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Effects of organic solvents on the activity of free and immobilised laccase from Rhus vernicifera
International Journal of Biological Macromolecules 47 (2010) 488–495
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Effects of organic solvents on the activity of free and immobilised laccase from Rhus vernicifera Yun-Yang Wan a,b,c , Rong Lu b , Ling Xiao a , Yu-Min Du a , Tetsuo Miyakoshi b , Chen-Loung Chen d , Charles J. Knill e , John F. Kennedy e,∗ a
College of Resource and Environmental Science, Wuhan University, Wuhan 430072, Hubei, China Department of Industrial Chemistry, Meiji University, Higashi-mita, Tama-ku, Kawasaki 214, Japan State Key Laboratory of Petroleum Resources and Exploration, Faculty of Natural Resources and Information Technology, China University of Petroleum, Beijing 102249, China d Department of Wood and Paper Science, North Carolina State University, Raleigh, NC 27695-8005, USA e Chembiotech Laboratories, Institute of Advanced Science & Technology, 5, The Croft, Buntsford Drive Stoke Heath, Bromsgrove, Worcestershire B60 4JE, UK b c
a r t i c l e
i n f o
Article history: Received 24 June 2010 Accepted 12 July 2010 Available online 18 July 2010 Keywords: Rhus laccase Immobilisation Chitosan Enzyme Catalysis Hydrophobicity
a b s t r a c t Rhus laccase (RL) was covalently immobilised onto chitosan, and the effects of immobilisation on pH optimum, enzyme activity, thermostability, and re-use evaluated, using either N,N-dimethyl-pphenylenediamine or 2,6-dimethoxyphenol as substrate. Immobilisation greatly enhanced enzyme thermostability, resulted in negligible loss of activity, and showed excellent re-use potential, with >80% relative activity retained after 15 cycles in aqueous solvent. Immobilised Rhus laccase (I-RL) was more catalytically active in both hydrophobic and hydrophilic organic solvents than free RL. With waterimmiscible organic solvents, both free RL and I-RL required a minimum water content to achieve activity. With water-miscible organic solvents, in general a water content of ∼20–50% (v/v) was required to achieve activity using free RL, whereas with I-RL less water was generally required to achieve enzyme activity, and therefore considerably higher relative activity was exhibited at lower water contents. Kinetic investigations showed that the rate of substrate disappearance generally followed a pseudofirst-order law, and for evaluated water-immiscible organic solvents rate constants generally increased with decrease of hydrophobicity, however, in water-miscible organic solvents no such relationship was observed. Some discussion of the potential interactions between organic solvent molecules and enzyme active centres was provided to explain obtained results. © 2010 Published by Elsevier B.V.
1. Introduction Lacquer trees, Rhus vernicifera, grow widely in Asian countries and regions, especially in China and Japan [1], and are a kind of abundant natural resource [2–4]. When xylems of lacquer trees are injured or cut, they produce a milk white sap, named raw lacquer or Chinese lacquer, which contains urushiols (60–65%, w/w), Rhus laccases (RL, 0.3–0.5%, w/w), polysaccharides (5–7%, w/w), glycoproteins (∼1–3%, w/w), the pseudo-glycoprotein stellacyanin (St, ∼1%, w/w) and water (20–30%, w/w), etc. [5]. All of these components have been separated and purified [1,6], some have been studied individually, and some of their complex interactions investigated [5,7]. The term laccase (EC 1.10.3.2, benzenediol:oxygen oxidoreductase), covers a group of multi-copper-containing proteins/enzymes that widely occur in higher plants, fungi and microorganisms
∗ Corresponding author. Tel.: +44 1527 576000. E-mail address: [email protected] (J.F. Kennedy). 0141-8130/$ – see front matter © 2010 Published by Elsevier B.V. doi:10.1016/j.ijbiomac.2010.07.003
[6,8,9], which catalyse the one-electron oxidation of various aromatic substrates, particularly substituted phenols and anilines, with concomitant reduction of molecular oxygen to water [10,11]. Such enzymes have great potential for industrial application, particularly in areas such as bioremediation, organic synthesis, and bio-bleaching [2,12–14]. Fungal laccases can exhibit activity in reaction media where all or most of the water has been replaced by organic solvents, where enzymes acquire novel properties, such as enhanced stability, altered inhibitor specificity, and the ability to catalyse reactions that cannot proceed in conventional aqueous environments [15–19]. Fungal laccases have been studied extensively [13,17,18,20], whereas the functions of plant laccases, such as Rhus laccases, have received far less attention [8]. There are significant differences between fungal and Rhus laccases [1,20,21], and therefore investigation of the enzymatic activity of the latter in various organic solvents is required, and should facilitate elucidation of the reaction mechanisms of phenolic substrates, such as urushiols and dioxins, which are poorly soluble/insoluble in water.
Y.-Y. Wan et al. / International Journal of Biological Macromolecules 47 (2010) 488–495
Previous studies have investigated the enzymatic activity of free and immobilised RL in aqueous systems. Employed immobilisation methodologies have included embedding in polysaccharides (such as carrageenan, sodium alginate, agar and chitosan) [5], adsorption and chemical derivatisation onto chitosan and modified chitosan [5,22], inorganic chelation using a zirconium salt [5], and transition metals [23,24]. Various enzyme immobilisation techniques and supports are available [25], however there is no single technique/support that is the best for immobilisation of all enzymes or all applications of a single given enzyme. In organic solvents, the support may be more important with respect to enzyme stability than the coupling methodology [26,27]. Chitosan is an inexpensive, inert and hydrophilic biopolymeric support [28,29], which can be responsive to changes in pH and/or temperature [30], has excellent biocompatibility and biodegradability [28,29,31], and is regularly used for enzyme immobilisation [5,22,32]. In this paper the effects of immobilisation of RL on chitosan, in terms of pH optimum, enzyme activity, and re-use performance are investigated, and the effects of water-immiscible and watermiscible organic solvent/water systems on the activity of free and immobilised RL are presented and discussed. 2. Materials and methods 2.1. Materials Dried acetone insoluble material (AIM), also known in the field of lacquer chemistry as acetone powder (AP), was isolated from Chinese lacquer trees, R. vernicifera, in the Cheng Kou region of China. Rhus laccase (RL) was isolated and purified according to previously reported methods [1,5,22]. Chitosan (MW ∼ 66 kDa) was generously donated by Prof. Yoshiro (Meiji University, Japan). All other chemicals were of suitable analytical grade and used as received unless otherwise stated. 2.2. Immobilisation of RL by chemical derivatisation onto chitosan Purified RL (4 mg) was mixed with sodium periodate solution (45 mmol/L in phosphate buffer, 100 mM, pH 6.0, 2 mL). The resulting solution was stored in the dark for 2 h under ambient conditions (∼20–25 ◦ C), followed by addition of ethylene glycol (200 L) and sodium phosphate buffer (200 mmol/L, pH 7.5, 2 mL). Chitosan (1.0 g) was added, and the enzyme immobilisation carried out for 24 h at 4 ◦ C. Excess enzyme was then removed by washing the suspended solid phase sequentially with deionised water, and sodium phosphate buffer (200 mmol/L, pH 7.0). The resulting immobilised RL (I-RL) was stored at 4 ◦ C until required. 2.3. Determination of enzyme activity in aqueous systems RL and I-RL activity was measured spectrophotometrically in triplicate at 25 ◦ C (unless different as detailed below) using either N,N-dimethyl-p-phenylenediamine (0.83 mmol/L in sodium phosphate buffer, 200 mmol/L, pH 7.0, unless different as detailed below) or 2,6-dimethoxyphenol (12.97 mmol/L in sodium phosphate buffer, 200 mmol/L, pH 7.0, unless different as detailed below) as substrates. RL or I-RL was added to the substrate solution (3 mL), mechanically stirred (5 min), and the increase in clear solution absorbance (A) measured at 323 nm (for N,N-dimethylp-phenylenediamine) and 468 nm (for 2,6-dimethoxyphenol). One unit of RL activity was defined as the change in optical density at the corresponding wavelength effected per min per mol of protein added to 3 mL substrate solution in a 1 cm cell path length and incubated at 25 ◦ C [6,22,24].
