EffectsofOrganicSolventsontheactivityofRhuslaccases.doc

effects of organic solvents on the activity of rhus laccasesyun-yang wana,b*, ling xiaoc, yu-min duc, tetsuo miyakoshib, chen-loung chend,a state key laboratory of petroleum resources and exploration, faculty of natural resources and information technology, china university of petroleum, beijing 102249, chinab department of industrial chemistry, meiji university, higashi-mita, tama-ku, kawasaki 214, japan.c school of resource and environment science, wuhan university, wuhan 430079, china.d department of wood and paper science, north carolina state university, raleigh, nc27695-8005, usaabstractthe optimum ph for the laccase-catalyzed oxidation of substrates was not appreciably changed when free rhus laccase (rl) was covalently immobilized on chitosan. in addition, the thermostability of the enzymes was improved appreciably after immobilization. the retained enzymatic activity was approximately 85 % of the original after it was continuously used for 15 times. studies were conducted on rl and immobilized rhus laccase (irl)-catalyzed oxidation of 2,6-dimethoxyphenol in water-organic solvent systems. these reactions proceeded well in water-immiscible organic solvent systems pre-saturated with water. in such solvent systems, the presence of enough water was necessary to ensure function of the laccases in full capacity. in water-miscible organic solvent systems, rl lost its enzymatic activity when more than 40 % of organic solvent was present. the studied solvents may be formally treated as the weak competitive or mixed inhibitors of rl for replaced the necessary water of enzyme. when the irl was used in such solvent systems, the enzymatic activity was detected after addition of 7 % water in the organic solvents. although it was still lower than that in pure water and did not have linear or absolute relationships with log p, the first order reaction rate constants were generally increased with decreasing log p. the following enzymatic activity sequence was discovered for alcohols: triol/polyalcohol diol monohydric alcohols; alcohols with short chains that with longer chains; straight chains alcohols that with side chains. a three-step reaction mechanism, especially among the rl and solvent molecules, was postulated to interpret the effects of variables.keywords: rhus laccase; immobilization; chitosan; laccase-catalyzed oxidation; organic solvents; 2,6-dimethoxyphenol; kinetics.1. introductionlaccases (ec , p-diphenol:dioxygen oxidoreductase), part of a larger group of enzymes among the multicopper enzymes, widely occur in higher plants and fungi 1;2. simply based on the function of catalyzing the oxidation of substituted phenols and/or anilines-type substrates with four-electron reduction of o2 to h2o 3-6, these enzymes constitute a very prospective class of enzymes for physiological development and industrial utility, such as bioremediation, organic catalyzed synthesis, pulp and paper biobleaching, and analytic uses3;7-9.it has been shown that fungal laccases can express their activity in reaction media where water or most of the water has been replaced by organic solvents, and where enzymes acquire corresponding author;4remarkably novel properties, such as greatly enhanced stability, drastically altered specificity toward inhibitors and the ability to catalyze new reactions which cannot be proceeded in conventional media 10-14. compared with fungal laccases which have been studied thoroughly and extensively 8;11;12;15, the function of plant laccases, such as rhus laccases (rl) which are isolated from the sap of chinese lacquer tree (rhus vernicifera), are only marginally evaluated 16. because there are significant differences between fungal and rl 15;17-19, its still worthwhile to study the enzymatic activity of rl in various solvents, especially in nonaqueous solvents with the appreciate advantages, and this will facilitate the investigation of the reactions mechanism of phenolic substrates, such as urushiol and dioxins, which are insoluble or poorly soluble in water. solvents play an important role in enzymatic reaction and thus led to the current investigation of the activity of rl in various solvents, especially in organic solvents.a recently study showed that the enzymatic activity of rl and/or immobilized rl on modified chitosan 20 and transition metal 21;22 in water solution and aot/n-octane/water reverse micelle 23 had very good properties. a variety of supports and techniques used for immobilization of enzyme are available 24;25, but there is no single support and immobilization technique, which is the best for immobilization of all enzymes or all applications of a single given enzyme. in organic solvents, the carrier may be more important for the stability of enzyme than the coupling method 24;26. chitosan is an inexpensive, inert and hydrophilic support, “intelligent” to ph and/or temperature etc. 27, especially for its good biocompatibility and biodegradability 28 and has received much attention for immobilization of enzymes 20;29.in the present study based on the previous research 20, rl was activated treated by sodium periodate to protect the active center sited in the protein part and then, the carbohydrate part of it was covalently coupled to natural chitosan. reactions in organic solvents catalyzed by free and immobilized rl were studied and compared in order to get the optimum conditions for the enzymes. furthermore, properties of dynamics on the catalytic efficiency of rl in water-miscible solutions were investigated. emphasis was