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Highly efficient and regioselective acylation of arbutin catalyzed by lipase from Candida sp.
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Short communication
Highly efficient and regioselective acylation of arbutin catalyzed by lipase from Candida sp. Liyan Jiang a,b , Xiaona Xie c , Hong Yue a , Zhuofu Wu a , Haoran Wang a , Fengjuan Yang a , Lei Wang a,b,∗ , Zhi Wang a,b,∗ a
Key Laboratory of Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun 130012, China College of Life Science, Jilin University, Changchun 130012, China c The First Hospital of Jilin University, Changchun 130021, China b
a r t i c l e
i n f o
Article history: Received 16 October 2014 Received in revised form 26 January 2015 Accepted 10 February 2015 Available online xxx Keywords: Arbutin Regioselective acylation Lipase from Candida sp. Transesterification
a b s t r a c t Arbutin, as a simple polyphenol, can be extracted from almost all the natural plants and has various important bioactivities. Regioselective acylation of arbutin can improve its cell membrane penetration and enhance its biological activity. The regioselective acylation of arbutin catalyzed by lipase from Candida sp. (CSL, expressed from Canadia sp. 99–125) has been successfully conducted in non-aqueous media. Under the optimum conditions (arbutin, 0.73 mmol; vinyl acetate, 29.71 mmol; organic solvent, 20 ml tetrahydrofuran; initial water activity, 0.63; temperature, 40 ◦ C; enzyme dosage, 100 mg enzyme powder in this reaction system), the highest enzyme activity (3.71 ± 0.13 mol/h/mg) could be obtained. The conversion could reach 91.42 ± 2.43% after 24 h and the regioselectivity of the CSL-catalyzed acylation was highly specific at the C-6 position in the glucose moiety of arbutin (>99%). CSL has been proven to be an excellent candidate as a catalyst for highly regioselective acylation of arbutin. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Plant polyphenols are very important compounds and can be found in almost all the natural plants [1–3]. Among these compounds, arbutin (hydroquinone-O--d-glucopyranoside) is one of the most attractive simple polyphenol. It can effectively inhibit the activity of tyrosinase [4] and have the ability to scavenge the free radicals [5]. Furthermore, arbutin can be applied as a cosmetic material due to its excellent skin-whitening property [6,7]. However, the application of arbutin has been strongly limited by its poor cell membrane penetration [8]. It is generally believed that acylation of arbutin can enhance its solubility in various media and improve its stability [8,9]. Its biological activity can also be adjusted by the acylation. For example, the acylated arbutin has been reported to have higher inhibitory effect on tyrosinase [10] and can inhibit the oxidation of low-density lipoproteins more
∗ Corresponding authors at: Key Laboratory of Molecular Enzymology and Engineering of Ministry of Education, College of Life Science, Jilin University, No. 2699 Qianjin Street, Changchun 130012, China. Tel.: +86 431 85155243; fax: +86 431 88980440. E-mail addresses: w [email protected] (L. Wang), [email protected] (Z. Wang).
efficiently than arbutin [11]. The acylated derivatives have also been reported to have higher anti-melanogenesis and anti-oxidant activities than its parent compound [12]. Therefore, acylation of arbutin has already attracted much attention in recent years. It is well known that enzymes are regioselective and the process can be conducted under mild conditions. Therefore, acylation of arbutin via the enzymatic approach will be a promising method due to the poly-hydroxylated nature of arbutin. However, only a few enzymes have been reported to have the ability for highly regioselective acylation of arbutin [8,9,13–17]. For example, arbutin 6 -laurate has been synthesized using the immobilized lipase from Candida antarctica as the catalyst [14]. An alkaline protease, Bioprase, has also been used to catalyze the transesterification of arbutin and undecylenic acid vinyl ester in dimethylformamide to get arbutin derivative [16]. Recently, Yang and his group have reported a highly regioselective route to arbutin esters by the immobilized lipase from Penicillium expansum [8,15]. However, the enzyme activity in their reports was still unsatisfactory and much work is still needed to find a novel enzyme with high enzyme activity and regioselectivity for acylation of arbutin. As a relatively cheaper commercial lipase, CSL was expressed from a novel constructed strain (Canadia sp. 99–125) established by Tan’s group and its preparation has been described in previous report [18]. It has
http://dx.doi.org/10.1016/j.procbio.2015.02.014 1359-5113/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Jiang L, et al. Highly efficient and regioselective acylation of arbutin catalyzed by lipase from Candida sp. Process Biochem (2015), http://dx.doi.org/10.1016/j.procbio.2015.02.014
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2
OR
OH HO
6'
4'
HO
O
5' 3'
1' O 1
CSL
2
2' OH
3
HO
4'
HO
Vinyl ester
4
6 5
6'
O
5' 3'
1' O 1 2
2' OH
4
6
OH
3
5
OH
Scheme 1. Regioselective acylation of arbutin catalyzed by CSL.
