of geraniol), a concentration ratio of geraniol and 556
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Figure 3 Effect of substrate concentration (acetic acid, A; geraniol B) on the esterification of geraniol by acetic acid. Reaction conditions: [Geraniol] 5 30 mM; [Acetic acid] 5 7.5 ; 60 mM (A); [Acetic acid] 5 15 mM; [Geraniol] 5 7.5 ; 75 mM (B) in 2 ml dried isooctane; reaction time, 24 h
acetic acid of 2/1 was thought to be optimum for esterification.
Reuse of surfactant-coated lipase Native lipase lost its enzymatic activity within several hours after exposure to an organic solvent. In contrast, the SCL prepared in this work was insoluble in organic solvents, allowing easy separation from the reaction medium by sedimentation. After removing the medium, new medium could be added to resume the esterification reaction. Iterative batch reactions with the same SCL at 12 and 24 h intervals demonstrated that the conversion remained unchanged for 8 –9 days. Esterification efficiencies of 41 and 83% were attained with the 12 and 24 h batch reactions, respectively, demonstrating that the lipase in the SCL maintained high enzyme activity. Figure 4 shows that the conversion fell by one half after 10 batch reactions (10 days). In summary, the SCL could be prepared with commercially available surfactant. The prepared SCL allowed a high esterification yield under the operational conditions investigated in this work and maintained good enzyme stability. The low solubility of SCL in organic solvents facilitated simple recovery and repeated use of the lipase. The SCL was also stable in organic solvents for prolonged periods. The lyophilized SCL was stable for at least seven months at 226°C. SCL appears to be promising for performing various lipolytic reactions in organic solvents.
Preparation of surfactant-coated lipase: S. Y. Huang et al.
Appendix 1. Constituent of reagent 1:15 ml of A (100 g Na2CO3 l21 0.5 n NaCl) 1 0.75 ml B (1 g CuSO4 z 5H2O (100 ml21) H2O) 1 0.75 ml C (2 g potassium tartrate (100 ml21) H2O) 2. Constituent of reagent 2:5 ml of 2 n Folin-phenol reagent 1 50 ml H2O
References 1. 2.
Figure 4 Time course of esterification yield in repeated batch reactions of SCL in isooctane: 12 hour intervals (A) and 24 hour intervals (B)
3.
Conclusions
5.
Lipolytic esterification of geraniol and acetic acid in an organic solvent was investigated. Surfactant-coated lipase (SCL) was prepared to minimize the inhibition of the acetic acid and the organic solvents to the lipase by preventing direct contact of organic solvent and substrates with the lipase. It was found that the catalytic behavior of the SCL was greatly affected by the origin of the enzyme, the kind of surfactant, the pH of the buffer employed to prepare the SCL, and the ratio of [enzyme]/[surfactant]. Some surfactants were unable to form SCL. The lipase from C. cylindracea interacted with the nonionic surfactant Span 85 in a pH 5.2 buffer to form SCL which possessed low solubility in organic solvents examined in this work. The low solubility of the SCL facilitated the recovery and reuse of the enzyme. With this SCL, the esterification yields at 24 and 36 h reached 83.1 and 97%, respectively, at a geraniol/acetic acid ratio of 2. The organic solvent had a crucial effect on the esterification yield. Solvent polarity, log P, together with an optimization policy in terms of |log Pcph 2 log Ps| and |log Pcph 2 log Pp| were successfully employed as a criterion for solvent selection for this purpose. The SCL prepared in this work exhibited long-term reusability. The esterification yield remained unchanged after 9 days of repeated use. The activity, however, dropped by one half after successive use for 10 days.
4.
6. 7.
8.
9.
10. 11. 12. 13. 14. 15. 16.
Acknowledgments
17.
The authors are grateful for the financial support from the National Science Council, ROC (Grant No NSC 83-0402E002-030 and NSC 84-2214-E002-031).
18.