put on some factors that affect enzymatic activity and, a three-step reaction mechanism was postulated based on the results.2. experimental2.1. materialsrl was isolated and purified from lacquer acetone powder produced from the exudate of lacquer trees in cheng kou region, china, according to the reported method 1;17. chitosan (mw = 66,000) was generously donated by professor yoshhiro of meiji university, japan. other reagents were analytical grade and purchased from commercial suppliers used as received except for special declaration.2,6-dimethoxyphenol (3.31 mmol) was dissolved in 1:1 (v/v) 20ml acetone/deionized water system at 4c. after completely dissolved, it was kept at room temperature 30min and then rl (1ml) and pcl (0.5 ml) respectively was added and reacted 24 hours under stirring. the produced polymers were acetone insoluble that was filtrated and washed by acetone, and the acetone soluble that was dissolved in ethyl acetate and was evaporated under reduced pressure to remove acetone. the residual part then was washed with saturated salt solution, dried with anhydrous sodium sulfate and concentrated under reduced pressure to yellow viscous liquid. this product was then eluted on silica gel with 3:2 (v/v) hexane/ethyl acetate and yellow powder collected after drying was analyzed by ft-ir, nmr, and gc/ms.2.2. activation and immobilization of rl on chitosan (irl)rl (4 mg) was mixed with 0.045 mol/l naio4 in 0.1 mol/l sodium phosphate buffer ph6.0 (2 ml). the resulting solution was stored in dark for 2 hrs at room temperature (20-25 c),followed by adding ethylene glycol (200l) and 0.1 mol/l sodium phosphate buffer solution ph 7.0 (2 ml). to this solution chitosan (1 g) was added, and the enzyme immobilization was carried out for 24 hrs at 4 c. the excess enzyme was removed by washing the solid phase with deionized h2o, 0.2 mol/l sodium phosphate buffer at ph 6.0 and then with 0.2 mol/l sodium phosphate buffer at ph 7.5. the resulted irl was stored at 4 c.2.3. enzyme assay2.3.1. determination of enzymatic activityboth rl and irl were assayed in 0.2 mmol/l sodium phosphate buffer solution at ph 7.5 using n,n-dimethyl-p-phenylenediamine (dmpda) as the substrate with the initial concentration of 0.83 mmol/l or in 0.2 mmol/l sodium phosphate buffer solution at ph 7.5 using 2,6-dimethoxyphenol (dmp) as substrate with the initial concentration of 12.97 mmol/l. all assays were carried out at 25 c. free and immobilized enzymes were added to the substrate solution, stirred mechanically for 1 minute, and increase in the absorbance (a) of the clear solution was measured at 323 nm, and 468 nm when dmpda, and dmp were used as substrate, respectively. one unit of rl activity was defined as the change of optical density at the according nm per min per mol of protein added to 3 ml substrate solution as measured in a 1 cm pathlength cell. one oxidase unit corresponds to a change in optical density of one a . determination of the effect of ph, thermostability, michael constants of rl and irlthe enzymatic activity was determined in the ph-range of 5.0 8.0 at 25 c using 12.97 mmol/l dmp as substrate.thermostability studies were performed by preincubation of rl and irl in 0.2 mol/l sodium phosphate buffer ph 7.5 in the temperature from 25 c to 60 c for every 15 mins with maximum of 1hr. after rapid cooling, the residual activities were assayed using dmp as substrate described in the enzyme assay above.km was determined at 25 c from linear lineweaver-burk plots in 0.2 mol/l sodium phosphate buffer at ph 7.5 using dmpda as substrate according to the method described by du et al. . reuse efficiency of irlthe irl was used in catalyzing the oxidation of dmpda with the initial concentration of0.83 mmol/l in 0.2 mol/l phosphate buffer solution ph 7.5 at 25 c. the irl was placed in substrate solution (3 ml), stirred for 1 min and then the uv absorbance of the solution was measured at 323 nm. the solid was washed thoroughly with the buffer solution until no uv absorbance and then repeated for 15 times.2.3.4. kinetic studies of irl-catalyzed oxidation of dmp in organic solventsthe irl-catalyzed oxidations of dmp in hydrophobic or hydrophilic organic solvent systems were carried out in a 50 ml reaction vessel at 25 c with stirring. to a mixture of the12.97 mmol/l substrate solution in an appropriate solvent system (25 ml) was added of laccase immobilized on chitosan (1.53 g). a sample solution (3 ml) of reaction mixture was taken at certain intervals for uv absorbance reading at 468 nm and then the solution was immediately transferred back to the reaction mixture.3. results and discussion3.1. immobilization and properties of rlrl is a glycoprotein and 45 % part of the molecular weight is carbohydrate group 17;18;20, and the active center of rl is just the copper sites situated in the protein part 5;9;30;31. although there were various reports on the effects of the carbohydrate part to the enzyme activity 32-36, binding of support molecules in the areas of the active center or its vicinity can lead to a decrease or complete loss of enzyme activity 37. it was reported that the carbohydrate of rl was oxidized with sodium periodate under mild conditions and the activity of it remained mostly 32;38. then sodium periodate was chosen to oxidize the carbohydrate part of the enzyme for the immobilization. as a result of immobilization of rl via its carbohydrate part, the stabilization of its structure was ensured, and the active center of rl oriented into the reaction solution was an important objective of this study.the laccase activity