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2.3.1. Effect of pH on enzyme activity RL and I-RL activity was measured spectrophotometrically in triplicate at 25 ◦ C using 2,6-dimethoxyphenol (12.97 mmol/L in sodium phosphate buffer, 200 mmol/L, pH 5.0–8.0, 0.5 pH unit intervals) as substrate, as detailed above. 2.3.2. Assessment of enzyme thermostability Assessment of enzyme thermostability was performed by preincubation of RL and I-RL in sodium phosphate buffer (200 mmol/L, pH 7.0) at 25–60 ◦ C (at 5 ◦ C intervals) for 1 h. Samples were removed at 15 min intervals, rapidly cooled to ambient temperature, and the residual activity determined as detailed above using 2,6-dimethoxyphenol (12.97 mmol/L in sodium phosphate buffer, 200 mmol/L, pH 7.0) as substrate. 2.3.3. Determination of Michaelis constants (Km ) Km values for RL and I-RL were determined at 25 ◦ C from linear Lineweaver–Burk plots using N,N-dimethyl-p-phenylenediamine as substrate in sodium phosphate buffer (200 mmol/L, pH 7.0), according to the method described by Du and Zhang [33]. 2.3.4. Evaluation of I-RL re-use efficiency I-RL activity was measured spectrophotometrically at 25 ◦ C using N,N-dimethyl-p-phenylenediamine (0.83 mmol/L in sodium phosphate buffer, 200 mmol/L, pH 7.0) as substrate (as detailed above). The I-RL solid was then repeatedly washed with sodium phosphate buffer (200 mmol/L, pH 7.0), until no absorbance (at 323 nm) was observed. The I-RL activity measurement (and subsequent washing) was then repeated to obtain 15 I-RL activity measurements in total. 2.4. Enzyme activity in organic solvent/water systems RL and I-RL activity was measured spectrophotometrically in triplicate at 25 ◦ C using 2,6-dimethoxyphenol (12.97 mmol/L in organic solvent) as substrate. RL or I-RL was added to the substrate solution (3 mL), mechanically stirred (5 min), and the increase in clear solution absorbance (A) measured at 468 nm. One unit of RL activity was defined as the change in optical density effected per min per mol of protein added to 3 mL substrate solution in a 1 cm cell path length and incubated at 25 ◦ C [6,22,24]. 2.5. Enzyme kinetics in water-miscible organic solvent/water systems I-RL activity in water-miscible organic solvent/water systems was measured spectrophotometrically at 25 ◦ C using 2,6-dimethoxyphenol (12.97 mmol/L in solvent, 25 mL, in a 50 mL volume reaction vessel) as substrate. I-RL (containing 1.53 g of enzyme) was added to the reaction under constant gentle stirring, and solution samples (3 mL aliquots) were removed at 3 min intervals (up to a maximum time of 21 min), and their absorbance measured at 468 nm. Samples were immediately transferred back to the reaction vessel after absorbance measurement. 3. Results and discussion 3.1. Effect of RL immobilisation on enzyme activity Laccase is a glycoprotein, with a carbohydrate content of ∼45% (w/w) [1,22,34], and its active centre is a three copper-containing site situated in the protein part of the molecule [2,11,35]. There are various reports on the effects of the carbohydrate part on enzyme activity [36], however, binding of support molecules too close to the active centres can result in a decrease in, or complete loss of, enzyme activity [37]. The activity of several enzymes remained
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Y.-Y. Wan et al. / International Journal of Biological Macromolecules 47 (2010) 488–495 Table 1 Thermostability of free (F) and immobilised (I) Rhus laccase (RL) using 2,6dimethoxyphenol (DMP) as substrate (13.0 mM in 0.2 M phosphate buffer, pH 7.0). Time (min)
15 30 45 60
Enzyme
F-RL I-RL F-RL I-RL F-RL I-RL F-RL I-RL
Relative activity (%) 25 ◦ C
30 ◦ C
35 ◦ C
45 ◦ C
55 ◦ C
60 ◦ C
100 88.0 88.1 77.6 82.3 75.9 81.6 75.9
79.9 80.0 71.6 79.8 63.4 76.2 58.7 75.2
58.6 80.2 33.5 76.6 28.3 68.9 21.4 70.7
36.7 75.5 22.1 70.4 16.0 66.7 6.52 65.3
15.7 69.3 7.26 62.8 NA 55.0 NA 55.0
2.1 55.5 NA 50.7 NA 48.1 NA 40.1
NA: no activity.
Fig. 1. Effect of pH on RL and I-RL relative activity using 2,6-dimethoxyphenol (12.97 mmol/L in sodium phosphate buffer solution, 200 mmol/L, at 25 ◦ C) as substrate (% variation for replicate analyses <5%).
mostly intact when the carbohydrate part of the glycoprotein was oxidised using sodium periodate under mild conditions [38], and therefore this immobilisation methodology was selected for use. The determined activity for RL-catalysed oxidation of N,Ndimethyl-p-phenylenediamine (0.83 mmol/L in sodium phosphate buffer solution, 200 mmol/L, pH 7.0) was ∼2.36 × 104 units/g, and for oxidation of 2,6-dimethoxyphenol (12.97 mmol/L in sodium phosphate buffer solution, 200 mmol/L, pH 7.0) was ∼7.14 × 103 units/g. The determined activity for I-RL-catalysed oxidation of N,N-dimethyl-p-phenylenediamine was ∼1.16 × 102 units/g, and for oxidation of 2,6-dimethoxyphenol was ∼3.5 × 101 units/g. For both substrates, I-RL displayed ∼0.5% of the activity of free RL, which indicates a high level of enzyme immobilisation and very little loss in activity, since in simplified terms 4 mg of RL was combined with 1000 mg of chitosan, giving an enzyme concentration change from 100 to 0.40%. 3.2. Effect of pH on enzyme activity The effect of pH (in the range 5.0–8.0) on RL and I-RL relative activity (activity values at pH 7.0 normalised to 100% and I-RL values corrected to compensate for reduced enzyme concentration) using 2,6-dimethoxyphenol (12.97 mmol/L in sodium phosphate buffer solution, 200 mmol/L) as substrate was investigated (Fig. 1). Immobilisation had no observable effect on the pH optimum for RL, which is in good agreement with the results of Wan et al. [6,22].
45 ◦ C, respectively. Indeed, free RL became irreversibly inactive after 45 min at 55 ◦ C, and 30 min at 60 ◦ C. Whereas I-RL on the other hand, retained ∼55% of its activity after 45 min at 55 ◦ C, and ∼40% activity even after incubation for 1 h at 60 ◦ C. Such increased stability could be due to immobilisation resulting in enhancement of the glycoprotein’s structural rigidity, which decreases the extent of enzyme distortion upon exposure to elevated temperatures [22,24]. Chitosan is therefore an excellent support matrix for the immobilisation of RL.
3.4. Michaelis constants (Km ) for RL and I-RL Km values were calculated as 0.16 mmol/L for RL, and 0.07 mmol/L for I-RL. The decrease in Km value after RL immobilisation shows that I-RL has a higher substrate specificity than RL.
3.5. I-RL re-use efficiency The effect of re-use on I-RL relative activity (initial enzyme activity normalised to 100%) using N,N-dimethyl-p-phenylenediamine (0.83 mmol/L in sodium phosphate buffer, 200 mmol/L, pH 7.0) as substrate was investigated (Fig. 2). The relative activity fluctuated significantly during repeated use; however, it never dropped below 80% of its initial activity during the repeated use evaluation (15 cycles), indicating good re-use potential/efficiency.
3.3. Effect of immobilisation on enzyme thermostability The in vitro stability of enzymes/proteins remains a critical issue in biotechnology and various methodologies, such as covalent attachment to polymer carriers and surface modification, have been employed to try and improve stability [37]. The method of immobilisation and the nature of the carrier should therefore be chosen according to the physical circumstances involved. The effect of temperature (in the range 25–60 ◦ C) on RL and I-RL relative activity (activity after 15 min at 25 ◦ C normalised to 100% and the I-RL values corrected to compensate for reduced enzyme concentration) as a function of time (up to 1 h) using 2,6-dimethoxyphenol (12.97 mmol/L in sodium phosphate buffer solution, 200 mmol/L, pH 7.0) as substrate was investigated (Table 1). The thermostability of RL was dramatically improved after immobilisation onto chitosan. Free RL retained good activity as a function of evaluated incubation time at 25 and 30 ◦ C, but exhibited drastic reductions with incubation at higher temperatures, going down to only ∼21 and ∼7% after 60 min at 35 and
Fig. 2. Effect of re-use on I-RL relative activity using N,N-dimethyl-pphenylenediamine (0.83 mmol/L in sodium phosphate buffer, 200 mmol/L, pH 7.0, at 25 ◦ C) as substrate (% variation for replicate analyses <2%).
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Fig. 3. Proposed reaction for the RL-catalysed oxidation of 2,6-dimethoxyphenol.