been successfully used to catalyze the esterification for fatty esters such as biodiesel and 2-ethylhexyl palmitate production from fatty acids [19,20]. In this paper, the lipase from Candida sp. (CSL) was successfully used to carry out the regioselective acylation of arbutin with vinyl ester as acyl donor (Scheme 1). This is a kinetically controlled synthesis [21] and its final yield depends not only on the thermodynamic constant of the reaction, but also on the kinetic properties of the enzyme [22]. The effects of acyl donor, organic solvent, water activity, temperature and enzyme dosage have been investigated. 2. Materials and methods 2.1. Enzyme and chemicals Arbutin was purchased from Wuhan Fude chemical. Co., Ltd. (Wuhan, China). The powder of CSL (lipase from Candida sp., expressed from Canadia sp. 99–125 [18]) was purchased from Beijing CTA New Century Biotechnology Co., Ltd. (Beijing, China). The protein content was quantified using the Branford protein assay with BSA as a standard [23]. Vinyl esters and other analytical grade reagents were purchased from Shanghai Chemical Reagent Company (Shanghai, China) and all the reagents were desiccated by 4 A˚ molecular sieve overnight.
(4.6 mm × 250 mm × 5 m, Agilent, USA) with a UV detector at 282 nm. The mobile phase is a mixture of water (A) and methanol (B) with a gradient program of 10–30% (B) for 0–5 min, 30% (B) for 5–25 min and 30–10% (B) for 25–30 min. The flow rate is 1.0 ml/min. The retention time of arbutin and its 6 -O-acetate were 5.689 min and 11.426 min, respectively. 2.5. Characterization of the acylated derivatives The chemical structure of the acylated derivatives were determined by 1 H NMR (500 MHz) and 13 C NMR (126 MHz) in DMSO-d6 using a Bruker AC500 spectrometer (Bruker, Courtaboeuf, France). The results of NMR analysis were listed below: 1 H NMR: ı ppm 2.01 (s, 3H, H ), 3.11–3.29 (m, 3H, H + H + 2 2 3 H4 ), 3.51 (ddd, 1H, H5 ), 4.08 (dd, 1H, H6 ), 4.26 (dd, 1H, H6 ), 4.68 (d, 1H, H1 ), 5.14 (d, 1H, OH4 ), 5.23 (d, 1H, OH3 ), 5.31 (d, 1H, OH2 ), 6.62–6.69 (m, 2H, H3 + H5 ), 6.81–6.86 (m, 2H, H2 + H6 ), 9.02 (s, 1H, OH4 ). 13 C NMR: ı ppm 21.13 (C ), 63.94 (C ), 70.45 (C ), 73.95 (C ), 2 6 4 2 74.31 (C5 ), 77.02 (C3 ), 102.12 (C1 ), 115.98 (C3 , C5 ), 118.15 (C2 , C6 ), 150.62 (C1 ), 152.87 (C4 ), 170.53 (C1 ). 3. Results and discussion 3.1. Effect of acyl donor and enzyme dosage
2.2. Regioselective acylation of arbutin The reaction was performed under the following conditions: arbutin (0.73 mmol), vinyl acetate (30 mmol), enzyme (100 mg), tetrahydrofuran (THF, 20 ml) and water activity (aw = 0.63) was incubated with shaking (100 rpm) at 40 ◦ C for 24 h. The control experiments were performed in the absence of the enzyme. The reaction was terminated by filtration to remove the enzyme and the solvent was evaporated under vacuum. The reaction was monitored by RPTLC and HPLC analysis. The enzyme activity (mol/h/mg) was defined as the amount (in micromoles) of the arbutin ester produced per hour per milligram of enzyme. The experiments were performed triplicate and all data were obtained based on the average values. 2.3. Water activity setting All the used reagents were previously dried in a vacuum of 133 Pa for 12 h. Then all reaction mixtures with specific water activity (aw ) were prepared by adding a specific amount of water. The aw was measured by the Hygrolab Humidity Detector (Rotronic, Switzerland) before being applied to the reaction. 2.4. Analysis methods The reaction was monitored by RPTLC and HPLC. For RPTLC analysis, samples were placed on Kieselgel 60 RP-18 F254 plates and eluted with chloroform/methanol (6/1, v/v). Arbutin and its acylated derivatives can be detected under UV light (254 nm). The HPLC analysis was carried out on a Thermo C18 column
Four different vinyl esters with various chain lengths were selected to examine the effect of acyl donors and the results were listed in Table 1. It could be found that CSL can catalyze all the reactions and the highest enzyme activity (3.71 ± 0.13 mol/h/mg) was achieved by using vinyl acetate as the acyl donor. Formation of the acyl-enzyme intermediate is believed to be the first step in lipasecatalyzed transesterification. Water or arbutin may then attack the acyl-enzyme intermediate. Longer acyl donor may produce more steric hindrances to the attack, which might decrease the enzyme activity [24,25]. Furthermore, the acetylated position was identified by NMR and we found that CSL displayed C-6 regioselectivity position excellently in the glucose moiety of arbutin (>99%) and no other compound could be detected. Therefore, vinyl acetate was chosen as the acyl donor for further study. The effects of molar ratio of substrates and enzyme dosage have also been investigated (data not shown). We found that the optimum molar ratio (vinyl acetate/arbutin) was about 40:1. The influence of the molar ratio of substrates might be attributed to a balance among the Table 1 Effect of acyl donor on the regioselective acylation of arbutin. Acyl donor
Enzyme activity (mol/h/mg)
Vinyl acetate Vinyl propionate Vinyl butyrate Vinyl valerate
3.71 2.84 2.02 1.35
± ± ± ±
0.13 0.09 0.15 0.07
6 -Regioselectivity (%) >99 >99 >99 >99
Reaction conditions: arbutin (200 mg, 0.73 mmol), vinyl esters (2.75 ml, 29.71 mmol), CSL (100 mg) and THF (20 ml, aw = 0.63) were performed at 40 ◦ C.