Goto, M., Goto, M., Kamiya, N., and Nakashio, F. Enzymatic interesterification of triglyceride with surfactant-coated lipase in organic media. Biotechnol. Bioeng. 1995, 45, 27–32 Goto, M., Kameyama, H., Goto, M., Miyata, M., and Nakashio, F. Design of surfactants suitable for surfactant-coated enzymes as biocatalysis in organic media. J. Chem. Eng. Jpn. 1993, 26, 109 –111 Goto, M., Goto, M., and Nakashio, F. Selective hydrolysis of triglycerides with surfactant-coated lipase. Kagaku Kogaku Ronbunshu 1993, 19, 424 – 430 Basheer, S., Mogi, K. I., and Nakajima, M. Surfactant-modified lipase for the catalysis of the interesterification of triglycerides and fatty acids. Biotechnol. Bioeng. 1995, 45, 187–195 Okahata, Y. and Ijiro, K. A lipid-coated lipase as a new catalyst for triglyceride synthesis in organic solvents. J. Chem. Soc. Chem. Commun. 1988, 20, 1392–1394 Okahata, Y., Fujimoto, Y., and Ijiro, K. Lipase-lipid complex as a resolution catalyst of racemic alcohol in organic solvents. Tetrahedron Lett. 1988, 29, 5133–5134 Takahashi, K., Ajima, A., Yoshimoto, T., Okada, M., Matsushima, A., Tamaura, Y., and Inada, Y. Chemical reactions by polyethyleneglycol modified enzymes in chlorinated hydrocarbons. J. Org. Chem. 1985, 50, 3414 –3415 Ajima, A., Yoshimoto, T., Takahashi, K., Tamaura, Y., Saito, Y., and Inada, Y. Polymerization of 10-hydroxydecanoic acid in benzene with polyethylene glycol modified lipase. Biotechnol. Lett. 1985, 17, 303–306 Yoshimoto, T., Takahashi, K., Nishimura, H., Ajima, A., Tamaura, Y., and Inada, Y. Modified lipase having high stability and various enzymic activities in benzene and its reuse by recovering from benzene solution. Biotechnol. Lett. 1984, 6, 337–340 Han, D. and Rhee, J. S. Characteristic of lipase-catalyzed hydrolysis of olive oil in AOT isooctane reversed micelles. Biotechnol. Bioeng. 1986, 28, 1250 –1255 Han, D., Walde, P., and Luisi, P. L. Dependence of lipase activity on water content and enzyme concentration in reverse micelles. Biocatalyst 1990, 4, 153–161 Hayes, D. G. and Gulari, E. Formation of poly-fatty acids by lipases in reverse micellar media. Biotechnol. Bioeng. 1990, 35, 793– 801 Langrand, G., Triantaphylides, C., and Baratti, J. Lipase-catalyzed formation of flavor esters. Biotechnol. Lett. 1988, 10, 549 –554 Segel, I. H. Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems. John Wiley & Sons, New York, 1975, 818 – 830 Laane, C., Boeren, S., Vos, K., and Veeger, C. Rules for optimization of biocatalysis in organic solvents. Biotechnol. Bioeng. 1987, 30, 81– 87 Rekker, R. F. and Mannhold, R. Calculation of Drug Lipophilicity— The Hydrophobic Fragmental Constant Approach. VCH Publisher, New York, 1992, 77– 82 Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein measurement with the Folin Phenol Reagent. J. Biol. Chem. 1951, 193, 265–275 Layne, E. Spectrophotometric and turbidimetric method for measuring proteins. Methods Enzymol. 1957, 3, 447– 454
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Introduction Enzymatic esterification, transesterification, and other lipolytic reactions in organic solvents have recently received much attention by bioindustries. Enzymes such as lipase often exhibit innovative catalytic functions in organic solvents including enhanced solubility of hydrophobic substrates, elimination of side reactions caused by water, facilitation of product recovery, and protection from microbial contamination. Modification of the surface of lipase to protect it from denaturation in an organic solvent has been achieved by some workers. Surfactant-coated lipase,1–3 surfactant-modified lipase,4 and lipid-coated lipase5,6 appear to be the most
Address reprint requests to Dr. S. Y. Huang, National Taiwan University, Department of Chemical Engineering, Taipei 10617, Taiwan The present address of H. L. Chang is Kao Yuan Junior College of Technology and Commerce Kaoshiung, Taiwan 82101 Received 15 October 1996; revised 23 September 1997; accepted 15 October 1997
Enzyme and Microbial Technology 22:552–557, 1998 © 1998 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