3.6. Enzyme activity in organic solvent/water systems For enzyme-catalysed reactions in non-conventional solvent systems, the primary factors that govern enzyme activity are thought to be: (a) the distribution of water in the system, (b) the specific properties of the organic solvents involved (such as hydrophobicity index, partition coefficient, polarity), and finally (c) the resulting solvation and distribution of reactants and products in the system [13,39,40]. The single product of RL-catalysed oxidation of 2,6dimethoxyphenol in aqueous solution has been identified as 3,3 ,5,5 -tetramethoxy-biphenyl-4,4 -diol (Fig. 3) [6,22,41], a single product being produced due to steric hindrance effects. 2,6-dimethoxyphenol undergoes a RL induced single-electronoxidation to produce a 2,6-dimethoxy-phenoxyl radical species ((a) in Fig. 3) that is in resonance with the corresponding para-radical species ((b) in Fig. 3). Combination of two such para-radical species produces the product (Fig. 3). The fact that only a single product is produced, makes 2,6-dimethoxyphenol a useful substrate for use in the study of RL-catalysed oxidation reactions, and hence it was used in these investigations. 3.6.1. Water-immiscible organic solvent/water systems RL was added to ethyl acetate containing 2,6-dimethoxylphenol, and the water content in the mixture adjusted by adding the desired amount of deionised water. RL was partially precipitated from the solvent system with the addition of 1% (v/v) water. Two distinct phases were formed when more than 3% (v/v) water was added, although an emulsion could be formed with vigorous mixing. RL activity was found to increase with increasing water content in the ethyl acetate/water solvent system, and reached its maximum when the water content was ∼2.5% (v/v). Other water-immiscible solvents evaluated, namely benzene, toluene and chloroform, showed similar behaviour, indicating that RL required a minimum water content to achieve its activity in such essentially non-aqueous systems. Such observations have been observed in other investigations using RL in organic solvent/water systems [42]. It is likely that the RL was absorbing water from the solvent system, and thereby functioning in an essentially aqueous microenvironment and thus retaining the characteristics of its activity in as it would in an aqueous system. These observations are comparable with those for tyrosinase [40], lipase [33,43], peroxidase [44], thermolysin [45], ␣-chymotrypsin [46] and subtilisin [47]. However, lipase, ␣-chymotrypsin and subtilisin required much less water to function in organic solvents. The required amount of water seemed to be dependent on the nature of enzyme and the type of solvents [48,49]. I-RL was added to benzene containing 2,6-dimethoxylphenol, and the water content in the mixture adjusted by adding the desired
amount of deionised water. I-RL activity was found to increase with increasing water content in the benzene/water solvent system, and reached its maximum when the water content was ∼0.5% (v/v), and then decreased readily with increasing water content (all required vigorous mixing). Other water-immiscible solvents evaluated, namely, toluene and chloroform, showed similar behaviour, with maximum I-RL activity achieved with water contents of 0.15% (v/v) and 0.5% (v/v), respectively. As in the case of free RL discussed above, with ethyl acetate maximum I-RL activity was achieved with a water content of 2.5% (v/v). In such solvent systems the I-RL became aggregated/entrapped in the water phase, leading to mass transfer limitations that would affect reaction catalysation. It appeared that the more hydrophilic the organic solvent, the lower the optimum water content required to achieve some enzyme activity with I-RL.
3.6.2. Free RL in water-miscible organic solvent/water systems The relative activity of RL (activity in sodium phosphate buffer, 200 mmol/L, pH 7.0 normalised to 100%) in a range of watermiscible organic solvent/water systems, as a function of increasing water content, using 2,6-dimethoxyphenol (12.97 mmol/L in solvent at 25 ◦ C) as substrate was investigated (Fig. 4(a)–(c)). Methanol, ethanol, 1-propanol, and 2-propanol exhibited no activity with water contents of <30% (v/v). This was followed by a slow gradual increase in activity (<10%) with water contents of ∼30–60% (v/v), up to ∼5–15% with water contents of ∼60–80% (v/v), and finally a dramatic increase up to ∼35–75% (depending on solvent system) with water contents of ∼80–90% (v/v) (Fig. 4(a)). For the same water contents the general trend was methanol = ethanol > 1propanol > 2-propanol. A threshold concentration of water was required to initiate activity, below which rapid enzyme inactivation occurred. These findings are in agreement with results reported for several other enzymes [50,51]. 1,2-Propanediol, 1,3-propanediol, 1,3-butanediol, and 1,4butanediol exhibited little (<5%) or no relative activity with water contents up to 50, 40, 40, and 20% (v/v), respectively (Fig. 4(b)). 1,2propanediol then exhibited an essentially linear activity increase up to ∼20% with a water content of 70% (v/v), followed by a more dramatic increase up to ∼70% with a water content of 90% (v/v). The profile of 1,3-butanediol was initially very similar to that of 1,2-propanediol, but did not increase as dramatically at the end, only reaching an activity of ∼45% with a water content of 90% (v/v). The profiles for 1,3-propanediol and 1,4-butanediol showed similar trends to each other, increasing to a relative activity of ∼30% (with water contents of ∼60 and ∼50% (v/v), respectively), maintaining this activity up to a water content of ∼70–80% (v/v), followed by a dramatic increase up to ∼80–85% activity with a water content of 90% (v/v) (Fig. 4(b)).
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Fig. 5. Effect of water content on I-RL relative activity in water-miscible organic solvent/water systems using 2,6-dimethoxyphenol (12.97 mmol/L, at 25 ◦ C) as substrate (% variation for replicate analyses <5%).
activity of 90% with a water content of 90% (v/v). Such observations were similar to others reported previously [51,52]. Organic solvents utilised in these investigations can be roughly divided into alcohols and non-alcohols. The following enzyme activity sequence has been observed in water-miscible alcohol/water systems: triols/polyalcohols > diols > monohydric alcohols; short chains alcohols > longer chain alcohols; straight chains alcohols > branched chain alcohols [51].
Fig. 4. Effect of water content on free RL relative activity in water-miscible organic solvent/water systems using 2,6-dimethoxyphenol (12.97 mmol/L, at 25 ◦ C) as substrate (% variation for replicate analyses <5%).
Acetone, acetonitrile, and 1,4-dioxane exhibited no activity with water contents up to 30, 40, and 40% (v/v), respectively (Fig. 4(c)). With acetonitrile and 1,4-dioxane this was followed by a very slow gradual increase in activity (<5%) with water contents of ∼40–60% (v/v), and finally a dramatic increase up to ∼80 and ∼70%, respectively with water contents of 90% (v/v). Acetone exhibited an essentially linear increase in relative activity from 0 to 95% with water contents from ∼30 to 80% (v/v), which was maintained at 90% (v/v). Glycerol/water systems demonstrated by far the highest free RL compatibility of all the solvent systems tested, with a relative activity of >10% with a water content of 10% (v/v), with rapid linear increase to ∼140% with a water content of 50% (v/v) (Fig. 4(c)). This was then followed by a sharp linear reduction down to a relative
3.6.3. I-RL in water-miscible organic solvent/water systems The relative activity of I-RL (activity in sodium phosphate buffer, 200 mmol/L, pH 7.0 normalised to 100%) in a range of watermiscible organic solvent/water systems, as a function of increasing water content, using 2,6-dimethoxyphenol (12.97 mmol/L in solvent at 25 ◦ C) as substrate was investigated (Fig. 5). Glycerol demonstrated essentially the same profile with I-RL as was observed for free RL, with a relative activity of >10% with a water content of 10% (v/v), with rapid linear increase to ∼140% with a water content of 50% (v/v). This was then followed by a sharp linear reduction down to a relative activity of ∼100% with a water content of 90% (v/v). Ethanol, 1,4-dioxane, and acetone all demonstrated considerably higher relative activity with I-RL at lower water contents (compared to free RL). This effect was greatest for ethanol, for example with free RL ethanol had an activity of <15% with a water content of <80% (v/v), whereas with I-RL an activity of >75% was achieved with a water content of only 20% (v/v) (Fig. 5). In the case of water-miscible organic solvent/water systems, a high organic solvent concentration normally results in suppression of enzyme activity due to the organic solvent replacing water molecules on the protein surface layer [16]. This results in distortion of enzyme molecular hydration to an extent that the active catalytic conformation is disrupted. It is assumed that the microenvironment of the enzyme immobilised on chitosan provides additional stability to aid in the preservation of the active enzyme conformation [22,23]. The hydrophilicity of chitosan, due to the amino and hydroxyl groups [28,29], should assist with retention of the necessary water molecules in the enzyme molecular microenvironment [32,53,54]. 3.6.4. Kinetics of I-RL catalysis in selected organic solvent/water systems In order to accurately study the effects of solvents, it is necessary to compare enzyme activity in different organic solvent whilst maintaining a constant degree of enzyme hydration. A suitable water activity towards a laccase-catalysed reaction should occur when the substrates and enzyme preparation in
Y.-Y. Wan et al. / International Journal of Biological Macromolecules 47 (2010) 488–495 Table 2 First-order reaction rate constants with respect to disappearance of 2,6dimethoxyphenol (DMP)a catalysed by immobilised Rhus laccase (I-RL) in a series of organic solvents (at 25 ◦ C). Solvent
Hydrophobicity (log P)
0.2 M phosphate buffer (pH 7.0) 0.2 M phosphate buffer (pH 7.5) Acetonitrileb Ethanolb Acetoneb Glycerineb 1,4-Dioxaneb Ethyl acetateb Tetrahydrofuranb Butyl acetatec Benzenec Toluenec Chloroformc N,N-Dimethylformamideb Cyclohexanec Hexanec
– – −0.21 −0.16 0.02 −1.76 −0.22 0.53 1.17 1.53 2.02 2.60 1.86 −0.46 3.15 3.83
a b c
First-order reaction rate constant k × 106 of I-RL (min−1 ) 154.41 115.35 37.58 46.37 46.32 35.83 4.92 2.17 1.86 1.23 1.22 1.19 0.78 0 0 0
Initial concentration 12.97 mmol/L. Water (7%, v/v) added to solvent. Solvent pre-saturated with distilled water at ambient temperature.