Please cite this article in press as: Jiang L, et al. Highly efficient and regioselective acylation of arbutin catalyzed by lipase from Candida sp. Process Biochem (2015), http://dx.doi.org/10.1016/j.procbio.2015.02.014
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Table 2 Effect of reaction medium on the regioselective acylation of arbutin. Medium
Enzyme activity (mol/h/mg)
6 -Regioselectivity (%)
Cyclohexane Methylenechloride Tertiary butanol THF Acetone Acetonitrile Dimethyl sulfoxide
– 0.57 ± 1.13 ± 3.71 ± 2.72 ± 2.69 ± –
– >99 >99 >99 >99 >99 –
0.05 0.18 0.13 0.16 0.20
Reaction conditions: arbutin (200 mg, 0.73 mmol), vinyl acetate (2.75 ml, 29.71 mmol), CSL (100 mg) and organic media (20 ml, aw = 0.63) were performed at 40 ◦ C.
alcoholysis with arbutin, the hydrolysis of vinyl acetate, and the denaturation of enzyme caused by the formed acetic acid and/or acetaldehyde. When the effect of enzyme dosage was investigated, it could be found that higher CSL dosage would increase the conversion. The conversion of 91.42 ± 2.43% could be obtained after 24 h when the enzyme dosage exceeded 100 mg. By increasing the enzyme dosage, more lipases would take part in the reaction and then increase the conversion [26]. However, high enzyme dosage may cause the aggregation of enzyme, decrease the stirring effect or disrupt the water activity, which may have some negative effects on the reaction process. So, there was no obvious difference between 100 mg and 140 mg CSL in this reaction system. 3.2. Effect of reaction medium
Fig. 2. Effect of temperature on the regioselective acylation of arbutin. The reactions were carried out in THF (20 ml, aw = 0.63) with arbutin (200 mg, 0.73 mmol), vinyl acetate (2.75 ml, 29.71 mmol) and CSL (100 mg) at different temperature.
“induced-fit” process of enzyme and decrease the enzyme activity [32]. On the contrary, higher aw tends to increase the aggregation of enzyme particle and then worsen the enzyme performance [33,34]. Furthermore, water is a competitor of arbutin by the acyl enzyme which may also explain the negative effects of higher aw . Therefore, the enzyme needs a suitable water activity to exhibit its optimum enzyme performance in organic solvents.
3.4. Effect of reaction temperature
The reaction medium is an important influencing factor. In this study, several organic solvents were selected to investigate the effect of the reaction medium. The results in Table 2 demonstrated that the highest enzyme activity (3.71 ± 0.13 mol/h/mg) was obtained when THF was used as the reaction medium. Various solvents have different dissolving capacity toward arbutin. Furthermore, the reaction media may fully alter the enzyme conformation and change the enzyme performance [27–30].
In the present study, the reaction temperature was changed from 20 to 50 ◦ C to investigate its effect. As shown in Fig. 2, the enzyme activity exhibited a bell-shaped curve with changing the temperature and the highest value was observed at 40 ◦ C. The possible explanation might be that high reaction temperature can increase the enzyme activity until is too high and produce enzyme inactivation [35–37].
3.3. Effect of initial water activity
3.5. Time course of the regioselective acylation of arbutin
Water activity (aw ) is another crucial parameter in nonaqueous enzymology [31]. In this study, the effect of initial water activities (aw ) on the regioselective acylation of arbutin was examined and the results were plotted in Fig. 1. The maximum enzyme activity (3.71 ± 0.13 mol/h/mg) was observed at aw = 0.63. Higher or lower aw values would decrease the enzyme activity. The excessively rigid conformation of the used enzyme at low aw can disturb the
Fig. 3 shows the progress of the regioselective acylation of arbutin catalyzed by CSL. It could be found that the conversion could reach 91.42 ± 2.43% at 24 h under the optimum conditions. The results demonstrated that CSL is an excellent candidate as a catalyst for regioselective acylation of arbutin due to its higher enzyme activity and its excellent regioselectivity.