was 2.36 104 units/g for the oxidation of dmpda (0.83 mmol/l) in sodium phosphate buffer (0.2 mol/l) at ph 7.5. for dmp (12.97 mmol/l) in 0.2 mol/l sodium phosphate buffer at ph 7.5, the laccase activity was 7.14 103 units/g. the immobilized laccase activity measured was initially 0.116 103 units/g for dmpda in same phosphate buffer. the recovery of free laccase activity after the immobilization of rl was2-7 % of the total laccase activity used in the immobilization. thus, in average, about 95% of the rl was immobilized on chitosan.80007000activity of irl (u min-1)6000500040003000200010000.00.81.0concentration of rl (mg ml-1)figure 1. concentration - activity relationship of rl on the irl using dmp as substrate in 0.2 m phosphate buffer at ph 7.0.the laccase activity of the immobilized enzyme increased obviously with increasing equilibrium concentration of the enzymes in the observed range (figure 1). this also proved that there was little effect of the sodium periodate to the enzyme molecule and the active center. the total activity of the enzyme was changed but not very appreciable in the process of immobilization (data not shown).100relative activity (%)80604020irl frl05.0 5.5 6.0 6.5 7.0 7.5 8.0phfigure 2. effect of ph on the relative activity of frl and irl. dmp concentration: 12.97 mmol/l in 0.2 mphosphate buffer at ph 7.0.the stability of enzymes and proteins in vitro remains a critical issue in biotechnology and various methods, such as covalent attachment to polymers and surface modification, were tried to amend and improve it 39 and to discover which method and/or carrier chosen according to the physical circumstance was very important 37.comparison of the properties of rl and irl showed that the optimum ph was not changed after immobilization (figure 2), in accord with our early results 1;20. in a small range, the ph of solution medium had negligible effect on the immobilization of the enzyme, which could be due to the intelligent response of chitosan 20;27 or the microenvironments of the enzymes changed 20;21.table 1. thermostability of frl and irl using dmp as substrate in 0.2 mol/l phosphate buffer at ph 7.0.relative activity (%)sample25 c30 c35 c45 c55 c60 cfrl10079.958.636.715.72.10irl88.080.080.275.569.355.5frl88.171.633.522.17.26nairl77.679.876.670.462.850.7frl82.363.428.316.0nanairl75.976.268.966.755.048.1frl81.658.721.46.52nanat (min)1530456017irl75.975.270.765.355.040.1na: no activity.the thermostability of rl was significantly improved after being immobilized to chitosan (table 1). the rl activity began to decrease as time and temperature increased and became completely inactive after 40 minutes at 55 c, in an irreversible manner. in contrast, the irl maintained its approximately 40% original activity after 1 hour at 60 c. it may be due to the strengthening of the protein molecules structural rigidity when it binds with the support. the structure rigidity of the protein molecule decreases of the extent of the distortion when the enzyme is exposed to heat 20;22. thus, chitosan is a good carrier for rl.the irl was very efficient as a catalyst for laccase-catalyzed oxidation, particularly when used repeatedly. in the irl-catalyzed oxidation of dmpda in phosphate buffer (ph 7.5) at 25c, the irl lost approximately 15% of the original enzymatic activity after using repeatedly for 15 times (figure 3). it was difficult to interpret this phenomenon for it might be the results of synergistic effects, including various influences, such as the stabilization of the support, the activation of enzyme and the suitable ph because of the support. it proved that, on the whole, the immobilization was very successful.relative laccaseactivity (%)105 b100959085800 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16relative laccase activity (%)frequency of use (times)0.12lacase activity(units min1, gl1)0.1150.110.1050.10.0950.09a0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16fraquency of use (times)figure 3. reuse efficiency of the rl immobilized on chitosan towards oxidation of dmpda (0.83 mmol/l)in 0.2 m phosphate buffer solution (ph 7.5) at 25 c. rl (15g) immobilized on 0.1g chitosan in3ml of 0.83mmol dmpda solution. experimental error: 2%.the oxidation spectra of substrates with rl and irl were the same. km of rl was calculated as 0.16 mmol/l and 0.07 mmol/l for the irl. the decrease in the km value showed that the irl had higher specificity to substrates than the rl did.3.2. factors of rl-catalyzed reactions in organic solventsit has been postulated 8;40;41 that in the enzyme-catalyzed reactions in non-conventional media,thefactorsgoverningtheenzymaticactivityprimarilyrelatedtothe structure-functional studies are (a) distribution of water in the biocatalytic reaction system, (b) the effect of organic solvents, like its solvent hydrophobicity log p (partition coefficient between water and n-octanol) and polarity, etc., and finally (c) the solvation and distribution of reactants and products in the reaction medium. therefore, the effects of water content andorganic solvents on the activity of the rl were investigated to clarify these postulations.4 h3co och3rhus laccase4 h3co och3oah3co2 hooch3ohoh o22 h2oh334 h co och obh3cooch3scheme 1. predicted reaction mechanism of 2,6-dimethoxyphenol catalyzed by rhus laccase in phosphate buffer and/or organic solvents.recently, the products of 2,6-dimethoxyphenol (dmp) catalyzed by rl has been identified both in aqueous and in organic solvents 1;20;42. two radical resonances of dmp were formed under rl catalysis, but only one product, 3,3,5,5-t