promising methods although some other methods for protecting lipases have been proposed, e.g., surface-modified lipase with activated polyethyleneglycol (PEG)7–9 and hosting lipases in reverse micelles.10 –12 Goto et al.1–3 studied the lipase-catalyzed esterification and interesterification by surfactant-coated lipase (SCL) in isooctane. With the nonionic surfactant, 2C18D 9GE, the esterification and interesterification reaction rates were improved 100-fold compared to unmodified lipase. The esterification yield after 72 h was as high as 94 –97%. Basheer et al.4 employed sorbitan esters to form a lipasesurfactant complex which could catalyze the interesterification of triglycerides and fatty acids in a microaqueous n-hexane system. Okahata et al.5,6 prepared a lipid-coated lipase (LCL) with synthetic dialkyl amphiphiles for the synthesis of triglycerides from monoglycerides and aliphatic acids in nonaqueous solvents. This LCL could also catalyze the enantioselective esterification of racemic alcohols in anhydrous organic solvents. The esterification yield from racemic 2-nonanol and dodecanoic acid was 63%. Fairly high enantioselectivity of racemic alcohol was ob-
0141-0229/98/$19.00 PII S0141-0229(97)00257-3
Preparation of surfactant-coated lipase: S. Y. Huang et al. tained. Inada et al.7–9 modified lipase with activated polyethyleneglycol. The modified lipase was dissolved in benzene and catalyzed various lipolytic reactions. The recovery of the modified enzyme was straightforward and enzyme activity was stable after repeated use. The preparation of the modified lipase, however, was fairly complicated. Han et al.10 investigated the lipase-catalyzed hydrolysis of olive oil in an [sodium bis(2-ethyl-1-hexyl)sulfosuccinate] AOTisooctane reverse micellar system. Hayes et al.12 also investigated esterification by lipases in reverse micelles. Factors affecting the activity of lipases were elucidated.10,12 The most serious problem of reverse micelles with lipases was rapid loss of enzyme activity.12 The esters obtained from some short-chain fatty acids (acetic, propionic, and butyric acids) and the naturally formed alcohols, e.g., geraniol, are important components of flavor and fragrance compounds.13 Langrand et al.13 reported that the esterification of propionic/butyric acids and geraniol/isoamylic alcohols could be facilitated with a commercially available lipase; however, the acetic acid esters were more difficult to produce. Acetic acid preferentially reacts with the serine residue at the active site of lipase to form an enzyme-acetic acid complex during the first step of geraniol/acetic acid esterification.14 During esterification, acetic acid is thought to strip the water on the surface of lipase due to its strong hydrophilicity. This results in the deactivation of lipase. It also causes a dead-end inhibition14; therefore, the yield of the ester is low.13 The aim of this work was to develop a lipase protection system to minimize the direct contact of organic solvents and substrates with lipase and to facilitate the lipolytic esterification of acetic acid and gerainol. A SCL was designed and employed for lipolytic esterification. An SCL system and the reaction conditions were found that greatly improved the esterification yield. The SCL exhibited long-term stability and reusability. The protection of enzymes with surfactant is considered to be widely applicable especially when organic solvents are employed in the reaction.
Materials and methods Enzymes and reagents Four kinds of lipase (EC 3.1.1.3) were used in this study. Lipase Cc from Candida cylindracea, lipase Pp from porcine pancreas, and lipase Wg from wheat germ were purchased from Sigma Chemical Co. (St. Louis, MO); lipase An from Aspergillus niger was purchased from Aldrich Chemical Co. (Milwaukee, WI).
Surfactants Span 85 and Tween 85 were purchased from Sigma. n-octanoyln-methyl-d-glucamine (OMG) was purchased from Aldrich. Cetyltrimethyl ammonium bromide (CTAB) was purchased from Tokyo Kasei Co. (Tokyo, Japan). The surfactants, 1C18D 9GE, 2C12GE, 2C16GE, and 2C18D 9GEC2QAC2PA were synthesized in the Goto laboratory.2 Bis (2-ethylhexyl)sulfosuccinate sodium salt (AOT) was purchased from Fluka AG Chemische Fabrik (Buchs, Switzerland).
Substrates Geraniol (98% purity) was purchased from Sigma. Acetic acid was from J. T. Baker Inc. Phillipsburg, (NJ).
Organic solvents The organic solvents used in this work (isooctane, n-hexane, n-heptane, toluene, benzene, chloroform, decane, n-butanol, acetonitrile, and dimethylsufoxide) were all analytical grade. Other chemicals used were reagent grade.
