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Biotechnology Letters

, Volume 30, Issue 1, pp 153–158 | Cite as

Tyrosinase activity in ionic liquids

Original Research Paper
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Abstract

Activity of mushroom tyrosinase was studied in three ionic liquids, 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIm][PF6]), 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIm][BF4]) and 1-butyl-3-methylimidazolium methylsulfate ([BMIm][MeSO4]), and was compared to that in chloroform. Kinetic parameters of the enzyme were determined and the results indicate that the enzyme in ionic liquids basically follows the same catalytic mechanism as in water, and that the ionic liquids may affect the enzyme activity by direct interacting with the enzyme and thus hindering the E–S binding due to their high hydrophilicity and polarity.

Keywords

Biocatalysis Ionic liquids Mushroom tyrosinase Polyphenol oxidase 

Introduction

Because of their negligible vapor pressures, ionic liquids have been generally recognized as green solvents and employed as substitutes for the traditional organic solvents in catalytic and synthetic reactions (Wasserscheid and Welton 2002; Sheldon 2001). Use of ionic liquids as media for performing biocatalytic reactions has also gained increasing attention (Park and Kazlauskas 2003; van Rantwijk et al. 2003; Yang and Pan 2005). As compared to those observed in conventional organic solvents, enzymes in ionic liquids are capable of presenting improved activity, stability, and selectivity.

To take full advantage of using enzymes in ionic liquids, it is important to understand the basic features and characteristics of this system. Lipases have been extensively studied in such reaction media (Kaar et al. 2003; Park and Kazlauskas 2001). Although a few oxidoreductases, such as laccase and peroxidase (Hinckley et al. 2002), chloroperoxidase (Sanfilippo et al. 2004), and d-amino acid oxidase (Lutz-Wahl et al. 2006), are active in ionic liquids, most of these studies were carried out in aqueous solution containing ionic liquids. In this paper, we report for the first time the catalytic performance of mushroom tyrosinase (polyphenol oxidase, EC 4.14.18.1) in ionic liquids. The enzyme catalyzes the oxidation of o-diphenols to o-quinones and has been shown active in organic solvents such as chloroform (Yang and Robb 1994). Three ionic liquids, [BMIm][PF6], [BMIm][BF4], and [BMIm][MeSO4], were selected. The aim of this study was to demonstrate the feasibility of using ionic liquids as the reaction media for tyrosinase, to study the kinetic behavior of the enzyme in different ionic liquids as compared to that in organic solvents, and to investigate the controlling factors, such as solvent properties, enzyme amount, substrate concentration, thermodynamic water activity, pH, and reaction temperature, that may affect the enzyme activity in ionic liquids.

Materials and methods

Materials

The three ionic liquids, 4-methylcatechnol (as the substrate), Celite 545, and 4 Å molecular sieves were purchased from Sigma.

Enzyme preparation

The enzyme was extracted into 100 ml phosphate buffer (50 mM, pH 6.0) and crudely purified from fresh mushrooms (50 g) and then adsorbed onto celite (2 g) and dried in a vacuum oven as indicated in (Yang and Robb 1994).

Partition coefficient of the product between [BMIm][PF6] and chloroform (Pp)

The product of the enzymatic reaction, 4-methyl-o-quinone, was produced by oxidation of the substrate with NaIO4 in CHCl3, and \( {\text{[P]}}_{{{\text{CHCl}}_{{\text{3}}} }} \) was determined from absorbance at 390 nm. By comparing the concentration of the product in CHCl3 before and after mixing with a known volume of [BMIm][PF6], Pp can be calculated to be 1.18 ± 0.03 according to the equation \( {\text{P}}_{{\text{p}}} {\text{ = [P]}}_{{{\text{[BMIm][PF}}_{{\text{6}}} {\text{]}}}} {\text{/[P]}}_{{{\text{CHCl}}_{{\text{3}}} }} \) .

Activity assay in chloroform

CHCl3 was pretreated by washing with deionized water and then drying over molecular sieves. Enzyme powders (10 mg) were added to a stoppered reaction vial containing 5 ml of 20 mM substrate in pretreated CHCl3. The reaction mixture was immediately placed in a constant temperature shaker (typically set at 25°C and 250 rpm) to initiate the reaction. Water activity was controlled by addition of a salt hydrate (Halling 1992). Periodically a sample (2 ml) was taken for absorbance reading at 390 nm and then transferred back to the reactor immediately. Molar extinction coefficient for the product in CHCl3 is 1.55 mM−1 cm−1 (Yang and Robb 1994).

Activity assay in ionic liquids

Reactions were carried out by dispersing enzyme powders in the substrate-containing [BMIm][PF6] in a stoppered vial to be placed in the constant temperature shaker (typically set at 30°C and 250 rpm). Periodically samples (0.1 ml) were taken and mixed thoroughly with CHCl3 (2.9 ml). After settling, the bottom CHCl3 phase was removed for absorbance reading at 390 nm. With the aid of the Pp obtained above, one was able to calculate the concentration of the product in the ionic liquid.

