Biochemical Engineering Journal 9 (2001) 85–89

Semi-continuous production of lauroyl kojic acid throughlipase-catalyzed condensation in acetonitrile

Takashi Kobayashi a, Shuji Adachi a,∗, Kazuhiro Nakanishi b, Ryuichi Matsuno a

a Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japanb Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Tsushima-naka, Okayama 700-8530, Japan

Received 4 April 2001; accepted 14 May 2001

Abstract

7-O-Lauroyl kojic acid was synthesized on some tens of grams scale through lipase-catalyzed condensation of kojic and lauric acids inacetonitrile using a semi-continuous system where unreacted substrates were reused after the product recovery. The acylation of kojic acidimproved the solubility in hexane and soybean oil. The absorption spectra of lauroyl kojic acid chelated with ferric ion were observed inethanol and soybean oil at various molar ratios. The color tone of the complex of lauroyl kojic acid and Fe3+ was similar to that of thecomplex of unmodified kojic acid and Fe3+ in ethanol. © 2001 Elsevier Science B.V. All rights reserved.

Keywords: Bioconversion; CSTR; Kojic acid; Lauric acid; Lipase

1. Introduction

Kojic acid (5-hydroxy-2-(hydroxymethyl)-1,4-pyrone)possesses some biological functions such as antimicrobialactivity, metal-chelating activity [1] and inhibitory activitytoward tyrosinase [2] and polyphenol oxidase [3]. It is un-stable even at ordinary temperature. To improve its stability,its enzymatic glycosylation has been reported [4–6]. How-ever, because kojic acid and its derivatives with saccharidesare water-soluble, the application of kojic acid to oily foodsor cosmetics is restricted.

The improvement in oil-solubility of kojic acid throughits chemical [7] or lipase-catalyzed [8] acylation with short-or medium-chain fatty acid or oleic acid has been reported.However, the acylation was carried out using a small batchreactor, and enzymatic production of acylated kojic acid ona gram scale has not been reported.

In this study, we developed a semi-continuous systemusing a continuous stirred tank reactor (CSTR) where anexcess of kojic acid was dispersed and unreacted substrateswere reused after simple recovery method for producinglauroyl kojic acid on some tens of grams scale. The solu-bility of the product in organic solvents and the color toneof the product chelated with Fe3+ in ethanol and soybeanoil were also measured.

∗ Corresponding author. Tel.: +81-75-753-6287; fax: +81-75-753-6285.E-mail address: adachi@kais.kyoto-u.ac.jp (S. Adachi).

2. Experimental section

2.1. Materials

Immobilized lipase from Candida antarctica, Chira-zyme® L-2 c.-f. C2, was purchased from Roche MolecularBiochemicals, Mannheim, Germany. Kojic and lauric acids,iron(III) chloride hexahydrate (ferric chloride), acetonitrileand soybean oil were purchased from Wako Pure ChemicalIndustries, Osaka, Japan. Other chemicals were obtainedfrom either Wako Pure Chemical Industries or NacalaiTesque, Kyoto.

2.2. CSTR

Acetonitrile was dehydrated over sodium sulfate. Lauricand kojic acids were dissolved or dispersed in the acetoni-trile at the concentrations of 40 and 100 mmol/l, respectively.Chirazyme® L-2 (35 g) was put in a 500 ml round-bottomedflask together with 200 ml of the substrate solution, andthe flask was tightly sealed with a silicon-stopper with at-tached influent and effluent tubes. The flask was immersedin a thermostated water-bath (Personal-11, Taitec, Tokyo)at 50◦C and shaken at 160 strokes/min. The reaction mix-ture was withdrawn with a peristaltic pump (MP-3, TokyoRikakikai) at a flow rate of 8–30 ml/min, and the sub-strate solution was fed by the pressure difference. After thesteady state was achieved, the product concentration in the

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86 T. Kobayashi et al. / Biochemical Engineering Journal 9 (2001) 85–89

effluent was determined to obtain the relationship betweenthe residence time and the product concentration.