Preparation of surfactant-coated lipase The method of preparation was similar to that presented by Goto et al.3 Lipase (250 mg) was solubilized in 125 ml of 100 mm phosphate buffer pH 5.2 and centrifuged at 1,250 g for 5 min at 4°C to remove insoluble residue. The supernatant was mixed with an aqueous dispersion of 125 mg Span 85 (125 ml) and sonicated in an ultrasonic bath at 80 W for 20 min. After incubating for 24 hours at 4°C, the precipitates were collected by centrifugation at 4°C (8,000 g for 5 min) and lyophilized. The yield and protein content of the SCL were about 27% and 10%, respectively. The yield of SCL is defined as the weight of SCL obtained divided by the weights of original lipase and surfactant. Unless otherwise stated, all the SCL employed in this study were prepared by mixing 1 mg ml21 lipase Cc and 0.5 mg ml21 Span 85 at pH 5.2.
Esterification SCL that was insoluble in organic solvent (mainly isooctane) was employed as a biocatalyst to perform the esterification of geraniol by acetic acid: CH3CHAC~CH3!~CH2!2CHAC~CH3! CH2OH 1 CH3COOH i CH3CHAC~CH3!~CH2!2CHA C~CH3!CH2COOCH3 1 H2O
(1)
An appropriate amount of SCL or unmodified enzyme was added into a screw-cap vial containing freshly prepared geraniol and acetic acid dissolved in an organic solvent. The organic solvent was dewatered overnight with molecular sieve 3. Esterification was conducted in a reciprocating waterbath at 200 rpm. Samples (0.4 ml) were periodically removed to determine the concentration of geranyl acetate. The esterification yield is defined as the concentration of geranyl acetate formed divided by the initial concentration of the acetic acid. Unless otherwise stated, all the esterification experiments were performed with 1.5 mg protein ml21 isooctane, 30 mm geraniol, and 15 mm acetic acid at 35°C.
Selection of organic solvent Log P was used as an index of solvent polarity where P is the distribution coefficient of an organic solvent between octanol and water, i.e., P5
@Solvent#octanol @Solvent#water
(2)
Ten organic solvents15,16 with log P values ranging betwen 21.3 and 5.6 were tested for esterification. The solvent which produced the highest conversion was selected. The strategy employed to select the optimal solvent was to maximize |log Pcph 2 log Ps| and to minimize |log Pcph 2 log Pp| where log Pcph, log Ps, and log Pp represent the log P of the continuous organic phase, substrate, and product, respectively.
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Lipolytic esterification in reverse micelles Isooctane containing 50 mm or 100 mm AOT [sodium bis (2-ethyl-1-hexyl)sulfosuccinate] were prepared. Geraniol (30 mm) and 15 mm acetic acid were added. Lipase dissolved in a trace amount of pH 5.2 buffer was added into the organic solvent containing surfactant. Various reverse micellar solutions with Wo (molar ratio of water and surfactant) ranging between 0;40 were prepared by vigorously shaking the liquid. Esterification was performed at 35°C. All experiments were performed in duplicate or triplicate.
Analysis of protein content of SCL A modified Lowry method was employed to determine the protein content in SCL.17,18 The SCL was vigorously shaken with 1 ml deionized water until the solution was homogeneous, and then reagent 1 and reagent 2 (see Appendix) were added with vigorous shaking. After the reaction was completed, suspended matter was removed by centrifugation and the optical density of the supernatant was measured at 750 nm. With the aid of a calibration curve obtained with lipase Cc, the concentration of protein was determined. The data obtained by the modified Lowry method was consistent with the results obtained using an elementary analyzer (Perkin-Elmer 2400-CHN, Perkin-Elmer, Norwalk, CT).
Determination of esterification yield Gas chromatography was used to determine the concentration of geranyl acetate and calculate the reaction yield. A DB-5 fusedsilica capillary column, 30 m 3 2.5 mm I.D., J&W Scientific (Folson, CA) was used with methanol employed as an internal standard. The temperatures at injection and FID was 250 and 260°C, respectively.