Fig. 1. Effect of water activity on the regioselective acylation of arbutin. The reactions were carried out in THF (20 ml) with arbutin (200 mg, 0.73 mmol), vinyl acetate (2.75 ml, 29.71 mmol) and CSL (100 mg) at 40 ◦ C. The initial water activity was varied from 0.04 to 0.97.
Fig. 3. Time course of the regioselective acylation of arbutin. The reactions were carried out in THF (20 ml, aw = 0.63) with arbutin (200 mg, 0.73 mmol), vinyl acetate (2.75 ml, 29.71 mmol) and CSL (100 mg) at 40 ◦ C.
Please cite this article in press as: Jiang L, et al. Highly efficient and regioselective acylation of arbutin catalyzed by lipase from Candida sp. Process Biochem (2015), http://dx.doi.org/10.1016/j.procbio.2015.02.014
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4. Conclusion From the results of this study it could be concluded that the regioselective acylation of arbutin could be easily carried out through transesterification catalyzed by CSL under mild reaction conditions. The regioselectivity of the CSL-catalyzed acylation was highly specific at the C-6 position in the glucose moiety of arbutin (>99%). CSL has been proved to be an excellent biocatalyst for highly regioselective acylation of arbutin. In order to improve the enzyme reusability and decrease the reaction cost further, a study adopting the technique of immobilization is currently in progress. Besides increasing enzyme reusability and stability, immobilization [22,38–43] can also greatly improve the enzyme activity and specificity. The work will be reported in due course. Acknowledgements The authors are grateful for the financial support from National Natural Science Foundation of China (Nos. 21172093, 31070708 and 21072075), the Special Fund for Basic Scientific Research of Jilin University (Nos. 450060326007 and 450060491559) and Natural Science Foundation of Jilin Province, China (No. 20140101141JC). References [1] Balandrin MF, Klocke JA, Wurtele ES, Bollinger WH. Natural plant chemicals: sources of industrial and medicinal materials. Science 1985;228: 1154–60. [2] Quideau S, Deffieux D, Douat-Casassus C, Pouységu L. Plant polyphenols: chemical properties, biological activities, and synthesis. Angew Chem Int Ed 2011;50:586–621. [3] Haslam E. Natural polyphenols (vegetable tannins) as drugs: possible modes of action. J Nat Prod 1996;59:205–15. [4] Jin YH, Lee SJ, Chung MH, Park JH, Park YI, Cho TH, et al. Aloesin and arbutin inhibit tyrosinase activity in a synergistic manner via a different action mechanism. Arch Pharm Res 1999;22:232–6. [5] Takebayashi J, Ishii R, Chen J, Matsumoto T, Ishimi Y, Tai A. Reassessment of antioxidant activity of arbutin: multifaceted evaluation using five antioxidant assay systems. Free Radic Res 2010;44:473–8. [6] Sakuma K, Ogawa M, Sugibayashi K, Yamada K-I, Yamamoto K. Relationship between tyrosinase inhibitory action and oxidation–reduction potential of cosmetic whitening ingredients and phenol derivatives. Arch Pharm Res 1999;22:335–9. [7] Chanchal D, Swarnlata S. Novel approaches in herbal cosmetics. J Cosmet Dermatol 2008;7:89–95. [8] Yang RL, Li N, Li RF, Smith TJ, Zong MH. A highly regioselective route to arbutin esters by immobilized lipase from Penicillium expansum. Bioresour Technol 2010;101:1–5. [9] Nagai M, Watanabe Y, Nomura M. Synthesis of acyl arbutin by an immobilized lipase and its suppressive ability against lipid oxidation in a bulk system and O/W emulsion. Biosci Biotechnol Biochem 2009;73:2501–5. [10] Tokiwa Y, Kitagawa M, Raku T. Enzymatic synthesis of arbutin undecylenic acid ester and its inhibitory effect on mushroom tyrosinase. Biotechnol Lett 2007;29:481–6. [11] Ma X, Yan R, Yu S, Lu Y, Li Z, Lu H. Enzymatic acylation of isoorientin and isovitexin from bamboo-leaf extracts with fatty acids and antiradical activity of the acylated derivatives. J Agric Food Chem 2012;60:10844–9. [12] Watanabe Y, Nagai M, Yamanaka K, Jose K, Nomura M. Synthesis of lauroyl phenolic glycoside by immobilized lipase in organic solvent and its antioxidative activity. Biochem Eng J 2009;43:261–5. [13] Nakajima N, Ishihara K, Matsumura S, Hamada H, Nakamura K, Furuya T. Lipasecatalyzed synthesis of arbutin cinnamate in an organic solvent and application of transesterification to stabilize plant pigments. Biosci Biotechnol Biochem 1997;61:1926–8. [14] Chigorimbo-Murefu NT, Riva S, Burton SG. Lipase-catalysed synthesis of esters of ferulic acid with natural compounds and evaluation of their antioxidant properties. J Mol Catal B: Enzym 2009;56:277–82. [15] Yang RL, Li N, Ye M, Zong MH. Highly regioselective synthesis of novel aromatic esters of arbutin catalyzed by immobilized lipase from Penicillium expansum. J Mol Catal B: Enzym 2010;67:41–4.
