Lipase-catalyzed enantiomeric synthesis of citronellyl butyrate

JOURNALOF FERMENTATION ANDBIOENGINEERING Vol. 80, No. 5, 473-477. 1995

Lipase-Catalyzed

Enantiomeric YONGXIANG

Laboratory

of Biotechnology

Synthesis of Citronellyl Butyrate

WANG8 AND YU-YEN LINKO*

and Food Engineering, Helsinki University of Technology, FIN-02150 Espoo, Finland

Received 12 July 1995IAccepted 21 September 1995

Lipases from Candida rugosa, Pseudomonas fluorescens and Rhizopus japonicus were employed in direct esterification of the different optical isomers of citronellol with butyric acid both with and without additional organic solvent. In systems with C. rugosa lipase and without addition of organic solvent, the highest ester yields in 18 h reaction were 98% for R( +)-citronellol at 12% water content and 67% for S( -)-citronellol at 18% water content, respectively. In comparison, a yield of only 65% was obtained with at otherwise similar conditions but with an optimum water content of about 18%. No ester synthesis took place in ‘dry’ n-hexane of about 1.2 ppm of water. When water content was increased to 0.1% in n-hexane system, rapid esterification occurred also in n-hexane with all three substrates, but there was little enantioselectivity. Both P. fruorescens and R. japonicus lipases exhibited somewhat higher initial rates for citronellyl butyrate synthesis, with greater than 80% yield with R( +)-citronellol in 4 h. [Key words:

Candida rugosa, citronellol,

citronellyl

butyrate,

chiral synthesis,

enantiomeric

synthesis,

enzyme, lipase]

solvent (Fig. 1). The effects of a number of parameters, and the kinetics of lipase-catalyzed esterification of (R)(+)-, (S)-( -)and (R,S)-( +)-citronellol with butyric acid were investigated.

Recently lipases (EC 3.1.1.3, triacylglycerol acyl hydrolases) have been intensively investigated both for the hydrolytic production of fatty acids (1, 2), and for the synthesis of esters (3, 4) and polyesters (5). Further, lipases have been successfully used in the production of enantiomerically pure esters either by specific hydrolysis of racemic substrates (6) or by direct stereoselective esterification (7). Lipases have been shown to be active in organic media, which has opened up novel applications (8, 9). There is an increasing interest in the use of lipases in the area of aroma and flavor chemistry to synthesize “natural” flavor esters (8-l 1). Short-chain fatty acid esters of the terpene alcohol, citronellol, are among the most important flavor and fragrance compounds used in the food, beverage, cosmetic and pharmaceutical industries (12). The two chiral forms, (R)-(+)- and (S)-( -)-citronellol esters have quite distinct, different flavors. After Yamaguchi et al. (13) had demonstrated that lipases catalyze stereospecific ester synthesis with racemic alcohols as substrates, the pioneering work of Iwai et al. (14) laid the ground for lipase-catalyzed chiral synthesis of citronellol esters by direct esterification. Nevertheless, nearly all published work on lipase catalyzed citronellol ester synthesis has been carried out with racemic substrates, aimed either at screening of the lipases, increasing the yield or esterification in various organic solvents (15-19). Fonteyn et al. (17) reported recently the production of citronellyl acetate in up to a 100 ml scale in a 74% yield using Candida antarctica lipase without an additional solvent. The yield could be further increased by using a dessicant. Very little details are, however, available on the optimal process conditions and the effects of chirality in lipase catalyzed esterification of citronellol. In the present work, chiral synthesis of butyric acid esters of the terpene alcohol citronellol was studied both in an organic solvent-free system, and using n-hexane as