Results and discussion
Figure 1 Comparison of geranyl acetate formation by surfactant-coated lipase, reverse micellar-entrapped lipase, and unmodified lipase (lipase from C. cylindracea)
observed after 24 h. The esterification yield tended to decrease at longer times. This likely was due to enhancement of ester hydrolysis by the small amount of water in the microemulsions. The SCL showed higher esterification yields than the other two enzyme systems. The yield increased almost linearly until 24 h. The yield obtained employing SCL with a lipase/surfactant ratio of 2 was 97% at 36 h (Figure 1) whereas a lipase/surfactant ratio of 4 produced 100% conversion after 36 h (data not shown). These results show that lipase coated with Span 85 was protected from denaturation in organic solvents and exhibited higher catalytic performance compared to the reverse micellar-entrapped lipase and the unmodified lipase.
Effect of lipase source
Effect of surfactant source
Preparation of SCL was conducted using Span 85 surfactant and four lipases, Cc, Pp, An, and Wg. The yield of SCL ranged from 21–28%. The SCL obtained with Wg lipase was dark green whereas the SCLs containing the other lipases were white. All SCLs contained about 10% protein. The four SCLs were insoluble or sparingly soluble in the ten organic solvents examined, similar to the results reported by Basheeer et al.4 Although the SCLs were insoluble in the organic solvents, the lipase was protected and worked effectively as a biocatalyst. The esterification of geraniol by acetic acid induced by the prepared SCLs, reverse micellar entrapped lipase, and unmodified lipase were compared. The conversions catalyzed by An, Cc, and Pp lipases in SCLs were 3.28-, 72.3-, and 6.24-fold greater, respectively, than the yield obtained with unmodified lipase. The lipase from Wg did not catalyze the esterification shown in Eq. (1) and was not studied further. SCL containing Cc produced the highest conversion (83.67% in 24 h) among the lipases tested in this work. The esterification kinetics of unmodified lipase, reverse micelles, and SCL with a lipase/surfactant ratio of 2/1 were compared (Figure 1). The esterification yields were identical during the first hour; however, the conversion leveled off to 10% after 5 h with the unmodified lipase. In the reverse system, a maximum conversion of 23% was
Various commercial surfactants were employed to produce SCLs containing Cc lipase. The surfactants used in this experiment were: (1) nonionic: Span 85, Tween 85, OMG, (2) cationic: CTAB, and (3) anionic: AOT. Table 1 shows the yields and protein content of SCLs as well as the
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Table 1 Effect of different type of surfactants on the preparation of SCL and esterification yield Preparation
Surfactant Span 85 Tween 85 OMG CTAB AOT 1C18D9GE 2C12GE 2C16GE 2C18D9GEC2QAG2PA
SCL yield (%)
Protein content
Esterification yield after 24 h (%)
27.4 15.6 0 21.12 0 49.5 35.3 44.8 39.7
10.4 8.8 – 10.3 – 11.1 10.6 9.8 10.0
83.7 0.5 – 9.7 – 30.6 42.5 28.2 23.8
N-octanoyl-N-methyl-d-glucamine (OMG); cetyltrimethylammonium bromide (CTAB); Bis (2-ethylhexyl) sulfosuccinate sodium salt (AOT)
Preparation of surfactant-coated lipase: S. Y. Huang et al. Table 2 Effect of pH on preparation of surfactant-coated lipase and the esterification yield
Table 3 Effect of lipase/surfactant ratio on protein content and esterification conversion of the surfactant-coated lipase
Preparation pH 3.0 4.0 5.2 6.0 7.2 7.6 8.0 9.0 10.0
Preparation
SCL yield (%)
Protein content (%)
Esterification yield in 24 h (%)
22.9 22.6 27.4 29.3 33.9 36.5 35.1 34.1 26.5
9.8 11.2 10.4 10.3 10.5 11.0 11.5 11.3 7.1
13.3 56.1 83.7 19.3 31.8 26.7 21.1 31.9 15.1
esterification yields. OMG and AOT were unable to coat the surface of lipase. The surfactants synthesized by one of the authors,2 1C18D 9GE, 2C12GE, 2C16GE, and 2C18D9GEC2QAC2PA in contrast, were able to form SCLs. The yields of the SCLs prepared with the synthesized surfactants were higher than those prepared with commercially available surfactants whereas the protein contents of the SCLs were similar. Esterification yields depended on the surfactants used to coat the lipase. Span 85 produced the highest esterification yield of 96.8% after 36 h (Figure 1) and 83.7% after 24 h. The SCLs prepared with the synthesized nonionic surfactants produced 30 ; 40% esterification yields. Tween 85 exhibited the poorest SCL in yield and esterification yield. The cationic surfactant, CTAB and the amphoteric surfactant, 2C18D 9GEC2QAG2PA produced relatively low yields of SCL. Charged surfactants interact through attractive and repulsive forces with the electric charges present on the surface of enzymes. This might alter the stereoconfiguration of the enzymes and result in the deterioration of the catalytic activity. It is suggested that the interaction between the nonionic surfactant and the lipase via a weak interaction like van der Waal forces favor maintainance of the stereoconfiguration of lipase for catalytic performance, thus producing the highest yield.