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Please cite this article in press as: Jiang L, et al. Highly efficient and regioselective acylation of arbutin catalyzed by lipase from Candida sp. Process Biochem (2015), http://dx.doi.org/10.1016/j.procbio.2015.02.014
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Short communication
Highly efficient and regioselective acylation of arbutin catalyzed by lipase from Candida sp. Liyan Jiang a,b , Xiaona Xie c , Hong Yue a , Zhuofu Wu a , Haoran Wang a , Fengjuan Yang a , Lei Wang a,b,∗ , Zhi Wang a,b,∗ a
Key Laboratory of Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun 130012, China College of Life Science, Jilin University, Changchun 130012, China c The First Hospital of Jilin University, Changchun 130021, China b
a r t i c l e
i n f o
Article history: Received 16 October 2014 Received in revised form 26 January 2015 Accepted 10 February 2015 Available online xxx Keywords: Arbutin Regioselective acylation Lipase from Candida sp. Transesterification
a b s t r a c t Arbutin, as a simple polyphenol, can be extracted from almost all the natural plants and has various important bioactivities. Regioselective acylation of arbutin can improve its cell membrane penetration and enhance its biological activity. The regioselective acylation of arbutin catalyzed by lipase from Candida sp. (CSL, expressed from Canadia sp. 99–125) has been successfully conducted in non-aqueous media. Under the optimum conditions (arbutin, 0.73 mmol; vinyl acetate, 29.71 mmol; organic solvent, 20 ml tetrahydrofuran; initial water activity, 0.63; temperature, 40 ◦ C; enzyme dosage, 100 mg enzyme powder in this reaction system), the highest enzyme activity (3.71 ± 0.13 mol/h/mg) could be obtained. The conversion could reach 91.42 ± 2.43% after 24 h and the regioselectivity of the CSL-catalyzed acylation was highly specific at the C-6 position in the glucose moiety of arbutin (>99%). CSL has been proven to be an excellent candidate as a catalyst for highly regioselective acylation of arbutin. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Plant polyphenols are very important compounds and can be found in almost all the natural plants [1–3]. Among these compounds, arbutin (hydroquinone-O--d-glucopyranoside) is one of the most attractive simple polyphenol. It can effectively inhibit the activity of tyrosinase [4] and have the ability to scavenge the free radicals [5]. Furthermore, arbutin can be applied as a cosmetic material due to its excellent skin-whitening property [6,7]. However, the application of arbutin has been strongly limited by its poor cell membrane penetration [8]. It is generally believed that acylation of arbutin can enhance its solubility in various media and improve its stability [8,9]. Its biological activity can also be adjusted by the acylation. For example, the acylated arbutin has been reported to have higher inhibitory effect on tyrosinase [10] and can inhibit the oxidation of low-density lipoproteins more
∗ Corresponding authors at: Key Laboratory of Molecular Enzymology and Engineering of Ministry of Education, College of Life Science, Jilin University, No. 2699 Qianjin Street, Changchun 130012, China. Tel.: +86 431 85155243; fax: +86 431 88980440. E-mail addresses: w [email protected] (L. Wang), [email protected] (Z. Wang).
efficiently than arbutin [11]. The acylated derivatives have also been reported to have higher anti-melanogenesis and anti-oxidant activities than its parent compound [12]. Therefore, acylation of arbutin has already attracted much attention in recent years. It is well known that enzymes are regioselective and the process can be conducted under mild conditions. Therefore, acylation of arbutin via the enzymatic approach will be a promising method due to the poly-hydroxylated nature of arbutin. However, only a few enzymes have been reported to have the ability for highly regioselective acylation of arbutin [8,9,13–17]. For example, arbutin 6 -laurate has been synthesized using the immobilized lipase from Candida antarctica as the catalyst [14]. An alkaline protease, Bioprase, has also been used to catalyze the transesterification of arbutin and undecylenic acid vinyl ester in dimethylformamide to get arbutin derivative [16]. Recently, Yang and his group have reported a highly regioselective route to arbutin esters by the immobilized lipase from Penicillium expansum [8,15]. However, the enzyme activity in their reports was still unsatisfactory and much work is still needed to find a novel enzyme with high enzyme activity and regioselectivity for acylation of arbutin. As a relatively cheaper commercial lipase, CSL was expressed from a novel constructed strain (Canadia sp. 99–125) established by Tan’s group and its preparation has been described in previous report [18]. It has
http://dx.doi.org/10.1016/j.procbio.2015.02.014 1359-5113/© 2015 Elsevier Ltd. All rights reserved.