Results and discussion

Comparison of tyrosinase activity in three ionic liquids and in chloroform

When conducted at 35°C and a water activity of 0.90, the enzyme-catalyzed reaction in [BMIm][PF6] showed an initial reaction rate which was 23% of that obtained in CHCl3, but 3.6- and 4.7-fold higher as compared to that obtained in [BMIm][BF4] and [BMIm][MeSO4], respectively.

It is not surprising that, like in organic solvents, an enzyme may exhibit variant activities in different ionic liquids. Hydrophobicity, polarity, and viscosity are the three that are commonly considered as the major solvent properties that affect enzyme activity in ionic liquids (Yang and Pan 2005). Table 1 compares the physical properties of [BMIm][PF6] and [BMIm][BF4]. Both ionic liquids possess the same polarity and O2 solubility (this parameter is worth noting as O2 is a co-substrate for tyrosinase), and the viscosities of both solvents fall in the same order of magnitude, taking into consideration the broad viscosity range of ionic liquids (35–500 cP) (Brennecke and Maginn 2001). The fact that [BMIm][PF6] yielded a much higher enzyme activity than [BMIm][BF4] despite the similar polarity, viscosity and oxygen solubility may be contributed to the significant difference in their hydrophobicity. [BMIm][BF4] and [BMIm][MeSO4] are highly hydrophilic, thus being able to enter the aqueous microenrivonments surrounding the enzyme molecules and have some direct interactions with the enzyme to inactivate it. Mushroom tyrosinase showed extremely low activity as well in hydrophilic organic solvents such as ethyl acetate and dioxane (Yang and Robb 1994). In particular, the ionic interaction between the solvent and the enzyme should be considered.
Table 1

A comparison of the solvent properties of the two ionic liquids

Solvent properties

[BMIm][BF4]

[BMIm][PF6]

Hydrophobicity

Hydrophilic

Hydrophobic (logP = −2.39d)

Density (g/cm3)a

1.17

1.36

Melting point (°C)a

−82

−8

Viscosity (cP)a

233

312

Polarity (EN T)b

0.68

0.68

O2 Solubility (mM)c

2.5

2.3

aThe data for density, melting point and viscosity were obtained from (Carda-Broch et al. 2003)

bThe polarity data were from (Park and Kazlauskas 2001)

cThe oxygen solubilities in [BMIm][BF4] and [BMIm][PF6] at the atmospheric pressure and ambient temperature were estimated from (Jacquemin et al. 2006) and (Kumelan et al. 2005), respectively

dThe log P value for [BMIm][PF6] was obtained from (Kaar et al. 2003)

The three ionic liquids used in this study contained the same cation and different anions. The isoelectric point of mushroom tyrosinase is 4.8 (Robb 1984), therefore the overall charge of the enzyme should be negative as the enzyme was prepared from a phosphate buffer of pH 6.0. The enzyme activity may then be altered by the repelling effect of the ionic liquid anions on the dynamics and conformation of the enzyme. Additionally, the polarity of the B–F bond in BF 4 − should be slightly higher than that for the P–F bond in PF 6 − according to the electronegativity theory (McMurry and Fay 2004), and this might result in a more disruptive interaction with the enzyme and therefore be responsible in part for the lower activity of tyrosinase in [BMIm][BF4]. The polarity of the C–Cl bond in CHCl3 is relatively much weaker.

The tolerance experiment (Fig. 1) revealed that compared to the enzyme being treated with chloroform or not, incubation of the enzyme in the three ionic liquids all led to a significant loss in the enzyme activity. This further confirmed the possible interactions of the ionic liquids with the enzyme molecules, leading to an irreversible inactivation to the enzyme. Chloroform was still the solvent to retain the highest enzyme activity. The enzyme treated with [BMIm][BF4] retained a higher activity than that with [BMIm][PF6], presumably due to the stabilizing effect caused by the more kosmotropic anion BF 4 − (Zhao 2005).
Fig. 1

Retained activity of the enzyme after being treated with different solvents. Enzyme powders (20 mg) were either untreated (✳) or immersed in 1 ml of CHCl3 (□), [BMIm][BF4] (△), [BMIm][PF6] (◯), [BMIm][MeSO4] (◊) at 30°C for 24 h. The precipitate was then washed 3 times with 1 ml CHCl3 before being vacuum-dried for overnight. Activity of the treated enzyme was detected in 2 ml CHCl3 containing 20 mM substrate at 25°C by following the absorbance of the reaction product at 390 nm. Water activity was controlled at 0.85 by using the pre-equilibrium method (Yang and Robb 1994)

Kinetic performance of tyrosinase in [BMIm][PF6]