Lipase/surfactant ratio (w/w) 4/1 2/1 1/1 0.5/1
SCL yield (%)
Protein content (%)
Esterification conversion in 24 h (%)
14.2 27.4 32.1 41.2
11.5 10.4 9.4 7.0
89.6 83.7 78.4 74.4
formed to determine the factors affecting the esterification efficiency with the SCL constituted with Span 85 and Cc lipase at pH 5.2.
Effect of lipase/surfactant ratio The weight ratio of lipase/surfactant produced a remarkable effect on the yield of SCL, protein recovery, and esterification yield (Table 3). The yield of SCL was only 14.2% for a lipase/surfactant ratio of 4 whereas at a ratio at 0.5, the yield increased to 41.2%. The yield of SCL increased as the lipase/surfactant ratio was decreased. Protein content, in contrast, increased with the lipase/ surfactant ratio. This might be attributed to the formation of a thicker surfactant layer on the lipase at higher surfactant loadings. Larger particles increase the yield of SCL.
Effect of reaction temperature The effect of reaction temperature on SCL-catalyzed esterification resembled the behavior of the native enzyme. Geranyl ester synthesis was performed at various temperatures in dried isooctane in the presence of SCL. The esterification yield was 71.6% after 24 h at 25°C. Yield increased at higher temperatures until a maximum yield was observed at 35°C. Beyond this temperature the conversion declined; the conversion dropped to 77.2% at 45°C; hence, the esterification temperature adopted in this work was 35°C.
Effect of aqueous pH on the preparation of SCL
Effect of organic solvent
pH values between 3.0 –10.0 were tested for preparation of SCL and esterification yield. As shown in Table 2, a wide distribution in SCL yield (23–36.5%) at various pH values was found. This is thought to be attributed to the different net charges possessed by the lipase (pI of lipase is 4.2). Protein content ranged from 10 –11% except for a lower protein content found at pH 10.0. A large variation in esterification yield was found at different pH values. The highest esterification yield after 24 h (83.7%) was achieved at a pH of 5.2. The esterification yield of pH 4.0 decreased to 56.1% whereas the yield was below 30% at other pH values. These results show that the pH of the microaqueous layer covering the surface of lipase inside the SCL plays a crucial role in maintaining lipase activity. A series of experiments were therefore per-
Ten organic solvents possessing different polarities were selected for the SCL-catalyzed esterification of geraniol. Figure 2 shows the esterification proceeded when the log P value of the solvents was greater than 2. n-Hexane (log P 5 3.5) and isooctane (log P 5 4.51) produced the highest yields of 82.2% and 83.7%, respectively. nHeptane, with a log P value of 4.0, also facilitated a high conversion (70.65%). These solvents are hydrophobic and do not penetrate the water layer surrounding the surface of lipase. This favored the maintenance of the stereoconfiguration of the enzyme and resulted in a high esterification yield. Esterification yield in decane was relatively low (20%) even though it is hydrophobic (log P 5 5.6). This result can be explained by the optimization policy for selecting a solvent,14,15 i.e., maximizing |log Pcph 2 log Ps| and Enzyme Microb. Technol., 1998, vol. 22, May 15
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Figure 2 Selection of organic solvent with the aid of log P values for enhancing esterification with surfactant-coated lipase. Solvents (log P): decane (5.6); isooctane (4.51); n-heptane (4.0); n-hexane (3.5); toluene (2.5); chloroform (2.0); benzene (2.0); n-butanol (0.8); acetonitrile (20.33); dimethylsulfoxide [DMSO] (21.3)
minimizing |log Pcph 2 log Pp|. The substrates were geraniol and acetic acid whereas the product was geranyl acetate. The log P values of these compounds were estimated by the method of the Rekker et al.16 as 3.17, 20.23, and 4.09, respectively. Acetic acid was a bottleneck to the esterification, indicating that maximizing |log Pcph 2 log Pacetic acid| was the first requisite. The three organic solvents mentioned above and decane all fulfill this requisite; however, the value of |log Pcph 2 log Pp| for decane and geranyl acetate is high (1.51); therefore, decane is not a suitable solvent for this reaction. The log P values of the three solvents selected above are around 4; this validates the experimental results.