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OR
OH HO
6'
4'
HO
O
5' 3'
1' O 1
CSL
2
2' OH
3
HO
4'
HO
Vinyl ester
4
6 5
6'
O
5' 3'
1' O 1 2
2' OH
4
6
OH
3
5
OH
Scheme 1. Regioselective acylation of arbutin catalyzed by CSL.
been successfully used to catalyze the esterification for fatty esters such as biodiesel and 2-ethylhexyl palmitate production from fatty acids [19,20]. In this paper, the lipase from Candida sp. (CSL) was successfully used to carry out the regioselective acylation of arbutin with vinyl ester as acyl donor (Scheme 1). This is a kinetically controlled synthesis [21] and its final yield depends not only on the thermodynamic constant of the reaction, but also on the kinetic properties of the enzyme [22]. The effects of acyl donor, organic solvent, water activity, temperature and enzyme dosage have been investigated. 2. Materials and methods 2.1. Enzyme and chemicals Arbutin was purchased from Wuhan Fude chemical. Co., Ltd. (Wuhan, China). The powder of CSL (lipase from Candida sp., expressed from Canadia sp. 99–125 [18]) was purchased from Beijing CTA New Century Biotechnology Co., Ltd. (Beijing, China). The protein content was quantified using the Branford protein assay with BSA as a standard [23]. Vinyl esters and other analytical grade reagents were purchased from Shanghai Chemical Reagent Company (Shanghai, China) and all the reagents were desiccated by 4 A˚ molecular sieve overnight.
(4.6 mm × 250 mm × 5 m, Agilent, USA) with a UV detector at 282 nm. The mobile phase is a mixture of water (A) and methanol (B) with a gradient program of 10–30% (B) for 0–5 min, 30% (B) for 5–25 min and 30–10% (B) for 25–30 min. The flow rate is 1.0 ml/min. The retention time of arbutin and its 6 -O-acetate were 5.689 min and 11.426 min, respectively. 2.5. Characterization of the acylated derivatives The chemical structure of the acylated derivatives were determined by 1 H NMR (500 MHz) and 13 C NMR (126 MHz) in DMSO-d6 using a Bruker AC500 spectrometer (Bruker, Courtaboeuf, France). The results of NMR analysis were listed below: 1 H NMR: ı ppm 2.01 (s, 3H, H ), 3.11–3.29 (m, 3H, H + H + 2 2 3 H4 ), 3.51 (ddd, 1H, H5 ), 4.08 (dd, 1H, H6 ), 4.26 (dd, 1H, H6 ), 4.68 (d, 1H, H1 ), 5.14 (d, 1H, OH4 ), 5.23 (d, 1H, OH3 ), 5.31 (d, 1H, OH2 ), 6.62–6.69 (m, 2H, H3 + H5 ), 6.81–6.86 (m, 2H, H2 + H6 ), 9.02 (s, 1H, OH4 ). 13 C NMR: ı ppm 21.13 (C ), 63.94 (C ), 70.45 (C ), 73.95 (C ), 2 6 4 2 74.31 (C5 ), 77.02 (C3 ), 102.12 (C1 ), 115.98 (C3 , C5 ), 118.15 (C2 , C6 ), 150.62 (C1 ), 152.87 (C4 ), 170.53 (C1 ). 3. Results and discussion 3.1. Effect of acyl donor and enzyme dosage
2.2. Regioselective acylation of arbutin The reaction was performed under the following conditions: arbutin (0.73 mmol), vinyl acetate (30 mmol), enzyme (100 mg), tetrahydrofuran (THF, 20 ml) and water activity (aw = 0.63) was incubated with shaking (100 rpm) at 40 ◦ C for 24 h. The control experiments were performed in the absence of the enzyme. The reaction was terminated by filtration to remove the enzyme and the solvent was evaporated under vacuum. The reaction was monitored by RPTLC and HPLC analysis. The enzyme activity (mol/h/mg) was defined as the amount (in micromoles) of the arbutin ester produced per hour per milligram of enzyme. The experiments were performed triplicate and all data were obtained based on the average values. 2.3. Water activity setting All the used reagents were previously dried in a vacuum of 133 Pa for 12 h. Then all reaction mixtures with specific water activity (aw ) were prepared by adding a specific amount of water. The aw was measured by the Hygrolab Humidity Detector (Rotronic, Switzerland) before being applied to the reaction. 2.4. Analysis methods The reaction was monitored by RPTLC and HPLC. For RPTLC analysis, samples were placed on Kieselgel 60 RP-18 F254 plates and eluted with chloroform/methanol (6/1, v/v). Arbutin and its acylated derivatives can be detected under UV light (254 nm). The HPLC analysis was carried out on a Thermo C18 column
Four different vinyl esters with various chain lengths were selected to examine the effect of acyl donors and the results were listed in Table 1. It could be found that CSL can catalyze all the reactions and the highest enzyme activity (3.71 ± 0.13 mol/h/mg) was achieved by using vinyl acetate as the acyl donor. Formation of the acyl-enzyme intermediate is believed to be the first step in lipasecatalyzed transesterification. Water or arbutin may then attack the acyl-enzyme intermediate. Longer acyl donor may produce more steric hindrances to the attack, which might decrease the enzyme activity [24,25]. Furthermore, the acetylated position was identified by NMR and we found that CSL displayed C-6 regioselectivity position excellently in the glucose moiety of arbutin (>99%) and no other compound could be detected. Therefore, vinyl acetate was chosen as the acyl donor for further study. The effects of molar ratio of substrates and enzyme dosage have also been investigated (data not shown). We found that the optimum molar ratio (vinyl acetate/arbutin) was about 40:1. The influence of the molar ratio of substrates might be attributed to a balance among the Table 1 Effect of acyl donor on the regioselective acylation of arbutin. Acyl donor
Enzyme activity (mol/h/mg)
Vinyl acetate Vinyl propionate Vinyl butyrate Vinyl valerate
3.71 2.84 2.02 1.35
± ± ± ±
0.13 0.09 0.15 0.07
6 -Regioselectivity (%) >99 >99 >99 >99
Reaction conditions: arbutin (200 mg, 0.73 mmol), vinyl esters (2.75 ml, 29.71 mmol), CSL (100 mg) and THF (20 ml, aw = 0.63) were performed at 40 ◦ C.