organic solvent are pre-equilibrated with water at lower temperature [55]. The solvents were therefore pre-saturated with water, and the resulting rate constants for I-RL-catalysed oxidation of 2,6-dimethoxylphenol in water–organic solvent systems were a relative measure of the reaction rates among the different solvent systems (Table 2). The rate of the disappearance of substrate followed a pseudo-first-order law. An increase in the water content in all the hydrophilic organic solvents investigated enhanced the efficiency of I-RL. For the evaluated water-immiscible organic solvents (namely benzene, toluene and chloroform), the first-order reaction rate constants generally increased with decrease of hydrophobicity (log P). However, in water-miscible organic solvents no apparent relationship between hydrophobicity and enzyme activity was observed, and an entirely different mechanism of activity is thought to occur
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[36]. When hexane and cyclohexane were used as solvent, the IRL-catalysed substrate oxidation occurred on the surface of the I-RL, however, no oxidation products were detectable in the organic phase of reaction mixture (by TLC and HPLC), due to the very low solubility of the products in these solvents. In the kinetic studies of I-RL in selected water-miscible organic solvents, the maximum rate constants for the disappearance of 2,6-dimethylphenol in three solvent systems, ethanol/water, acetone/water and 1,4-dioxane/water were 1.42 × 10−4 , 2.15 × 10−4 , 1.2 × 10−4 min−1 , with water content of 80% (v/v), respectively (Table 3). 3.7. Pseudo-mechanism for interactions between enzyme, solvent and support The existence of a hydrophobic shell (HS) on the surface of the active centre of the enzyme molecule is postulated (Fig. 6), where hydrogen bonding and hydrophobic interactions work together to balance the conformation of the enzyme, and the associated enzyme activity. Organic solvent molecules may disrupt this HS and displace water molecules from the enzyme surface. If the new solvent molecules are unable to maintain the hydrogen bonding/hydrophobic interactions, then the required enzyme conformation is lost and therefore so is the enzyme activity. Clearly some organic solvents are able to successfully maintain such interactions, however they may to a greater or lesser extent block the enzyme active centre and prevent access to the substrate, thereby reducing enzyme activity. On the other hand, some organic solvents may allow (or even enhance) substrate access to the enzyme active centre. Interactions between organic solvent molecules and the enzyme active centre thus determine overall enzyme activity (Fig. 6). Polyalcohols have at least two potential hydrogen bonding centres, and are hence most likely to be capable of forming threedimensional hydrogen bond network structures. Some organic solvents have been classified according to their solvophobic interaction [39]. Water and glycerin belong to the group of strongest solvophobic solvents. Hydrophilic solvent hydroxyl groups are obviously incompatible with the hydrophobic regions of enzymes, but can participate in hydrogen bonding to produce rigid inter-
Fig. 6. Pseudo-mechanism for the interaction between enzyme, solvent and/or support molecules.
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Table 3 First-order reaction rate constants with respect to disappearance of 2,6-dimethoxyphenol (DMP)a catalysed by immobilised Rhus laccase (I-RL) in a series of water-miscible organic solvent/water systems (at 25 ◦ C). Solvent system
Ethanol/water Acetone/water 1,4-Dioxane/water a b
First-order reaction rate constant k × 106 of I-RL (min−1 ) 7% (v/v)b
20% (v/v)b
40% (v/v)b
60% (v/v)b
80% (v/v)b
46.37 46.32 4.92
126.06 147.59 37.10
136.42 184.86 74.41
139.70 203.05 97.82
141.94 214.97 120.35
Initial concentration 12.97 mmol/L. Water content.
molecular frameworks, which are very important in maintaining enzyme conformation and thus enzyme activity. However, the size and conformation of the solvent molecules themselves is important, since steric hindrance of the active centre by the solvent (and disruption of the formation of inter- and/or intra-molecular hydrogen bonded networks) can also reduce enzyme activity. For example in this work, enzyme activity in ethanol/water systems was greater than both 1-propanol and 2-propanol, with 1-propanol having higher activity than 2-propanol. Differences in reaction rates in various organic solvents can also be attributed to differences in the solvation of the substrate [56], differences in the partition of substrate and products between the bulk solvent and biocatalyst phases [40], and the aforementioned differences in interactions between substrate, solvent and/or enzyme molecules (Fig. 6). 4. Conclusions The effects of organic solvents on the enzyme activity of free RL and I-RL (on chitosan) were investigated. A pH of ∼7.0 was determined to be the optimum pH for both free RL and I-RL, and immobilisation on chitosan greatly enhanced the enzyme thermostability, resulted in negligible loss of activity, and demonstrated excellent re-use potential, with >80% relative activity retained even after 15 use cycles (as evaluated in aqueous solvent). I-RL was more catalytically active in both hydrophobic and hydrophilic organic solvents than free RL. With water-immiscible organic solvents, both free RL and I-RL required a minimum water content to achieve activity in such essentially non-aqueous systems, with maximum activity achieved with a water content in the region of ∼0.5–3.0% (v/v) for the solvents evaluated. With watermiscible organic solvents, in general a water content of ∼20–50% (v/v) was required to achieve activity using free RL (depending on the organic solvent), the exception being glycerol, which exhibited activity at low water contents and the highest overall relative activity with free RL. With I-RL less water was generally required to achieve enzyme activity, and therefore considerably higher relative activity was exhibited at lower water contents, compared with free RL. Kinetic investigations showed that the rate of the disappearance of substrate generally followed a pseudo-first-order law, and for the evaluated water-immiscible organic solvents the firstorder reaction rate constants generally increased with decrease of hydrophobicity, however, in water-miscible organic solvents no such relationship was observed. Some discussion of the potential interactions between organic solvent molecules and enzyme active centres was provided to explain the obtained experimental results. Finally, chitosan is an excellent support for immobilisation of Rhus laccase and therefore has potential for a wide range of applications, particularly in the lacquer industry. Acknowledgements This work was partly supported by the Academic Frontier Project for Private Universities, matching fund subsidy from the