Effect of concentration ratio of geraniol and acetic acid The effect of the substrate concentration on the esterification yield was investigated. The esterification was conducted in dewatered isooctane containing 1.5 mg protein ml21 at 35°C for 24 h. Figure 3A shows the effect of acetic acid concentration on the ester formation at a geraniol concentration of 30 mm. A maximum yield of geranyl acetate (13 mm, esterification yield of 86.7%) was achieved at an acetic acid concentration of 15 mm. Higher acetic acid concentrations inhibited the esterification reaction and decreased the yield of geranyl acetate; at 60 mm acetic acid, the concentration of geranyl acetate decreased to less than 3 mm. Figure 3B shows the effect of geraniol concentration on ester formation at an acetic acid concentration of 15 mm. The formation of geranyl acetate was relatively insensitive to geraniol concentrations between 15–75 mm, although the product formation slightly declined at 75 mm geraniol. Based on the esterification yield (amount of product formed) and economic considerations (cost of geraniol), a concentration ratio of geraniol and 556
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Figure 3 Effect of substrate concentration (acetic acid, A; geraniol B) on the esterification of geraniol by acetic acid. Reaction conditions: [Geraniol] 5 30 mM; [Acetic acid] 5 7.5 ; 60 mM (A); [Acetic acid] 5 15 mM; [Geraniol] 5 7.5 ; 75 mM (B) in 2 ml dried isooctane; reaction time, 24 h
acetic acid of 2/1 was thought to be optimum for esterification.
Reuse of surfactant-coated lipase Native lipase lost its enzymatic activity within several hours after exposure to an organic solvent. In contrast, the SCL prepared in this work was insoluble in organic solvents, allowing easy separation from the reaction medium by sedimentation. After removing the medium, new medium could be added to resume the esterification reaction. Iterative batch reactions with the same SCL at 12 and 24 h intervals demonstrated that the conversion remained unchanged for 8 –9 days. Esterification efficiencies of 41 and 83% were attained with the 12 and 24 h batch reactions, respectively, demonstrating that the lipase in the SCL maintained high enzyme activity. Figure 4 shows that the conversion fell by one half after 10 batch reactions (10 days). In summary, the SCL could be prepared with commercially available surfactant. The prepared SCL allowed a high esterification yield under the operational conditions investigated in this work and maintained good enzyme stability. The low solubility of SCL in organic solvents facilitated simple recovery and repeated use of the lipase. The SCL was also stable in organic solvents for prolonged periods. The lyophilized SCL was stable for at least seven months at 226°C. SCL appears to be promising for performing various lipolytic reactions in organic solvents.
Preparation of surfactant-coated lipase: S. Y. Huang et al.
Appendix 1. Constituent of reagent 1:15 ml of A (100 g Na2CO3 l21 0.5 n NaCl) 1 0.75 ml B (1 g CuSO4 z 5H2O (100 ml21) H2O) 1 0.75 ml C (2 g potassium tartrate (100 ml21) H2O) 2. Constituent of reagent 2:5 ml of 2 n Folin-phenol reagent 1 50 ml H2O
References 1. 2.
Figure 4 Time course of esterification yield in repeated batch reactions of SCL in isooctane: 12 hour intervals (A) and 24 hour intervals (B)
3.
Conclusions
5.
Lipolytic esterification of geraniol and acetic acid in an organic solvent was investigated. Surfactant-coated lipase (SCL) was prepared to minimize the inhibition of the acetic acid and the organic solvents to the lipase by preventing direct contact of organic solvent and substrates with the lipase. It was found that the catalytic behavior of the SCL was greatly affected by the origin of the enzyme, the kind of surfactant, the pH of the buffer employed to prepare the SCL, and the ratio of [enzyme]/[surfactant]. Some surfactants were unable to form SCL. The lipase from C. cylindracea interacted with the nonionic surfactant Span 85 in a pH 5.2 buffer to form SCL which possessed low solubility in organic solvents examined in this work. The low solubility of the SCL facilitated the recovery and reuse of the enzyme. With this SCL, the esterification yields at 24 and 36 h reached 83.1 and 97%, respectively, at a geraniol/acetic acid ratio of 2. The organic solvent had a crucial effect on the esterification yield. Solvent polarity, log P, together with an optimization policy in terms of |log Pcph 2 log Ps| and |log Pcph 2 log Pp| were successfully employed as a criterion for solvent selection for this purpose. The SCL prepared in this work exhibited long-term reusability. The esterification yield remained unchanged after 9 days of repeated use. The activity, however, dropped by one half after successive use for 10 days.