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Table 2 Effect of reaction medium on the regioselective acylation of arbutin. Medium
Enzyme activity (mol/h/mg)
6 -Regioselectivity (%)
Cyclohexane Methylenechloride Tertiary butanol THF Acetone Acetonitrile Dimethyl sulfoxide
– 0.57 ± 1.13 ± 3.71 ± 2.72 ± 2.69 ± –
– >99 >99 >99 >99 >99 –
0.05 0.18 0.13 0.16 0.20
Reaction conditions: arbutin (200 mg, 0.73 mmol), vinyl acetate (2.75 ml, 29.71 mmol), CSL (100 mg) and organic media (20 ml, aw = 0.63) were performed at 40 ◦ C.
alcoholysis with arbutin, the hydrolysis of vinyl acetate, and the denaturation of enzyme caused by the formed acetic acid and/or acetaldehyde. When the effect of enzyme dosage was investigated, it could be found that higher CSL dosage would increase the conversion. The conversion of 91.42 ± 2.43% could be obtained after 24 h when the enzyme dosage exceeded 100 mg. By increasing the enzyme dosage, more lipases would take part in the reaction and then increase the conversion [26]. However, high enzyme dosage may cause the aggregation of enzyme, decrease the stirring effect or disrupt the water activity, which may have some negative effects on the reaction process. So, there was no obvious difference between 100 mg and 140 mg CSL in this reaction system. 3.2. Effect of reaction medium
Fig. 2. Effect of temperature on the regioselective acylation of arbutin. The reactions were carried out in THF (20 ml, aw = 0.63) with arbutin (200 mg, 0.73 mmol), vinyl acetate (2.75 ml, 29.71 mmol) and CSL (100 mg) at different temperature.
“induced-fit” process of enzyme and decrease the enzyme activity [32]. On the contrary, higher aw tends to increase the aggregation of enzyme particle and then worsen the enzyme performance [33,34]. Furthermore, water is a competitor of arbutin by the acyl enzyme which may also explain the negative effects of higher aw . Therefore, the enzyme needs a suitable water activity to exhibit its optimum enzyme performance in organic solvents.
3.4. Effect of reaction temperature
The reaction medium is an important influencing factor. In this study, several organic solvents were selected to investigate the effect of the reaction medium. The results in Table 2 demonstrated that the highest enzyme activity (3.71 ± 0.13 mol/h/mg) was obtained when THF was used as the reaction medium. Various solvents have different dissolving capacity toward arbutin. Furthermore, the reaction media may fully alter the enzyme conformation and change the enzyme performance [27–30].
In the present study, the reaction temperature was changed from 20 to 50 ◦ C to investigate its effect. As shown in Fig. 2, the enzyme activity exhibited a bell-shaped curve with changing the temperature and the highest value was observed at 40 ◦ C. The possible explanation might be that high reaction temperature can increase the enzyme activity until is too high and produce enzyme inactivation [35–37].
3.3. Effect of initial water activity
3.5. Time course of the regioselective acylation of arbutin
Water activity (aw ) is another crucial parameter in nonaqueous enzymology [31]. In this study, the effect of initial water activities (aw ) on the regioselective acylation of arbutin was examined and the results were plotted in Fig. 1. The maximum enzyme activity (3.71 ± 0.13 mol/h/mg) was observed at aw = 0.63. Higher or lower aw values would decrease the enzyme activity. The excessively rigid conformation of the used enzyme at low aw can disturb the
Fig. 3 shows the progress of the regioselective acylation of arbutin catalyzed by CSL. It could be found that the conversion could reach 91.42 ± 2.43% at 24 h under the optimum conditions. The results demonstrated that CSL is an excellent candidate as a catalyst for regioselective acylation of arbutin due to its higher enzyme activity and its excellent regioselectivity.
Fig. 1. Effect of water activity on the regioselective acylation of arbutin. The reactions were carried out in THF (20 ml) with arbutin (200 mg, 0.73 mmol), vinyl acetate (2.75 ml, 29.71 mmol) and CSL (100 mg) at 40 ◦ C. The initial water activity was varied from 0.04 to 0.97.