The enzyme followed the classic Michaelis–Menten kinetics in [BMIm][PF6] as it did in chloroform. The enzymatic reaction rate increased proportionally with the amount of enzyme powders that were added to the reaction system. The apparent K m value of the enzyme in [BMIm][PF6] (33 mM) was much higher than those obtained in both CHCl3 (0.5 mM) and aqueous solution (0.3 mM) (Yang and Robb 1993). Like in the latter two reaction media, the enzyme in [BMIm][PF6] was also subjected to substrate inhibition, reaching a maximum activity when the substrate concentration was 60 mM (Fig. 2).
Fig. 2

Variation of enzyme activity upon the change in substrate concentration in [BMIm][PF6]. The reactions were carried out by adding 20 mg of enzyme powders and 0.5 g Na2HPO4·12H2O into 2 ml [BMIm][PF6] containing 20–80 mM substrate under the conditions of 30°C and 250 rpm. The 100% relative reaction rate referred to the initial reaction rate obtained at the substrate concentration of 60 mM, that is, 0.53 mM/h

Water activity has been known to play an important role in affecting enzyme activity in non-aqueous environments (Halling 1994). Figure 3 illustrates that the enzyme presented a higher activity at a higher water activity. This is consistent with our previous findings about the effect of water activity on the activity of the same enzyme in CHCl3 (Yang and Robb 1991).
Fig. 3

Progress curves of tyrosinase-catalyzed reactions at two water activities, 0.85 (✳) and 0.52 (◯), given by addition of two salt hydrates, Na2HPO4·12H2O and Na4P2O7·10H2O, at 30°C, respectively (Halling 1992). 60 mg of enzyme powders were added to 2 ml of [BMIm][PF6] containing 20 mM substrate

Although pH memory has been considered as one of the novel properties exhibited by enzymes in organic solvents, it did not look so obvious for tyrosinase in our ionic liquid system. This weak pH memory might be a result of the buffering effect to the enzyme contributed by both the cation and anion of the ionic liquid.

Effect of the reaction temperature on the enzyme activity has also been investigated (Fig. 4). The rate of the tyrosinase-catalyzed reaction increased with the increase in the reaction temperature from 30 to 50°C. Unfortunately, no further reactions at a higher temperature were carried out, as 50°C is the maximum that the constant temperature shaker can reach. Obviously, one can expect that the optimal reaction temperature of the enzyme in [BMIm][PF6] should be higher than 50°C, which is therefore higher than the one for the enzyme in chloroform (20°C) and in aqueous solution (45°C) (Yang and Robb 1993).
Fig. 4

Effect of reaction temperature on enzyme activity in [BMIm][PF6]. Reactions were conducted at different temperatures by adding 40 mg of enzyme powders and 0.5 g Na2HPO4·12H2O into 2 ml [BMIm][PF6] containing 20 mM substrate

By using the data in Fig. 4, one can calculate the activation energy of the enzymatic reaction in [BMIm][PF6] to be 78.7 kJ/mol according to the Arrhenius equation. This activation energy is 2.7 times as high as that obtained in chloroform (29.1 kJ/mol)), whereas the latter is only slightly higher than the value for the aqueous system (28.2 kJ/mol) (Yang and Robb 1993). This is in consistence with the K m values obtained in all the three media, suggesting that the nonaqueous solvents affect the enzyme activity basically by hindering the binding of the substrate to the enzyme’s active site, thereby resulting in both a higher K m and a higher activation energy, and this situation is much more serious when an ionic liquid is used as the reaction medium. Indeed, in the presence of the same substrate concentration (20 mM), the enzyme in [BMIm][PF6] showed only 23% of the initial reaction rate that was obtained in CHCl3; and, by using the K m values listed above, one might expect that the enzyme in [BMIm][PF6] could present a V max value that was 59% of the V max obtained for the same enzyme in CHCl3. A simultaneous increase in K m and decrease in V max were also observed for laccase when the reaction medium was switched from aqueous solution to ionic liquids such as [BMIm][PF6] and [BMIm][BF4] (Hinckley et al. 2002).

Conclusions

Our experiments have demonstrated that mushroom tyrosinase remains catalytically active in ionic liquids, capable of presenting an enzyme activity that is comparable to that obtained in organic solvents. Like in chloroform, the enzyme basically follows the same catalytic mechanism as in aqueous solution. However, the activity of the enzyme may vary depending upon which ionic liquid is used as the reaction medium, indicating that ionic liquids have some direct effects on the enzyme. Because of their high polarity and high hydrophilicity, they can inactivate the enzyme by penetrating into the aqueous microphase surrounding the enzyme molecules so as to interact directly with the enzyme and in turn to hinder the binding between the enzyme and the substrate. The cation and anion of the ionic liquid may have a pH buffering effect, hence weakening the pH memory of the enzyme. Moreover, enzymatic reactions in ionic liquids can be conducted at a higher temperature, thus offering a higher catalytic efficiency. Further investigation regarding the effects of ionic liquids on enzyme activity is being carried out in our laboratory.

Notes

Acknowledgement

This project was sponsored by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, Ministry of Education, PRC.

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Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  1. 1.College of Life SciencesShenzhen UniversityShenzhenChina

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