2.3. Semi-continuous production of lauroyl kojic acid

Fig. 1 schematically shows a system for semi-continuousproduction of lauroyl kojic acid, where unreacted substrateswere reused. Twenty-four grams (0.12 mol) of lauric acidand 42 g (0.30 mol) of kojic acid were dissolved or dispersedin 3-l of the dehydrated acetonitrile. The substrate solutionwas magnetically stirred to prevent the sedimentation ofundissolved kojic acid. The reaction vessel was the same asthe above-mentioned CSTR; the amount of the immobilizedlipase was 35 g and the working volume of the reactor was200 ml. The reaction mixture was withdrawn at a flow rateof 20 ml/min through a settler and the substrate solutionwas fed at the same flow rate, which corresponded to theresidence time of 10 min. The most of the unreacted kojicacid had settled down in the settler as the solid particles.

The solutions in the product reservoir and in the settlerwere put together and filtered on a G4 glass filter to removeunreacted kojic acid. The recovered kojic acid was reusedas a substrate in the next run. The filtrate was kept at 5◦Cfor 2 days to deposit the product. The precipitated productwas recovered by filtration on a Büchner funnel with a ToyoNo. 2 filter paper. The filtrate including unreacted lauric acidwas dehydrated and reused as the reaction medium in thenext run. The concentration of lauric acid in the mediumafter product recovery was analyzed, and lauric acid wassupplemented to adjust its concentration to 40 mmol/l. Theproduct on the paper was washed five times with water toremove kojic acid contaminated in the product. The wash-ings were evaporated to reuse the unreacted kojic acid as thesubstrate.

The product was dissolved with 500 ml of chloroform. Analiquot of the solution was diluted 10 times with chloroform,

Fig. 1. Schematic drawing of a semi-continuous system for producinglauroyl kojic acid: ( ) substrate reservoir; ( ) magnetic stirrer; ( ) aCSTR; ( ) thermostated water-bath; ( ) settler; ( ) peristaltic pump;( ) product reservoir. The CSTR was shaken at 160 strokes/min in thebath kept at 50◦C.

and the product concentration was determined by the HPLC.Based on the analysis, the yield of the lauroyl kojic acid wasestimated.

The semi-continuous production of lauroyl kojic acid wasrepeated four times without replacing the immobilized en-zyme. The product was recrystallized twice in acetonitrileand once in methanol and was analyzed by 1H NMR.

2.4. Solubility of substrates and product

To find a method for recovering the product from theeffluent, the solubilities of the substrates and the productwere measured at various temperatures. Lauric acid (1 g),kojic acid (50 mg) or lauroyl kojic acid (0.1 g) was put ina vial with 5 ml of the dehydrated acetonitrile. The solutionwas warmed at 50◦C to dissolve the solute and then kept ata given temperature for 2 or 3 days with occasional stirringto reach the equilibrium solubility at that temperature. Aportion of the supernatant was taken and filtered on a Toyo13HP020AN membrane. The solute concentration of thefiltrate was determined by the HPLC to obtain the solubility.

Fifty milligrams of unmodified or lauroyl kojic acid wasdispersed in 5 ml of hexane or soybean oil. The dispersionwas warmed at 50◦C for 1 h, and then kept at 20◦C for 2 days.The solubility was then determined by the above-mentionedprocedures.

2.5. Absorption spectra of unmodified orlauroyl kojic acid–Fe3+ complex

The absorption spectra of unmodified and lauroyl kojicacids chelated with Fe3+ were observed in ethanol. The spec-tra of lauroyl kojic acid–Fe3+ complex were also measuredin soybean oil.

Ferric chloride was dissolved in ethanol at a concentrationof 0.2 mmol/l. Kojic acid or lauroyl kojic acid was dissolvedin the solution at a concentration of 0.1–2 mmol/l, and theabsorbance from 200 to 800 nm was measured at room tem-perature using a Shimadzu UV-1600 spectrophotometer.

Ferric chloride was dissolved in acetone at a concentra-tion of 0.2 mmol/l. Lauroyl kojic acid was dissolved in thesolution at a concentration of 0.1–2 mmol/l. Ten millilitersof the solution was mixed with 10 ml of soybean oil, andacetone was then removed under reduced pressure. The ab-sorbance from 350 to 800 nm of the soybean oil includingthe complex of lauroyl kojic acid and Fe3+ was measuredat room temperature.

2.6. Analysis

The concentrations of unmodified and lauroyl kojicacids were determined using a Shimadzu LC-3A HPLCequipped with an ODS column (Cadenza CD-C18,3 × 75 mm, Imtakt, Kyoto) and an SPD-2A UV detector(Shimadzu, 272 nm). The eluents used were mixtures ofmethanol/water/acetic acid = 50/50/0.1 by vol. for kojic

T. Kobayashi et al. / Biochemical Engineering Journal 9 (2001) 85–89 87

acid and =95/5/0.1 by vol. for lauroyl kojic acid. The flowrate was 0.5 ml/min.