Ministry of Education, Culture, Sports, Science and Technology, a Research Project Grant B from the Institute of Science & Technology, Meiji University, Japan, and the National Natural Science Foundation of China (no. 30170757). References [1] Y.-Y. Wan, Y.-M. Du, F.-X. Yang, Y. Xu, R.-Z. Chen, J.F. Kennedy, Int. J. Biol. Macromol. 38 (2006) 232–240. [2] Y.-M. Du, Hua Xue Tong Bao 10 (1986) 1–9. [3] T. Miyakoshi, K. Nagase, T. Yoshida, Progress of Lacquer Chemistry, IPC Publishing, Tokyo, Japan, 2000. [4] J.-H. Yang, Y.-M. Du, R.-H. Huang, Y.-Y. Wan, T.-Y. Li, Int. J. Biol. Macromol. 31 (2002) 55–62. [5] Y.-Y. Wan, R. Lu, K. Akiyama, K. Okamoto, T. Honda, Y.-M. Du, T. Yoshida, T. Miyakoshi, C.J. Knill, J.F. Kennedy, Int. J. Biol. Macromol. 47 (2010) 76–81. [6] Y.-Y. Wan, Y.-M. Du, J.-H. Yang, R.-Z. Chen, L. Xiao, Y. Zhang, J. Wuhan Univ. (Nat. Sci. Ed.) 49 (2003) 201–204. [7] J. Kumanotani, Prog. Org. Coat. 34 (1998) 309–315. [8] D.M. O’Malley, R. Whetten, W.-L. Bao, C.-L. Chen, R.R. Sederoff, Plant J. 4 (1993) 751–757. [9] P. Baldrian, FEMS Microbiol. Rev. 30 (2006) 215–242. [10] T.E. Machonkin, L. Quintanar, A.E. Palmer, R. Hassett, S. Severance, D.J. Kosman, E.I. Solomon, J. Am. Chem. Soc. 123 (2001) 5507–5517. [11] S.-K. Lee, S. DeBeer George, W.E. Antholine, B. Hedman, K.O. Hodgson, E.I. Solomon, J. Am. Chem. Soc. 124 (2002) 6180–6193. [12] T. Shiba, L. Xiao, T. Miyakoshi, C.-L. Chen, J. Mol. Catal. B: Enzym. 10 (2000) 605–615. [13] J. Rodakiewicz-Nowak, S.M. Kasture, B. Dudek, J. Haber, J. Mol. Catal. B: Enzym. 11 (2000) 1–11. [14] P. Widsten, A. Kandelbauer, Enzyme Microb. Technol. 42 (2008) 293–307. [15] R.Z. Kazandjian, A.M. Klibanov, J. Am. Chem. Soc. 107 (1985) 5448–5450. [16] A. Zaks, A.M. Klibanov, J. Biol. Chem. 263 (1988) 8017–8021. [17] S. Nicotra, A. Intra, G. Ottolina, S. Riva, B. Danieli, Tetrahedron–Asymm. 15 (2004) 2927–2931. [18] R. Mustafa, L. Muniglia, G. Rovel, M. Girardin, Food Res. Int. 38 (2005) 995–1000. [19] J. Michizoe, S. Ichinose, N. Kamiya, T. Maruyama, M. Goto, J. Biosci. Bioeng. 99 (2005) 642–647. [20] A.M. Mayer, R.C. Staples, Phytochemistry 60 (2002) 551–565. [21] K. Nitta, K. Kataoka, T. Sakurai, J. Inorg. Biochem. 91 (2002) 125–131. [22] Y.-Y. Wan, Y.-M. Du, X.-W. Shi, J. Li, R. Lu, T. Miyakoshi, B. Chen, Process Biochem. 41 (2006) 1378–1382. [23] D.-F. Zhan, Y.-M. Du, B.-G. Qian, Chem. J. Chin. Univ. 11 (1990) 71–74. [24] L. Xiao, Y.-M. Du, Funct. Polym. 4 (1991) 218–222. [25] R.S. Freire, N. Durán, L.T. Kubota, Talanta 54 (2001) 681–686. [26] N. Durán, M.A. Rosa, A. D’Annibale, L. Gianfreda, Enzyme Microb. Technol. 31 (2002) 907–931. [27] R. Ulbrich-Hofmann, B. Selisko, Enzyme Microb. Technol. 15 (1993) 33–41. [28] J.F. Kennedy, C.J. Knill, M. Thorley, in: J.F. Kennedy, G.O. Philliips, P.A. Williams, H. Hatakeyama (Eds.), Recent Advances in Environmentally Compatible Polymers, Woodhead Publishing Ltd., Cambridge, UK, 2001, pp. 97–104. [29] J.F. Kennedy, C.J. Knill, in: S.C. Anand, J.F. Kennedy, M. Miraftab, S. Rajendran (Eds.), Medical Textiles & Biomaterials for Healthcare, Woodhead Publishing Ltd., Cambridge, UK, 2006, pp. 3–22. [30] L.-Y. Chen, Z.-G. Tian, Y.-M. Du, Biomaterials 25 (2004) 3725–3732. [31] C.-Q. Qin, Y.-M. Du, L. Xiao, Z. Li, X.-H. Gao, Int. J. Biol. Macromol. 31 (2002) 111–117. [32] A. D’Annibale, S.R. Stazi, V. Vinciguerra, E. Di Mattia, G.G. Sermanni, Process Biochem. 34 (1999) 697–706. [33] Y.-M. Du, J.-M. Zhang, Chem. J. Chin. Univ. 16 (1995) 715–721. [34] B. Reinhammar, Biochim. Biophys. Acta 205 (1970) 35–47. [35] E.I. Solomon, U.M. Sundaram, T.E. Machonkin, Chem. Rev. 96 (1996) 2563–2606. [36] D.-F. Zhan, Y.-M. Du, B.-G. Qian, Chem. Indust. Forest Prod. 11 (1991) 111– 116. [37] Z. Bílková, M. Slováková, A. Lyˇcka, D. Horák, J. Lenfeld, J. Turková, J. Churáˇcek, J. Chromatogr. B 770 (2002) 25–34. [38] J.H. Pazur, H.R. Knull, D.L. Simpson, Biochem. Biophys. Res. Commun. 40 (1970) 110–116. [39] A. Ray, Nature 231 (1971) 313–314.
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Effects of organic solvents on the activity of free and immobilised laccase from Rhus vernicifera Yun-Yang Wan a,b,c , Rong Lu b , Ling Xiao a , Yu-Min Du a , Tetsuo Miyakoshi b , Chen-Loung Chen d , Charles J. Knill e , John F. Kennedy e,∗ a
College of Resource and Environmental Science, Wuhan University, Wuhan 430072, Hubei, China Department of Industrial Chemistry, Meiji University, Higashi-mita, Tama-ku, Kawasaki 214, Japan State Key Laboratory of Petroleum Resources and Exploration, Faculty of Natural Resources and Information Technology, China University of Petroleum, Beijing 102249, China d Department of Wood and Paper Science, North Carolina State University, Raleigh, NC 27695-8005, USA e Chembiotech Laboratories, Institute of Advanced Science & Technology, 5, The Croft, Buntsford Drive Stoke Heath, Bromsgrove, Worcestershire B60 4JE, UK b c
a r t i c l e
i n f o
Article history: Received 24 June 2010 Accepted 12 July 2010 Available online 18 July 2010 Keywords: Rhus laccase Immobilisation Chitosan Enzyme Catalysis Hydrophobicity
a b s t r a c t Rhus laccase (RL) was covalently immobilised onto chitosan, and the effects of immobilisation on pH optimum, enzyme activity, thermostability, and re-use evaluated, using either N,N-dimethyl-pphenylenediamine or 2,6-dimethoxyphenol as substrate. Immobilisation greatly enhanced enzyme thermostability, resulted in negligible loss of activity, and showed excellent re-use potential, with >80% relative activity retained after 15 cycles in aqueous solvent. Immobilised Rhus laccase (I-RL) was more catalytically active in both hydrophobic and hydrophilic organic solvents than free RL. With waterimmiscible organic solvents, both free RL and I-RL required a minimum water content to achieve activity. With water-miscible organic solvents, in general a water content of ∼20–50% (v/v) was required to achieve activity using free RL, whereas with I-RL less water was generally required to achieve enzyme activity, and therefore considerably higher relative activity was exhibited at lower water contents. Kinetic investigations showed that the rate of substrate disappearance generally followed a pseudofirst-order law, and for evaluated water-immiscible organic solvents rate constants generally increased with decrease of hydrophobicity, however, in water-miscible organic solvents no such relationship was observed. Some discussion of the potential interactions between organic solvent molecules and enzyme active centres was provided to explain obtained results. © 2010 Published by Elsevier B.V.
1. Introduction Lacquer trees, Rhus vernicifera, grow widely in Asian countries and regions, especially in China and Japan [1], and are a kind of abundant natural resource [2–4]. When xylems of lacquer trees are injured or cut, they produce a milk white sap, named raw lacquer or Chinese lacquer, which contains urushiols (60–65%, w/w), Rhus laccases (RL, 0.3–0.5%, w/w), polysaccharides (5–7%, w/w), glycoproteins (∼1–3%, w/w), the pseudo-glycoprotein stellacyanin (St, ∼1%, w/w) and water (20–30%, w/w), etc. [5]. All of these components have been separated and purified [1,6], some have been studied individually, and some of their complex interactions investigated [5,7]. The term laccase (EC 1.10.3.2, benzenediol:oxygen oxidoreductase), covers a group of multi-copper-containing proteins/enzymes that widely occur in higher plants, fungi and microorganisms
∗ Corresponding author. Tel.: +44 1527 576000. E-mail address: [email protected] (J.F. Kennedy). 0141-8130/$ – see front matter © 2010 Published by Elsevier B.V. doi:10.1016/j.ijbiomac.2010.07.003
[6,8,9], which catalyse the one-electron oxidation of various aromatic substrates, particularly substituted phenols and anilines, with concomitant reduction of molecular oxygen to water [10,11]. Such enzymes have great potential for industrial application, particularly in areas such as bioremediation, organic synthesis, and bio-bleaching [2,12–14]. Fungal laccases can exhibit activity in reaction media where all or most of the water has been replaced by organic solvents, where enzymes acquire novel properties, such as enhanced stability, altered inhibitor specificity, and the ability to catalyse reactions that cannot proceed in conventional aqueous environments [15–19]. Fungal laccases have been studied extensively [13,17,18,20], whereas the functions of plant laccases, such as Rhus laccases, have received far less attention [8]. There are significant differences between fungal and Rhus laccases [1,20,21], and therefore investigation of the enzymatic activity of the latter in various organic solvents is required, and should facilitate elucidation of the reaction mechanisms of phenolic substrates, such as urushiols and dioxins, which are poorly soluble/insoluble in water.