MATERIALS

AND METHODS

Chemicals (R,s)-( &)-Citronellol, (R)-( +)-citronellol and (S)-(-)-citronellol (all of 95% purity) were purchased from Sigma Chemical Co. (St. Louis, MO, USA) and butyric acid (>99%) from Riedel-de Hahn, A. G. (Seelze b. Hannover, Germany). Acetic acid, n-hexane (0.01% water), petroleum ether, and diethyl ether were of HPLC grade, and obtained from E. Merck (Darmstadt, Germany), Enzymes Powdered lipases from Candida rugosa (98 U/mg, 6.0% water) and Pseudomonas fluorescens (8 U/mg, 3.1% water) were obtained from Biocatalysts Ltd. (Pontypridd, UK), and lipase F-AP-15 from Rhizopus japonicus (78 U/mg, 5.2% water) was obtained from Amano Pharmaceutical Co. (Nagoya, Japan). Lipase activity assay Hydrolytic activity of lipases was determined according to Sigma Bulletin no. 800, using 50% olive oil emulsion as substrate. Free fatty acids liberated were titrated with 0.05 M sodium hydroxide. One unit of lipase activity was defined as the amount of enzyme which liberated 1 micromole of free fatty acid per minute at 37°C and pH 7.0. Esterlfication Reaction mixture for direct esterification of (R)-(f)-, (s)-(-)or (R,S)-(*)-citronellol with butyric acid consisted of 10mM of the alcohol, 7.0mM of butyric acid, and 500U of lipase. Ester syntheses were carried out in screw capped flasks incubated at 37°C under constant agitation at 300rpm by a magnetic stirrer. Two different systems were used. System 1 was solvent-free, with varying amounts of water to solubilize the enzyme before its addition to the reaction mixture. In such a way, the water content in the mixture could be controlled at the desired level. In system 2, n-hexane was used as a solvent, either with 0.1% or no added water. In both cases the alcohol and acid were mixed thorough-

* Corresponding author. 8 Current address: Michigan Biotechnology Institute, 3900 Collins Road, Lansing, Michigan 48910. Visiting scientist from MB1 at HUT. 473

474

WANG

J. FERMENT. BIOENG.,

AND LINK0

H,C

\

H&

...H ‘: +

‘:

7

00

H-F-F-F-C, H

H

H

.,“.H ‘:

OH

c

butyric

acid

H

I O

CH,+H

R(-l-)-citronellol

‘:

‘:

‘:

H

H

H

$-0-F-F-+-H

m

+

H/o\

H

3

R(+)-citronellyl

butyrate

1-1~0

H3C H =/ +

79’: H-F-y-F-C+ H

H3C

1.

‘OH

H

H H3C

CH3

butyric

S(-)-citronellol FIG.

H

‘1w’: ;-O-y-F-F-t-H

0

Reaction

schemes

acid

+ H

butyrate

of (R)-( +)- and (S’)( p)-citronellol

ly before the addition of lipase, as suggested by Claon and Akoh (15) to minimize lipase inhibition by terpene alcohol for the best overall ester yield. At a certain Estimation of the yield of synthesis time, a sample was taken, the reaction was stopped by the addition of 20ml of ethanol, mixed for one minute, and the residual free acid was titrated with 0.05 M sodium hydroxide. The yield (%) was calculated from the amount of acid consumed in the reaction mixture. The esters were extracted Identification of esters with diethyl ether, and identified by thin-layer chromatography (TLC) and Fourier transform infrared spectroscopy (Nicolet Magna FTIR 750). Silica gel TLC plates (type 60, Merck Co., Germany) were used, and developed in petroleum ether : diethyl ether : acetic acid (70 : 30 : 1, per volume). The spots were visualised by spraying with 50% sulfuric acid, and heating in 105°C oven for 20min. The IR-spectra of esters separated by TLC were scanned from a potassium bromide pellet. The strong absorptions at 1170, 1195 and 1740 l/cm were characteristic of ester bonds.

H/o\

H

H

CH3

S(-)-citronellyl

for the esterification

H

13~0 with butyric

acid.

RESULTS AND DISCUSSION The effect of water content Effect of water content on the ester formation was first examined using an organic solvent-free system with C. rugosu (ex. cylindrucea) lipase, which has previously been found to have a high ester synthesis activity (4, 20), and has been successfully used in stereoselective esterifications (7, 21) and interesterification of (R,s)-( *)-menthol (22). The C. rugosa enzyme is also currently one of the most cost efficient among the about 50 or more commercially available lipase preparations (24, 25). Water content of the reaction mixture was varied from 5.5 to 30%. The yield of ester was determined after 18 h of reaction. Figure 2 shows the results. With all three forms of citronellol used as substrate the amount of water had a clear effect on ester formation, with the highest yields obtained at 12 to 18% water. The highest yield of about 98% with the (R)-(+)-citronellol was obtained with 12% of water.