Fig. 3. Time course of the regioselective acylation of arbutin. The reactions were carried out in THF (20 ml, aw = 0.63) with arbutin (200 mg, 0.73 mmol), vinyl acetate (2.75 ml, 29.71 mmol) and CSL (100 mg) at 40 ◦ C.
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4. Conclusion From the results of this study it could be concluded that the regioselective acylation of arbutin could be easily carried out through transesterification catalyzed by CSL under mild reaction conditions. The regioselectivity of the CSL-catalyzed acylation was highly specific at the C-6 position in the glucose moiety of arbutin (>99%). CSL has been proved to be an excellent biocatalyst for highly regioselective acylation of arbutin. In order to improve the enzyme reusability and decrease the reaction cost further, a study adopting the technique of immobilization is currently in progress. Besides increasing enzyme reusability and stability, immobilization [22,38–43] can also greatly improve the enzyme activity and specificity. The work will be reported in due course. Acknowledgements The authors are grateful for the financial support from National Natural Science Foundation of China (Nos. 21172093, 31070708 and 21072075), the Special Fund for Basic Scientific Research of Jilin University (Nos. 450060326007 and 450060491559) and Natural Science Foundation of Jilin Province, China (No. 20140101141JC). References [1] Balandrin MF, Klocke JA, Wurtele ES, Bollinger WH. Natural plant chemicals: sources of industrial and medicinal materials. Science 1985;228: 1154–60. [2] Quideau S, Deffieux D, Douat-Casassus C, Pouységu L. Plant polyphenols: chemical properties, biological activities, and synthesis. Angew Chem Int Ed 2011;50:586–621. [3] Haslam E. Natural polyphenols (vegetable tannins) as drugs: possible modes of action. J Nat Prod 1996;59:205–15. [4] Jin YH, Lee SJ, Chung MH, Park JH, Park YI, Cho TH, et al. Aloesin and arbutin inhibit tyrosinase activity in a synergistic manner via a different action mechanism. Arch Pharm Res 1999;22:232–6. [5] Takebayashi J, Ishii R, Chen J, Matsumoto T, Ishimi Y, Tai A. Reassessment of antioxidant activity of arbutin: multifaceted evaluation using five antioxidant assay systems. Free Radic Res 2010;44:473–8. [6] Sakuma K, Ogawa M, Sugibayashi K, Yamada K-I, Yamamoto K. Relationship between tyrosinase inhibitory action and oxidation–reduction potential of cosmetic whitening ingredients and phenol derivatives. Arch Pharm Res 1999;22:335–9. [7] Chanchal D, Swarnlata S. Novel approaches in herbal cosmetics. J Cosmet Dermatol 2008;7:89–95. [8] Yang RL, Li N, Li RF, Smith TJ, Zong MH. A highly regioselective route to arbutin esters by immobilized lipase from Penicillium expansum. Bioresour Technol 2010;101:1–5. [9] Nagai M, Watanabe Y, Nomura M. Synthesis of acyl arbutin by an immobilized lipase and its suppressive ability against lipid oxidation in a bulk system and O/W emulsion. Biosci Biotechnol Biochem 2009;73:2501–5. [10] Tokiwa Y, Kitagawa M, Raku T. Enzymatic synthesis of arbutin undecylenic acid ester and its inhibitory effect on mushroom tyrosinase. Biotechnol Lett 2007;29:481–6. [11] Ma X, Yan R, Yu S, Lu Y, Li Z, Lu H. Enzymatic acylation of isoorientin and isovitexin from bamboo-leaf extracts with fatty acids and antiradical activity of the acylated derivatives. J Agric Food Chem 2012;60:10844–9. [12] Watanabe Y, Nagai M, Yamanaka K, Jose K, Nomura M. Synthesis of lauroyl phenolic glycoside by immobilized lipase in organic solvent and its antioxidative activity. Biochem Eng J 2009;43:261–5. [13] Nakajima N, Ishihara K, Matsumura S, Hamada H, Nakamura K, Furuya T. Lipasecatalyzed synthesis of arbutin cinnamate in an organic solvent and application of transesterification to stabilize plant pigments. Biosci Biotechnol Biochem 1997;61:1926–8. [14] Chigorimbo-Murefu NT, Riva S, Burton SG. Lipase-catalysed synthesis of esters of ferulic acid with natural compounds and evaluation of their antioxidant properties. J Mol Catal B: Enzym 2009;56:277–82. [15] Yang RL, Li N, Ye M, Zong MH. Highly regioselective synthesis of novel aromatic esters of arbutin catalyzed by immobilized lipase from Penicillium expansum. J Mol Catal B: Enzym 2010;67:41–4.
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Please cite this article in press as: Jiang L, et al. Highly efficient and regioselective acylation of arbutin catalyzed by lipase from Candida sp. Process Biochem (2015), http://dx.doi.org/10.1016/j.procbio.2015.02.014