Y.-Y. Wan et al. / International Journal of Biological Macromolecules 47 (2010) 488–495
Previous studies have investigated the enzymatic activity of free and immobilised RL in aqueous systems. Employed immobilisation methodologies have included embedding in polysaccharides (such as carrageenan, sodium alginate, agar and chitosan) [5], adsorption and chemical derivatisation onto chitosan and modified chitosan [5,22], inorganic chelation using a zirconium salt [5], and transition metals [23,24]. Various enzyme immobilisation techniques and supports are available [25], however there is no single technique/support that is the best for immobilisation of all enzymes or all applications of a single given enzyme. In organic solvents, the support may be more important with respect to enzyme stability than the coupling methodology [26,27]. Chitosan is an inexpensive, inert and hydrophilic biopolymeric support [28,29], which can be responsive to changes in pH and/or temperature [30], has excellent biocompatibility and biodegradability [28,29,31], and is regularly used for enzyme immobilisation [5,22,32]. In this paper the effects of immobilisation of RL on chitosan, in terms of pH optimum, enzyme activity, and re-use performance are investigated, and the effects of water-immiscible and watermiscible organic solvent/water systems on the activity of free and immobilised RL are presented and discussed. 2. Materials and methods 2.1. Materials Dried acetone insoluble material (AIM), also known in the field of lacquer chemistry as acetone powder (AP), was isolated from Chinese lacquer trees, R. vernicifera, in the Cheng Kou region of China. Rhus laccase (RL) was isolated and purified according to previously reported methods [1,5,22]. Chitosan (MW ∼ 66 kDa) was generously donated by Prof. Yoshiro (Meiji University, Japan). All other chemicals were of suitable analytical grade and used as received unless otherwise stated. 2.2. Immobilisation of RL by chemical derivatisation onto chitosan Purified RL (4 mg) was mixed with sodium periodate solution (45 mmol/L in phosphate buffer, 100 mM, pH 6.0, 2 mL). The resulting solution was stored in the dark for 2 h under ambient conditions (∼20–25 ◦ C), followed by addition of ethylene glycol (200 L) and sodium phosphate buffer (200 mmol/L, pH 7.5, 2 mL). Chitosan (1.0 g) was added, and the enzyme immobilisation carried out for 24 h at 4 ◦ C. Excess enzyme was then removed by washing the suspended solid phase sequentially with deionised water, and sodium phosphate buffer (200 mmol/L, pH 7.0). The resulting immobilised RL (I-RL) was stored at 4 ◦ C until required. 2.3. Determination of enzyme activity in aqueous systems RL and I-RL activity was measured spectrophotometrically in triplicate at 25 ◦ C (unless different as detailed below) using either N,N-dimethyl-p-phenylenediamine (0.83 mmol/L in sodium phosphate buffer, 200 mmol/L, pH 7.0, unless different as detailed below) or 2,6-dimethoxyphenol (12.97 mmol/L in sodium phosphate buffer, 200 mmol/L, pH 7.0, unless different as detailed below) as substrates. RL or I-RL was added to the substrate solution (3 mL), mechanically stirred (5 min), and the increase in clear solution absorbance (A) measured at 323 nm (for N,N-dimethylp-phenylenediamine) and 468 nm (for 2,6-dimethoxyphenol). One unit of RL activity was defined as the change in optical density at the corresponding wavelength effected per min per mol of protein added to 3 mL substrate solution in a 1 cm cell path length and incubated at 25 ◦ C [6,22,24].
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2.3.1. Effect of pH on enzyme activity RL and I-RL activity was measured spectrophotometrically in triplicate at 25 ◦ C using 2,6-dimethoxyphenol (12.97 mmol/L in sodium phosphate buffer, 200 mmol/L, pH 5.0–8.0, 0.5 pH unit intervals) as substrate, as detailed above. 2.3.2. Assessment of enzyme thermostability Assessment of enzyme thermostability was performed by preincubation of RL and I-RL in sodium phosphate buffer (200 mmol/L, pH 7.0) at 25–60 ◦ C (at 5 ◦ C intervals) for 1 h. Samples were removed at 15 min intervals, rapidly cooled to ambient temperature, and the residual activity determined as detailed above using 2,6-dimethoxyphenol (12.97 mmol/L in sodium phosphate buffer, 200 mmol/L, pH 7.0) as substrate. 2.3.3. Determination of Michaelis constants (Km ) Km values for RL and I-RL were determined at 25 ◦ C from linear Lineweaver–Burk plots using N,N-dimethyl-p-phenylenediamine as substrate in sodium phosphate buffer (200 mmol/L, pH 7.0), according to the method described by Du and Zhang [33]. 2.3.4. Evaluation of I-RL re-use efficiency I-RL activity was measured spectrophotometrically at 25 ◦ C using N,N-dimethyl-p-phenylenediamine (0.83 mmol/L in sodium phosphate buffer, 200 mmol/L, pH 7.0) as substrate (as detailed above). The I-RL solid was then repeatedly washed with sodium phosphate buffer (200 mmol/L, pH 7.0), until no absorbance (at 323 nm) was observed. The I-RL activity measurement (and subsequent washing) was then repeated to obtain 15 I-RL activity measurements in total. 2.4. Enzyme activity in organic solvent/water systems RL and I-RL activity was measured spectrophotometrically in triplicate at 25 ◦ C using 2,6-dimethoxyphenol (12.97 mmol/L in organic solvent) as substrate. RL or I-RL was added to the substrate solution (3 mL), mechanically stirred (5 min), and the increase in clear solution absorbance (A) measured at 468 nm. One unit of RL activity was defined as the change in optical density effected per min per mol of protein added to 3 mL substrate solution in a 1 cm cell path length and incubated at 25 ◦ C [6,22,24]. 2.5. Enzyme kinetics in water-miscible organic solvent/water systems I-RL activity in water-miscible organic solvent/water systems was measured spectrophotometrically at 25 ◦ C using 2,6-dimethoxyphenol (12.97 mmol/L in solvent, 25 mL, in a 50 mL volume reaction vessel) as substrate. I-RL (containing 1.53 g of enzyme) was added to the reaction under constant gentle stirring, and solution samples (3 mL aliquots) were removed at 3 min intervals (up to a maximum time of 21 min), and their absorbance measured at 468 nm. Samples were immediately transferred back to the reaction vessel after absorbance measurement. 3. Results and discussion 3.1. Effect of RL immobilisation on enzyme activity Laccase is a glycoprotein, with a carbohydrate content of ∼45% (w/w) [1,22,34], and its active centre is a three copper-containing site situated in the protein part of the molecule [2,11,35]. There are various reports on the effects of the carbohydrate part on enzyme activity [36], however, binding of support molecules too close to the active centres can result in a decrease in, or complete loss of, enzyme activity [37]. The activity of several enzymes remained
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Y.-Y. Wan et al. / International Journal of Biological Macromolecules 47 (2010) 488–495 Table 1 Thermostability of free (F) and immobilised (I) Rhus laccase (RL) using 2,6dimethoxyphenol (DMP) as substrate (13.0 mM in 0.2 M phosphate buffer, pH 7.0). Time (min)
15 30 45 60
Enzyme
F-RL I-RL F-RL I-RL F-RL I-RL F-RL I-RL
Relative activity (%) 25 ◦ C
30 ◦ C
35 ◦ C
45 ◦ C
55 ◦ C
60 ◦ C
100 88.0 88.1 77.6 82.3 75.9 81.6 75.9
79.9 80.0 71.6 79.8 63.4 76.2 58.7 75.2
58.6 80.2 33.5 76.6 28.3 68.9 21.4 70.7
36.7 75.5 22.1 70.4 16.0 66.7 6.52 65.3
15.7 69.3 7.26 62.8 NA 55.0 NA 55.0
2.1 55.5 NA 50.7 NA 48.1 NA 40.1
NA: no activity.
Fig. 1. Effect of pH on RL and I-RL relative activity using 2,6-dimethoxyphenol (12.97 mmol/L in sodium phosphate buffer solution, 200 mmol/L, at 25 ◦ C) as substrate (% variation for replicate analyses <5%).
mostly intact when the carbohydrate part of the glycoprotein was oxidised using sodium periodate under mild conditions [38], and therefore this immobilisation methodology was selected for use. The determined activity for RL-catalysed oxidation of N,Ndimethyl-p-phenylenediamine (0.83 mmol/L in sodium phosphate buffer solution, 200 mmol/L, pH 7.0) was ∼2.36 × 104 units/g, and for oxidation of 2,6-dimethoxyphenol (12.97 mmol/L in sodium phosphate buffer solution, 200 mmol/L, pH 7.0) was ∼7.14 × 103 units/g. The determined activity for I-RL-catalysed oxidation of N,N-dimethyl-p-phenylenediamine was ∼1.16 × 102 units/g, and for oxidation of 2,6-dimethoxyphenol was ∼3.5 × 101 units/g. For both substrates, I-RL displayed ∼0.5% of the activity of free RL, which indicates a high level of enzyme immobilisation and very little loss in activity, since in simplified terms 4 mg of RL was combined with 1000 mg of chitosan, giving an enzyme concentration change from 100 to 0.40%. 3.2. Effect of pH on enzyme activity The effect of pH (in the range 5.0–8.0) on RL and I-RL relative activity (activity values at pH 7.0 normalised to 100% and I-RL values corrected to compensate for reduced enzyme concentration) using 2,6-dimethoxyphenol (12.97 mmol/L in sodium phosphate buffer solution, 200 mmol/L) as substrate was investigated (Fig. 1). Immobilisation had no observable effect on the pH optimum for RL, which is in good agreement with the results of Wan et al. [6,22].
45 ◦ C, respectively. Indeed, free RL became irreversibly inactive after 45 min at 55 ◦ C, and 30 min at 60 ◦ C. Whereas I-RL on the other hand, retained ∼55% of its activity after 45 min at 55 ◦ C, and ∼40% activity even after incubation for 1 h at 60 ◦ C. Such increased stability could be due to immobilisation resulting in enhancement of the glycoprotein’s structural rigidity, which decreases the extent of enzyme distortion upon exposure to elevated temperatures [22,24]. Chitosan is therefore an excellent support matrix for the immobilisation of RL.