‘lo 0

5

10

15

Water content

20

25

30

35

[%]

FIG. 2. Effect of water content on esterification of different isomers of citronellyl with butyric acid [5OOU of C. rugosa lipase, 37”C, 300rpm, 18h; + (I?)-(+)-, n (s)-(-)-, and A @,A’)(?)citronellyl butyrate].

FIG. 3. Time course of optically active citronellyl butyrate synthesis in a solvent-free system [500 U of C. rugosa lipase, 12% water, 300 rpm, 36 h; + (R)-(t)-, n (s>-(-)-, and A (R,S’)-(t)-citronellyl butyrate].

VOL. 80, 1995

LIPASE

The yields with both (s)-(-)and @$)-(*)-substrates were about equal, 65%. It is of interest to note, however, that with the (S)-(-)-citronellol the optimum water content was higher, about 18%. We have previously found with a number of lipases, including that from C. rugosa, that about 14% water content is optimal for n-butyl oleate synthesis. Yamaguchi et al. (19) demonstrated that both the type of acid and water content markedly influences enantioselectivity in chiral esterification. West (23) reported a considerably higher optimum water content of about 26% for (R,S)-(-t)-citronellyl butyrate synthesis with about 53% conversion using Aspergillus niger lipase and 2.6-fold molar excess of citronellol. They emphasized that it is important to determine the optimal conditions for each individual system separately. In the early work of Iwai et al. (14) a 33% yield of (R,S)-( *)-citronellyl butyrate was reported with A. niger lipase in 18 h at a higher water content of about 75% and no additional organic solvent. It appears quite clear that high conversions can be obtained in lipase catalyzed ester syntheses in solvent-free systems at relatively high water levels, although the optimal water content depends highly on the process conditions in each given case. Interestingly, Fonteyn et al. (17) produced citronellyl acetate in a high purity and yield by using immobilized Candida antarctica lipase as biocatalyst with stepwise addition of acetic acid in a solvent-free system containing only 0.1% water. Effect of chirality of citronellol Under the conditions employed in the esterifications, the rate of synthesis was found to be different with different isomers. With C. rugosa lipase, (R)-(+)-citronellol was found to be the most preferred substrate in an organic solvent-free system, with a nearly complete conversion in 18 h as described above. The time courses of the lipase-catalyzed citronellol ester synthesis in a solvent-free system for the different isomers are shown in Fig. 3. The time courses further illustrate the ability of C. rugosa lipase to catalyze chiral ester synthesis, with a preference to the

CATALYZED

TABLE

SYNTHESIS

1.

OF CITRONELLYL

BUTYRATE

Esterification of different isomers of citronellol butyric acid by C. rugosa lipase Synthesis

Ester of

Solvent-free

(R)-( +)-Citronellol (R,s)-( k)-Citronellol (S)-( -)-Citronellol

svstem

475

with

(%) n-Hexane

96.6 60.5 66.6

as solvent 85.3 84.5 93.4

Reaction mixture contained 0.4 g of butyric acid, 1 .O g of terpene alcohol, and 500 U of lipase. In solvent-free reaction system, the enzyme was dissolved in 0.18 ml of water before addition. Same amount of powdered enzyme (7.5% moisture) was added in n-hexane solvent system. The reaction was carried out for 24 h under the conditions described in the text.

+)-substrate. A 90% conversion of (R)-( +)-citronellol was reached already in about 16 h, at which time the conversion of (S)-(-)-citronellol was about 40%. An about 75% conversion was reached with (s)-( -)-citronellol only after 36 h. It has also been shown that C. rugosa lipase preferentially catalyzes the esterification of (R)-( +)2-bromopropionic acid with n-butanol (7), (-)-menthol with lauric acid (24), and @)-(+)-ibuprofen with primary alcohols (26). It should be stressed, that the enantioselectivity of C. rugosa lipase may vary markedly between different production lots (27) and for a given lipase be reversed even within a structurally similar series of substrates (9). Further, the ester synthesis activity in relation to the hydrolysis (lipolytic) activity of lipases

R(

----98 96 94

78 76 74 1

721.-..-4000

3500

2500

:

2000

1500

Wavenumbers (cm-‘) T

IOO95 90. 85.

ii/ FIG. 4. Time course of optically active citronellyl butyrate synthesis in n-hexane. To 2.0ml of n-hexane (water content 0.1%) 10 mM of citronellol and 7 mM of butyric acid were successively added and allowed to mix for 1 min before the addition of 500U of C. rugosa lipase [+ (R)-(f)-, w (.S)-(-)-, and A (R,S)-(+)-citronellyl butyrate].