The concentration of lauric acid was determined with theHPLC equipped with a YMC Pack-SIL A-102 column (6 ×150 mm, Kyoto) and the UV detector (220 nm). The eluentwas a mixture of hexane/2-propanol = 99/1 by vol., andits flow rate was 1.2 ml/min.

3. Results and discussion

3.1. Relationship between the residence time andthe product concentration

The steady-state concentration of product in the CSTRwas measured at various residence times (Fig. 2). At resi-dence times shorter than 10 min, the product concentrationincreased almost proportionally to the time, although it onlygradually increased at times longer than 10 min. The productconcentration at the residence time = 10 min correspondedto a conversion of 30% based on the feed concentration oflauric acid, which was the limiting substrate. The productconcentration was converted to the productivity and is alsoplotted versus the residence time in the figure.

3.2. Solubility of kojic, lauric and lauroylkojic acids in acetonitrile

The solubilities of kojic, lauric and lauroyl kojic acids inacetonitrile were measured at various temperatures (Fig. 3).Lauroyl kojic acid was least soluble in acetonitrile amongthe substrates and product at any temperature. The solubilityof each solute was lowered as the temperature decreased.The difference in the solubility between the product andthe substrates became larger at the lower temperature.This indicated that the product, lauroyl kojic acid, could

Fig. 2. Dependencies of the product concentration in effluent of the reactorand the productivity on the residence time. The curves were empiricallydrawn.

Fig. 3. Temperature dependencies of the solubilities of (�) lauric acid,(�) kojic acid and (�) lauroyl kojic acid in acetonitrile.

be recovered as a solid by chilling the effluent withoutsolidifying lauric acid.

As shown in Fig. 2, the product concentration in the ef-fluent was ca. 12 mmol/l at the residence time = 10 min.Because its solubility at 5◦C was ca. 1.5 mmol/l, more than85% of the product could be recovered as a solid at thattemperature. Therefore, the product was recovered at thattemperature in the following semi-continuous production oflauroyl kojic acid.

3.3. Semi-continuous production of lauroyl kojic acid

Using the system shown in Fig. 1, lauroyl kojic acid wassemi-continuously produced. The production was repeatedfour times. Fig. 4 shows the product concentration in theproduct reservoir at the end of each run. The product concen-tration at the residence time = 10 min was ca. 11.5 mmol/l in

Fig. 4. Semi-continuous production of lauroyl kojic acid using the pro-posed system. Symbols (�) and (�) indicate the product concentrationin the product reservoir at the end of each run and the cumulative amountof product recovered, respectively.

88 T. Kobayashi et al. / Biochemical Engineering Journal 9 (2001) 85–89

Fig. 5. Structure of lauroyl kojic acid.

Fig. 1, although it was ca. 9.5 mmol/l in the semi-continuousproduction (Fig. 4). One possible reason for the differencemight be the difference in the water content of the reactionmedium among the runs, although the medium was dehy-drated with sodium sulfate before each run.

The cumulative amount of product recovered as a solid isalso shown in Fig. 4. The total amount of lauric acid used forfour runs was 43 g (0.215 mol); the amount loaded in the firstruns was 24 g and that loaded in each of the following runswas ca. 6 g. The amount of the product after four runs was37 g (0.114 mol). Therefore, the overall yield for the runswas 53%. The yield would become higher if the productionis further repeated.

The recrystallized product was analyzed by 1H NMR(300 MHz, CDCl3, TMS, 298 K): δ = 0.88 (12′-Me, 3H, t,J = 6.7 Hz); 1.26 (4′-11′-H, 16H, m); 1.65 (3′-H, 2H, m),2.40 (2′-H, 2H, t, J = 7.5 Hz); 4.93 (7-H, 2H, s); 6.50 (3-H,1H, s); 7.85 (6-H, 1H, s). The spectrum indicated that theproduct was 7-O-lauroyl kojic acid, as shown in Fig. 5. Liuand Shaw [8] reported that lipases from Psuedomonas cepa-cia and Penicillium camembertii catalyzed the acylation atC-5 of kojic acid with lauric or oleic acid. In our case, kojicacid was acylated at C-7.