3.4. Michaelis constants (Km ) for RL and I-RL Km values were calculated as 0.16 mmol/L for RL, and 0.07 mmol/L for I-RL. The decrease in Km value after RL immobilisation shows that I-RL has a higher substrate specificity than RL.
3.5. I-RL re-use efficiency The effect of re-use on I-RL relative activity (initial enzyme activity normalised to 100%) using N,N-dimethyl-p-phenylenediamine (0.83 mmol/L in sodium phosphate buffer, 200 mmol/L, pH 7.0) as substrate was investigated (Fig. 2). The relative activity fluctuated significantly during repeated use; however, it never dropped below 80% of its initial activity during the repeated use evaluation (15 cycles), indicating good re-use potential/efficiency.
3.3. Effect of immobilisation on enzyme thermostability The in vitro stability of enzymes/proteins remains a critical issue in biotechnology and various methodologies, such as covalent attachment to polymer carriers and surface modification, have been employed to try and improve stability [37]. The method of immobilisation and the nature of the carrier should therefore be chosen according to the physical circumstances involved. The effect of temperature (in the range 25–60 ◦ C) on RL and I-RL relative activity (activity after 15 min at 25 ◦ C normalised to 100% and the I-RL values corrected to compensate for reduced enzyme concentration) as a function of time (up to 1 h) using 2,6-dimethoxyphenol (12.97 mmol/L in sodium phosphate buffer solution, 200 mmol/L, pH 7.0) as substrate was investigated (Table 1). The thermostability of RL was dramatically improved after immobilisation onto chitosan. Free RL retained good activity as a function of evaluated incubation time at 25 and 30 ◦ C, but exhibited drastic reductions with incubation at higher temperatures, going down to only ∼21 and ∼7% after 60 min at 35 and
Fig. 2. Effect of re-use on I-RL relative activity using N,N-dimethyl-pphenylenediamine (0.83 mmol/L in sodium phosphate buffer, 200 mmol/L, pH 7.0, at 25 ◦ C) as substrate (% variation for replicate analyses <2%).
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Fig. 3. Proposed reaction for the RL-catalysed oxidation of 2,6-dimethoxyphenol.
3.6. Enzyme activity in organic solvent/water systems For enzyme-catalysed reactions in non-conventional solvent systems, the primary factors that govern enzyme activity are thought to be: (a) the distribution of water in the system, (b) the specific properties of the organic solvents involved (such as hydrophobicity index, partition coefficient, polarity), and finally (c) the resulting solvation and distribution of reactants and products in the system [13,39,40]. The single product of RL-catalysed oxidation of 2,6dimethoxyphenol in aqueous solution has been identified as 3,3 ,5,5 -tetramethoxy-biphenyl-4,4 -diol (Fig. 3) [6,22,41], a single product being produced due to steric hindrance effects. 2,6-dimethoxyphenol undergoes a RL induced single-electronoxidation to produce a 2,6-dimethoxy-phenoxyl radical species ((a) in Fig. 3) that is in resonance with the corresponding para-radical species ((b) in Fig. 3). Combination of two such para-radical species produces the product (Fig. 3). The fact that only a single product is produced, makes 2,6-dimethoxyphenol a useful substrate for use in the study of RL-catalysed oxidation reactions, and hence it was used in these investigations. 3.6.1. Water-immiscible organic solvent/water systems RL was added to ethyl acetate containing 2,6-dimethoxylphenol, and the water content in the mixture adjusted by adding the desired amount of deionised water. RL was partially precipitated from the solvent system with the addition of 1% (v/v) water. Two distinct phases were formed when more than 3% (v/v) water was added, although an emulsion could be formed with vigorous mixing. RL activity was found to increase with increasing water content in the ethyl acetate/water solvent system, and reached its maximum when the water content was ∼2.5% (v/v). Other water-immiscible solvents evaluated, namely benzene, toluene and chloroform, showed similar behaviour, indicating that RL required a minimum water content to achieve its activity in such essentially non-aqueous systems. Such observations have been observed in other investigations using RL in organic solvent/water systems [42]. It is likely that the RL was absorbing water from the solvent system, and thereby functioning in an essentially aqueous microenvironment and thus retaining the characteristics of its activity in as it would in an aqueous system. These observations are comparable with those for tyrosinase [40], lipase [33,43], peroxidase [44], thermolysin [45], ␣-chymotrypsin [46] and subtilisin [47]. However, lipase, ␣-chymotrypsin and subtilisin required much less water to function in organic solvents. The required amount of water seemed to be dependent on the nature of enzyme and the type of solvents [48,49]. I-RL was added to benzene containing 2,6-dimethoxylphenol, and the water content in the mixture adjusted by adding the desired
amount of deionised water. I-RL activity was found to increase with increasing water content in the benzene/water solvent system, and reached its maximum when the water content was ∼0.5% (v/v), and then decreased readily with increasing water content (all required vigorous mixing). Other water-immiscible solvents evaluated, namely, toluene and chloroform, showed similar behaviour, with maximum I-RL activity achieved with water contents of 0.15% (v/v) and 0.5% (v/v), respectively. As in the case of free RL discussed above, with ethyl acetate maximum I-RL activity was achieved with a water content of 2.5% (v/v). In such solvent systems the I-RL became aggregated/entrapped in the water phase, leading to mass transfer limitations that would affect reaction catalysation. It appeared that the more hydrophilic the organic solvent, the lower the optimum water content required to achieve some enzyme activity with I-RL.
3.6.2. Free RL in water-miscible organic solvent/water systems The relative activity of RL (activity in sodium phosphate buffer, 200 mmol/L, pH 7.0 normalised to 100%) in a range of watermiscible organic solvent/water systems, as a function of increasing water content, using 2,6-dimethoxyphenol (12.97 mmol/L in solvent at 25 ◦ C) as substrate was investigated (Fig. 4(a)–(c)). Methanol, ethanol, 1-propanol, and 2-propanol exhibited no activity with water contents of <30% (v/v). This was followed by a slow gradual increase in activity (<10%) with water contents of ∼30–60% (v/v), up to ∼5–15% with water contents of ∼60–80% (v/v), and finally a dramatic increase up to ∼35–75% (depending on solvent system) with water contents of ∼80–90% (v/v) (Fig. 4(a)). For the same water contents the general trend was methanol = ethanol > 1propanol > 2-propanol. A threshold concentration of water was required to initiate activity, below which rapid enzyme inactivation occurred. These findings are in agreement with results reported for several other enzymes [50,51]. 1,2-Propanediol, 1,3-propanediol, 1,3-butanediol, and 1,4butanediol exhibited little (<5%) or no relative activity with water contents up to 50, 40, 40, and 20% (v/v), respectively (Fig. 4(b)). 1,2propanediol then exhibited an essentially linear activity increase up to ∼20% with a water content of 70% (v/v), followed by a more dramatic increase up to ∼70% with a water content of 90% (v/v). The profile of 1,3-butanediol was initially very similar to that of 1,2-propanediol, but did not increase as dramatically at the end, only reaching an activity of ∼45% with a water content of 90% (v/v). The profiles for 1,3-propanediol and 1,4-butanediol showed similar trends to each other, increasing to a relative activity of ∼30% (with water contents of ∼60 and ∼50% (v/v), respectively), maintaining this activity up to a water content of ∼70–80% (v/v), followed by a dramatic increase up to ∼80–85% activity with a water content of 90% (v/v) (Fig. 4(b)).
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Fig. 5. Effect of water content on I-RL relative activity in water-miscible organic solvent/water systems using 2,6-dimethoxyphenol (12.97 mmol/L, at 25 ◦ C) as substrate (% variation for replicate analyses <5%).
activity of 90% with a water content of 90% (v/v). Such observations were similar to others reported previously [51,52]. Organic solvents utilised in these investigations can be roughly divided into alcohols and non-alcohols. The following enzyme activity sequence has been observed in water-miscible alcohol/water systems: triols/polyalcohols > diols > monohydric alcohols; short chains alcohols > longer chain alcohols; straight chains alcohols > branched chain alcohols [51].
Fig. 4. Effect of water content on free RL relative activity in water-miscible organic solvent/water systems using 2,6-dimethoxyphenol (12.97 mmol/L, at 25 ◦ C) as substrate (% variation for replicate analyses <5%).