4000

. 3500

3000

2500

2000

1500

1000

500

Wavenumbers (cm-‘] 5. Infrared spectra of (top) (R)-(+)-citronellol and (botthe corresponding ester citronellyl butyrate obtained by C. rugosa lipase biocatalysis. FIG.

tom)

476

WANG

0

J. FERMENT. BIOENG.,

AND LINK0

6

15

IO

Time

20

25

[h]

FIG. 6. Time course of lipase-catalyzed reaction of (R)-(+)citronellol and butyric acid in n-hexane by (+) C. rugosa, ( n) P. fluorescens, and (A) R. japonicus lipases. The experimental conditions were the same as in Fig. 4.

FIG. 7. Time course of lipase-catalyzed reaction of (s)-(p)citronellol and butyric acid in n-hexane by ( l ) C. rugosa, ( n ) P. jluorescens,and (A) R. japonicus lipases. The experimental conditions were the same as in Fig. 4.

may vary widely (28). Effect of organic solvent In n-hexane as solvent the results were quite different. No ester synthesis with the C. rugosa lipase was observed during 12 h in a ‘water-free’ system of about 1.2 ppm of water, which originated only from the lipase preparation and the solvent. Clearly, a minimum quantity of water is required for efficient lipase catalyzed ester synthesis. When the water content was increased to 0.1% a rapid ester synthesis took place with all three substrates, as shown in Fig. 4. Claon and Akoh (15) also reported a nearly complete molar conversion in (racemic) citronellyl butyrate synthesis catalyzed by immobilized C. antarctica lipase (Novo Nordisk) in n-hexane in 24 h. Table 1 also shows that, unlike in the aquaeous system, in n-hexane the rate of ester production was similar in all cases, with a yield of about 85% in 24 h and only a slight preference toward the (S)-(-)-substrate. Contrary to our findings Kamiya et al. (24) with surfactant coated lipase AY (Amano Pharmaceutical Co.) from C. rugosa and Koshiro et al. (20) with PU-3 polyurethane gel entrapped C. rugosa lipase OF 360 (Meito Sangyo Co.) reported a high stereoselectivity with these lipases toward (-)-menthol. Figure 5 shows the FTIR-spectra of citronellol and of the citronellyl butyrate obtained as purified by TLC. Effect of type of lipase Figures 6 and 7 show that both (R)-( +)-, and (s)-( -)-citronellol, respectively, was esterified in 24 h in n-hexane in a relatively high yield. This agrees well with the observation of Claon and Akoh (15), and of Langrand et al. (28) who found that both immobilized C. antarctica and Rhizomucor miehei lipases were most active in catalyzing the synthesis of citronellyl and geranyl butyrate in n-hexane. They did not, however, investigate the enantiomeric preference of the different lipases. In the present work, unlike with the organic solvent-free system, C. rugosa lipase showed no marked preference for either isomer under the experimental conditions. Both P. fruorescens and R. japonicus lipases exhibited a somewhat higher initial rate for citronellyl butyrate synthesis, with some preference for the (R)-(+)-substrate. With both of these lipases the

yield of (R)-(+)-citronellol in 4 h of reaction.