4.
6. 7.
8.
9.
10. 11. 12. 13. 14. 15. 16.
Acknowledgments
17.
The authors are grateful for the financial support from the National Science Council, ROC (Grant No NSC 83-0402E002-030 and NSC 84-2214-E002-031).
18.
Goto, M., Goto, M., Kamiya, N., and Nakashio, F. Enzymatic interesterification of triglyceride with surfactant-coated lipase in organic media. Biotechnol. Bioeng. 1995, 45, 27–32 Goto, M., Kameyama, H., Goto, M., Miyata, M., and Nakashio, F. Design of surfactants suitable for surfactant-coated enzymes as biocatalysis in organic media. J. Chem. Eng. Jpn. 1993, 26, 109 –111 Goto, M., Goto, M., and Nakashio, F. Selective hydrolysis of triglycerides with surfactant-coated lipase. Kagaku Kogaku Ronbunshu 1993, 19, 424 – 430 Basheer, S., Mogi, K. I., and Nakajima, M. Surfactant-modified lipase for the catalysis of the interesterification of triglycerides and fatty acids. Biotechnol. Bioeng. 1995, 45, 187–195 Okahata, Y. and Ijiro, K. A lipid-coated lipase as a new catalyst for triglyceride synthesis in organic solvents. J. Chem. Soc. Chem. Commun. 1988, 20, 1392–1394 Okahata, Y., Fujimoto, Y., and Ijiro, K. Lipase-lipid complex as a resolution catalyst of racemic alcohol in organic solvents. Tetrahedron Lett. 1988, 29, 5133–5134 Takahashi, K., Ajima, A., Yoshimoto, T., Okada, M., Matsushima, A., Tamaura, Y., and Inada, Y. Chemical reactions by polyethyleneglycol modified enzymes in chlorinated hydrocarbons. J. Org. Chem. 1985, 50, 3414 –3415 Ajima, A., Yoshimoto, T., Takahashi, K., Tamaura, Y., Saito, Y., and Inada, Y. Polymerization of 10-hydroxydecanoic acid in benzene with polyethylene glycol modified lipase. Biotechnol. Lett. 1985, 17, 303–306 Yoshimoto, T., Takahashi, K., Nishimura, H., Ajima, A., Tamaura, Y., and Inada, Y. Modified lipase having high stability and various enzymic activities in benzene and its reuse by recovering from benzene solution. Biotechnol. Lett. 1984, 6, 337–340 Han, D. and Rhee, J. S. Characteristic of lipase-catalyzed hydrolysis of olive oil in AOT isooctane reversed micelles. Biotechnol. Bioeng. 1986, 28, 1250 –1255 Han, D., Walde, P., and Luisi, P. L. Dependence of lipase activity on water content and enzyme concentration in reverse micelles. Biocatalyst 1990, 4, 153–161 Hayes, D. G. and Gulari, E. Formation of poly-fatty acids by lipases in reverse micellar media. Biotechnol. Bioeng. 1990, 35, 793– 801 Langrand, G., Triantaphylides, C., and Baratti, J. Lipase-catalyzed formation of flavor esters. Biotechnol. Lett. 1988, 10, 549 –554 Segel, I. H. Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems. John Wiley & Sons, New York, 1975, 818 – 830 Laane, C., Boeren, S., Vos, K., and Veeger, C. Rules for optimization of biocatalysis in organic solvents. Biotechnol. Bioeng. 1987, 30, 81– 87 Rekker, R. F. and Mannhold, R. Calculation of Drug Lipophilicity— The Hydrophobic Fragmental Constant Approach. VCH Publisher, New York, 1992, 77– 82 Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein measurement with the Folin Phenol Reagent. J. Biol. Chem. 1951, 193, 265–275 Layne, E. Spectrophotometric and turbidimetric method for measuring proteins. Methods Enzymol. 1957, 3, 447– 454
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