Acetone, acetonitrile, and 1,4-dioxane exhibited no activity with water contents up to 30, 40, and 40% (v/v), respectively (Fig. 4(c)). With acetonitrile and 1,4-dioxane this was followed by a very slow gradual increase in activity (<5%) with water contents of ∼40–60% (v/v), and finally a dramatic increase up to ∼80 and ∼70%, respectively with water contents of 90% (v/v). Acetone exhibited an essentially linear increase in relative activity from 0 to 95% with water contents from ∼30 to 80% (v/v), which was maintained at 90% (v/v). Glycerol/water systems demonstrated by far the highest free RL compatibility of all the solvent systems tested, with a relative activity of >10% with a water content of 10% (v/v), with rapid linear increase to ∼140% with a water content of 50% (v/v) (Fig. 4(c)). This was then followed by a sharp linear reduction down to a relative
3.6.3. I-RL in water-miscible organic solvent/water systems The relative activity of I-RL (activity in sodium phosphate buffer, 200 mmol/L, pH 7.0 normalised to 100%) in a range of watermiscible organic solvent/water systems, as a function of increasing water content, using 2,6-dimethoxyphenol (12.97 mmol/L in solvent at 25 ◦ C) as substrate was investigated (Fig. 5). Glycerol demonstrated essentially the same profile with I-RL as was observed for free RL, with a relative activity of >10% with a water content of 10% (v/v), with rapid linear increase to ∼140% with a water content of 50% (v/v). This was then followed by a sharp linear reduction down to a relative activity of ∼100% with a water content of 90% (v/v). Ethanol, 1,4-dioxane, and acetone all demonstrated considerably higher relative activity with I-RL at lower water contents (compared to free RL). This effect was greatest for ethanol, for example with free RL ethanol had an activity of <15% with a water content of <80% (v/v), whereas with I-RL an activity of >75% was achieved with a water content of only 20% (v/v) (Fig. 5). In the case of water-miscible organic solvent/water systems, a high organic solvent concentration normally results in suppression of enzyme activity due to the organic solvent replacing water molecules on the protein surface layer [16]. This results in distortion of enzyme molecular hydration to an extent that the active catalytic conformation is disrupted. It is assumed that the microenvironment of the enzyme immobilised on chitosan provides additional stability to aid in the preservation of the active enzyme conformation [22,23]. The hydrophilicity of chitosan, due to the amino and hydroxyl groups [28,29], should assist with retention of the necessary water molecules in the enzyme molecular microenvironment [32,53,54]. 3.6.4. Kinetics of I-RL catalysis in selected organic solvent/water systems In order to accurately study the effects of solvents, it is necessary to compare enzyme activity in different organic solvent whilst maintaining a constant degree of enzyme hydration. A suitable water activity towards a laccase-catalysed reaction should occur when the substrates and enzyme preparation in
Y.-Y. Wan et al. / International Journal of Biological Macromolecules 47 (2010) 488–495 Table 2 First-order reaction rate constants with respect to disappearance of 2,6dimethoxyphenol (DMP)a catalysed by immobilised Rhus laccase (I-RL) in a series of organic solvents (at 25 ◦ C). Solvent
Hydrophobicity (log P)
0.2 M phosphate buffer (pH 7.0) 0.2 M phosphate buffer (pH 7.5) Acetonitrileb Ethanolb Acetoneb Glycerineb 1,4-Dioxaneb Ethyl acetateb Tetrahydrofuranb Butyl acetatec Benzenec Toluenec Chloroformc N,N-Dimethylformamideb Cyclohexanec Hexanec
– – −0.21 −0.16 0.02 −1.76 −0.22 0.53 1.17 1.53 2.02 2.60 1.86 −0.46 3.15 3.83
a b c
First-order reaction rate constant k × 106 of I-RL (min−1 ) 154.41 115.35 37.58 46.37 46.32 35.83 4.92 2.17 1.86 1.23 1.22 1.19 0.78 0 0 0
Initial concentration 12.97 mmol/L. Water (7%, v/v) added to solvent. Solvent pre-saturated with distilled water at ambient temperature.
organic solvent are pre-equilibrated with water at lower temperature [55]. The solvents were therefore pre-saturated with water, and the resulting rate constants for I-RL-catalysed oxidation of 2,6-dimethoxylphenol in water–organic solvent systems were a relative measure of the reaction rates among the different solvent systems (Table 2). The rate of the disappearance of substrate followed a pseudo-first-order law. An increase in the water content in all the hydrophilic organic solvents investigated enhanced the efficiency of I-RL. For the evaluated water-immiscible organic solvents (namely benzene, toluene and chloroform), the first-order reaction rate constants generally increased with decrease of hydrophobicity (log P). However, in water-miscible organic solvents no apparent relationship between hydrophobicity and enzyme activity was observed, and an entirely different mechanism of activity is thought to occur
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[36]. When hexane and cyclohexane were used as solvent, the IRL-catalysed substrate oxidation occurred on the surface of the I-RL, however, no oxidation products were detectable in the organic phase of reaction mixture (by TLC and HPLC), due to the very low solubility of the products in these solvents. In the kinetic studies of I-RL in selected water-miscible organic solvents, the maximum rate constants for the disappearance of 2,6-dimethylphenol in three solvent systems, ethanol/water, acetone/water and 1,4-dioxane/water were 1.42 × 10−4 , 2.15 × 10−4 , 1.2 × 10−4 min−1 , with water content of 80% (v/v), respectively (Table 3). 3.7. Pseudo-mechanism for interactions between enzyme, solvent and support The existence of a hydrophobic shell (HS) on the surface of the active centre of the enzyme molecule is postulated (Fig. 6), where hydrogen bonding and hydrophobic interactions work together to balance the conformation of the enzyme, and the associated enzyme activity. Organic solvent molecules may disrupt this HS and displace water molecules from the enzyme surface. If the new solvent molecules are unable to maintain the hydrogen bonding/hydrophobic interactions, then the required enzyme conformation is lost and therefore so is the enzyme activity. Clearly some organic solvents are able to successfully maintain such interactions, however they may to a greater or lesser extent block the enzyme active centre and prevent access to the substrate, thereby reducing enzyme activity. On the other hand, some organic solvents may allow (or even enhance) substrate access to the enzyme active centre. Interactions between organic solvent molecules and the enzyme active centre thus determine overall enzyme activity (Fig. 6). Polyalcohols have at least two potential hydrogen bonding centres, and are hence most likely to be capable of forming threedimensional hydrogen bond network structures. Some organic solvents have been classified according to their solvophobic interaction [39]. Water and glycerin belong to the group of strongest solvophobic solvents. Hydrophilic solvent hydroxyl groups are obviously incompatible with the hydrophobic regions of enzymes, but can participate in hydrogen bonding to produce rigid inter-
Fig. 6. Pseudo-mechanism for the interaction between enzyme, solvent and/or support molecules.
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Table 3 First-order reaction rate constants with respect to disappearance of 2,6-dimethoxyphenol (DMP)a catalysed by immobilised Rhus laccase (I-RL) in a series of water-miscible organic solvent/water systems (at 25 ◦ C). Solvent system
Ethanol/water Acetone/water 1,4-Dioxane/water a b
First-order reaction rate constant k × 106 of I-RL (min−1 ) 7% (v/v)b
20% (v/v)b
40% (v/v)b
60% (v/v)b
80% (v/v)b
46.37 46.32 4.92
126.06 147.59 37.10
136.42 184.86 74.41
139.70 203.05 97.82
141.94 214.97 120.35
Initial concentration 12.97 mmol/L. Water content.
molecular frameworks, which are very important in maintaining enzyme conformation and thus enzyme activity. However, the size and conformation of the solvent molecules themselves is important, since steric hindrance of the active centre by the solvent (and disruption of the formation of inter- and/or intra-molecular hydrogen bonded networks) can also reduce enzyme activity. For example in this work, enzyme activity in ethanol/water systems was greater than both 1-propanol and 2-propanol, with 1-propanol having higher activity than 2-propanol. Differences in reaction rates in various organic solvents can also be attributed to differences in the solvation of the substrate [56], differences in the partition of substrate and products between the bulk solvent and biocatalyst phases [40], and the aforementioned differences in interactions between substrate, solvent and/or enzyme molecules (Fig. 6). 4. Conclusions The effects of organic solvents on the enzyme activity of free RL and I-RL (on chitosan) were investigated. A pH of ∼7.0 was determined to be the optimum pH for both free RL and I-RL, and immobilisation on chitosan greatly enhanced the enzyme thermostability, resulted in negligible loss of activity, and demonstrated excellent re-use potential, with >80% relative activity retained even after 15 use cycles (as evaluated in aqueous solvent). I-RL was more catalytically active in both hydrophobic and hydrophilic organic solvents than free RL. With water-immiscible organic solvents, both free RL and I-RL required a minimum water content to achieve activity in such essentially non-aqueous systems, with maximum activity achieved with a water content in the region of ∼0.5–3.0% (v/v) for the solvents evaluated. With watermiscible organic solvents, in general a water content of ∼20–50% (v/v) was required to achieve activity using free RL (depending on the organic solvent), the exception being glycerol, which exhibited activity at low water contents and the highest overall relative activity with free RL. With I-RL less water was generally required to achieve enzyme activity, and therefore considerably higher relative activity was exhibited at lower water contents, compared with free RL. Kinetic investigations showed that the rate of the disappearance of substrate generally followed a pseudo-first-order law, and for the evaluated water-immiscible organic solvents the firstorder reaction rate constants generally increased with decrease of hydrophobicity, however, in water-miscible organic solvents no such relationship was observed. Some discussion of the potential interactions between organic solvent molecules and enzyme active centres was provided to explain the obtained experimental results. Finally, chitosan is an excellent support for immobilisation of Rhus laccase and therefore has potential for a wide range of applications, particularly in the lacquer industry. Acknowledgements This work was partly supported by the Academic Frontier Project for Private Universities, matching fund subsidy from the
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