ester was of higher than 80%

ACKNOWLEDGMENT The authors are grateful to CIMO (Center for International Mobility, Ministry of Education, Finland) for financial support. REFERENCES 1. Linko, Y.-Y., Koivisto, U.-M., and Kautola, H.: Optimization of enzymic production of oleic acid. Ann. N.Y. Acad. Sci., 613, 691-696 (1990). 2. Marangoni, A. G.: Candida and Pseudomonas lipase-catalyzed hydrolysis of butteroil in the absence of organic solvents. J. Food Sci., 59, 1096-1099 (1994). 3. Linko, Y.-Y. and Yu, H.-C.: Enzymic synthesis of oleic acid esters by various lipases. Ann. N.Y. Acad. Sci., 612, 492-496 (1992). 4. Linko, Y.-Y., Liimsii, M., Huhtala, A., and Rantanen, 0.: Lipase biocatalysis in the production of esters. J. Am. Oil Chem. Sot. (1995). in press 5. Linko, Y.-Y., Wang, Z.-L., and SeppBIB, J.: Lipase catalyzed synthesis of linear aliphatic polyester synthesis in organic solvent. Enzyme Microb. Technol., 17, 506-511 (1995). H., Furui, M., Shibatani, T., and Tosa, T.: 6. Matsumae, Production of optically active 3-phenylglycidic acid ester by the lipase from Serratiu marcescens on a hollow-fiber membrane reactor. J. Ferment. Bioeng., 78, 59-63 (1994). 7. Kirchner, G., Scollar, M. P., and Klibanov, A. M.: Resolution of racemic mixtures via lipase catalysis in organic solvents. J. Am. Chem. Sot., 107, 7072-2076 (1985). 8. Klibanov, A.M.: Advances in enzymes, p. 25-43. Bioflavour ‘87. Walter de Gruyter & Co., Berlin (1988). 9. Sautaniello, E., Ferraboschi, P., and Grisenti, P.: Lipasecatalyzed transesterification in organic solvent: applications to the preparation of enantiomerically pure compounds. Enzyme Microb. Technol., 15, 367-382 (1993). 10. Gillies, B., Yamazaki, H., and Armatrong, D. W.: Production of flavor esters by immobilized Iipase. Biotechnol. Lett., 9, 709-714 (1987). 11. Triantaphylides, C., Landgrand, G., Millet, H., Rangheard, M. S., Buono, G., and Baratti, J.: On the use of lipase specificity. Application to flavour chemistry, p. 531-542. In Bioflavour ‘87. Walter de Gruyter & Co., Berlin (1988).

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LIPASE CATALYZED SYNTHESIS OF CITRONELLYL

12. Brauer, K., Garbe, D., and Surburg, H.: Common frangrance and flavor materials, 2nd ed. VCH Publishers, New York (1990). 13. Yamagucbi, Y., Komatsu, A.S., and Moroe, T.: Microbial transformation of odorous compounds. II. Esterification of racemic alcohols by Can&da cylindracea lipase. J. Agric. Chem. Sot. Japan, 51, 123-125 (1977). 14. Iwai, M., Okumura, S., and Tsujisaka, Y.: Synthesis of terpene alcohol esters by lipase. Agric. Biol. Chem., 44, 27312732 (1980). 15. Claon, P. A. and Akoh, C. C.: Enzymatic synthesis of geraniol and citronellol esters by direct esterification in n-hexane. Biotechnol. Lett., 15, 1211-1216 (1993). 16. Claon, P. A. and Akoh, C. C.: Effect of reaction parameters on SP435 lipase-catalyzed synthesis of citronellyl acetate in organic solvent. Enzyme Microb. Technol., 16, 835-838 (1994). 17. Fonteyn, F., Blecker, C., Lognay, G., Marller, M., and Severin, M.: Optimization of Iipase catalyzed synthesis of citronellyl acetate in solvent-free medium. Biotechnol. Lett., 16, 693-696 (1994). 16. Nishio, T., Takahashi, T., Yoshimoto, T., Kodera, Y., Saito, Y., and Inada, Y.: Terpene alcohol ester synthesis by polyethylene glycol-modified lipase. Biotechnol. Lett., 9, 187-190 (1987). 19. Okumura, S., Iwal, M., and Tsujisaka, Y.: Synthesis of various kinds of esters by four microbial lipases. Biochim. Biophys. Acta, 575, 156-165 (1979). 20. Linko, Y.-Y. and Yu, H.-C.: Enzymic synthesis of oleic acid esters by various lipases. Ann. N.Y. Acad. Sci., 672, 492-496 (1992). 21. Koshiro, S., Sonomoto, K., Tanaka, A., and Fukui, S.:

22.

23. 24.

25.

26. 27. 28. 29.

BUTYRATE

477

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