3.4. Solubility of kojic and lauroyl kojic acidin hexane and soybean oil

Kojic acid is water-soluble but is hard to dissolve in or-ganic solvent. Its acylation would be expected that the solu-bility in organic solvent would be improved. The solubilitiesat 20◦C of unmodified and lauroyl kojic acids in hexanewere 0.0034 and 0.641 mmol/l, respectively. Introductionof a lauroyl residue into kojic acid increased the solubilityin hexane by 190 times. The solubilities at 20◦C of unmod-ified and lauroyl kojic acids in soybean oil were 0.127 and1.29 mmol/l, respectively. The solubility of lauroyl kojicacid was about 10 times that of kojic acid. Thus, acylation ofkojic acid improved the solubility in a hydrophobic medium.

3.5. Absorption spectra of unmodified orlauroyl kojic acid–Fe3+ complex

Fig. 6(a) and (b) shows the absorption spectra of unmod-ified and lauroyl kojic acids, respectively, chelated withFe3+ in ethanol at various molar ratios. The concentrationof ferric chloride was fixed at 0.2 mmol/l. Lauroyl kojic acidshowed coloration similar to that of unmodified kojic acid

Fig. 6. Absorption spectra of (a) kojic acid and (b) lauroyl kojic acidchelated with Fe3+ in ethanol. The figures indicate the molar ratio ofunmodified or lauroyl kojic acid to ferric chloride.

in ethanol, although the absorption intensity of the complexof lauroyl kojic acid and Fe3+ in the visible portion of thespectra was slightly smaller than that for unmodified kojicacid. The solutions were reddish-purple.

The absorption spectra of lauroyl kojic acid chelated withFe3+ were also measured in soybean oil (Fig. 7). The spectrain the oil shifted to shorter wavelengths, and the color wasyellowish compared with that in ethanol.

Fig. 7. Absorption spectra of lauroyl kojic acid chelated with Fe3+ insoybean oil. The figures indicate the molar ratio of lauroyl kojic acidto ferric chloride. E1 is the spectrum for lauroyl kojic acid mixed withferric chloride at the molar ratio of 1:1 in ethanol.

T. Kobayashi et al. / Biochemical Engineering Journal 9 (2001) 85–89 89

Fig. 8. Dependencies of absorbance at 510 nm for (�) kojic acid and (�)lauroyl kojic acid chelated with Fe3+ in ethanol and (�) absorbance at400 nm for lauroyl kojic acid chelated with Fe3+ in soybean oil on themolar ratio of unmodified or lauroyl kojic acid to ferric chloride.

The absorbances at 510 and 400 nm are plotted versusthe molar ratio of unmodified or lauroyl kojic acid to ferricchloride in ethanol and soybean oil, respectively, in Fig. 8.The absorbance A was empirically correlated with the molarratio R by Eq. (1):

A = A∞R

B + R(1)

where A∞ is the absorbance at R → ∞, and B a parameterreflecting the binding affinity between kojic acid and Fe3+.The parameters A∞ and B were estimated by a non-linearregression using the software, Origin Ver. 6.1J, OriginLab,Northampton, MA. The A∞ for lauroyl kojic acid in ethanolwas 0.377, which was smaller than that for the unmodifiedacid (=0.603). The B value for lauroyl kojic acid (=0.537)was also smaller than that of the unmodified acid (=1.14),i.e., the binding affinity of the product to Fe3+ was slightlystronger than that of unmodified kojic acid.

The A∞ and B values of lauroyl kojic acid in soybeanoil were 1.09 and 0.818, respectively. The color intensity at400 nm in soybean oil was much stronger than that at 510 nmin ethanol.

4. Conclusions

As mentioned above, lauroyl kojic acid of 37 g wasproduced through four runs repeatedly using immobilizedlipase in the semi-continuous system where most of theunreacted kojic and lauric acids was effectively reused. Theoverall yield of the product for four runs was ca. 53% basedon the amount of used lauric acid, which was the limit-ing substrate. The acylation of kojic acid with lauric acidgreatly improved the solubility in hexane and soybean oil.Chelating activity with Fe3+ was slightly affected by theacylation.

Acknowledgements

This study was supported by the Program for the Promo-tion of Basic Research Activities for Innovative Biosciences(PROBRAIN), Japan.

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