Chinese Chemical Letters  2014, Vol.25 Issue (07):983-988   PDF    
Infl uence of counteranions on catalytic ability of immobilized laccase in Cu-alginate matrices:Inhibition of chloride and activation of acetate
Ting Pan, Yao-Jin Sun, Xiao-Lei Wang, Ting Shi, Yi-Lei Zhao     
State Key Laboratory of Microbial Metabolism, School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China
Abstract: Laccase is a promising oxidase with environmental applications, such as lignin degradation and chlorophenol detoxification. Laccase immobilization can significantly improve physiochemical stability and reusability compared to the free enzymes. In this work, anion effect was investigated in entrapment of Cu-alginate matrix with five types of anions, including perchlorate (ClO4-), nitrate (NO3-), sulfate (SO42- ), chloride (Cl-), and acetate (CH3CO2-). Accordingly, chloride inhibition and acetate activation were detected in the o-tolidine kinetic experiments, while effects of the other three anions were much smaller. Such counteranion effects were also observed in the laccase-catalyzed biodegradation of 2,4-dichlorophenol. The results indicated that counteranions in the enzyme immobilization process are crucial for catalytic capacity, probably due to the competition with the carboxylate groups in alginate. Our results also imply that these anions might coordinate the copper cations in laccase.
Key words: Counterion effect     Cu-alginate matrix     Laccase immobilization     Enzyme activity    
1. Introduction

Laccase,a type of phenoloxidase that catalyzes the oxidation of a large scope of phenolic and non-phenolic aromatic compounds,is very promising in environment-friendly bioremediation,and industrially employed in textile finishing,pulp bleaching,and food treatment [1]. It was first discovered in the lacquer treeRhus vernicifera,and then in various plant species,bacteria and insects [2]. For convenient accessibility,most studies were carried out using laccase from fungi,in particular from wood-rotting fungi such as Trametes versicolor. Besides diphenols,laccase is also characterized for its wide spectrum of substrates,including aminophenols,methoxyphenols,aromatic amine and lignin [3, 4]. The active site of laccase contains four copper atoms, classified as types 1,2,and 3 [5]. Three of the copper ions (types 2 and 3) form a trinuclear cluster that binds the electron acceptor dioxygen (O2),and the substrate transfers electrons to the trinuclear cluster via the fourth copper center (Type 1) [6]. Dioxygen is reduced into water after accepting four electrons,so that the typical laccase reaction is fairly clean and does not need additional reagent such as H2O2and other assistant co-factors [7]. Owing to the different redox potentials in the sequential four electron transfer,certain small molecules can enhance the ability of laccase oxidation via a cooperative pathway,namely laccase mediator systems (LMSs) [8]. Thus,a dramatic range of oxidizable compounds makes laccase applicable in many fields of environmental biotechnology such as delignification and detoxification of wastewater [9].

In order to use the expensive biocatalysts more economically and efficiently,many enzyme immobilization techniques have been developed,including entrapment,encapsulation,adsorption,covalent binding,and self-immobilization [10]. The basic concept for these methods is to entrap the functional protein in a semipermeable support material and allow quick exchanges of substrates and products. The easiest immobilization method is entrapment, which reserves enzyme in a super-molecular matrix and induces no structural alterations of the protein. Alginate,a seaweed extract that composes of alternating α-L-guluronic acid and &bate;-D-mannuronic acid residues,is the most widely used biopolymer [11, 12]. As shown in Scheme 1,alginate molecules can aggregate into a matrix structure when the carboxyl group ina-L-guluronic acid crosslinks high-valent cations such as Ca2+ ,Zn2+ ,and even poly(L-lysine) [13].Immobilized laccase,like other immobilized enzymes,has the advantages of increased resistance,easy operation and separation, as well as repetitive use. Since laccase is a multicopper oxidase,it is reasonable to replace the classic divalent ion with copper ion in alginate matrices. In 2008,Niladevi and Prema have reported that Cu-alginate system retains a higher activity than the classic Caalgniate under the same conditions [14].

Scheme 1. Diagram for the laccase immobilization and the plausible structures of Cu-alginate matrix.

Since alginate has a broad range of application in medicine and materials,ion effect has long been noticed in the divalent gelation [15]. Dragetet al. have reported that potassium and sodium cations involve in the Ca2+ -mediated formation of alginate matrices,and potassium leads to faster sol/gel transition at the low concentrations of Ca2+ [16]. Donatiet al. have developed a counterion condensation theory to interpret thermodynamic processes,based on the interactions between the cross-linking ions and the urinate moieties [17]. [M+H]+agnesium ions,previously considered as non-gelling ions,do induce a slow gelation at a relative high concentration [18]. Recently, a careful molecular dynamic simulation has been carried out for alginate molecules in aqueous solution,indicating that large water pools are present between the polysaccharide chains [19]. Indeed, much literature focused on divalent cation effect in the crosslinking network of the biopolymer. However,to our knowledge,counteranion effect has rarely been reported yet,though anion obviously exists in the water pool of cation-alginate matrices.

In particular,copper coordination in blue protein became extremely important for biological and inorganic chemists [20]. It has been reported that copper reduction potential is tuned to match the particular function of a given protein like laccase,by regulation of chemical environment surrounding the metal site,such as exclusion of water and control of neighboring ligands. In this work, we prepared a series of Cu-alginate matrices with entrapment ofT. versicolor laccase,in different counteranion solutions,including ClO4-,Cl-,SlO42-,NO3-,andCH3CO2-. The resulting soft matters were in different colors even after they were thoroughly washed. This indicated that the ligands were adsorbed in the Cu-alginate matrices during enzyme entrapment. The enzyme activity of immobilized laccase was measured with the traditional o-tolidine spectrophotometric assay and also evaluated with 2,4-dichlorophenol (2,4-DCP),a common environmental pollutant. The results demonstrated that the absorbed anions in Cu-alginate matrices can regulate the enzyme activity,as it was,consistent with the coordination ability of the counteranions to the cupric ions. 2. Experimental

2.1. Laccase immobilization

Copper salts such as Cu(ClO4)2·6H2O (98%),CuCl2·2H2O (99.0%), CuSO4·5H2O (99.0%),Cu(NO3)2·3H2O (99.0%),Cu(CH3CO2)2·H2O (99.0%) and other chemicals were purchased from Sinopharm Chemical Reagent Co.,Ltd. Laccase (3.7 g/L,10 mL) from T. versicolor (activity·0.5 U/mg,purchased from Sigma-Aldrich company) was added to a 2% 190 mL sodium alginate solution (after boiling and cooling) and mixed thoroughly under magnetic stirring. The 10 mL viscous alginate-enzyme mixture was taken in a syringe fitted with a luer-lock needle and the solution was extruded drop wise from the syringe into a series of 100 mL divalent cupric solutions ([Cu2+] = 0.05 mol/L,unless otherwise noted) with different counter anions (0.1,0.2,0.3,0.4,and 0.5 mol/ L,after adding certain amounts of corresponding sodium salts), under magnetic stirring to produce Cu-alginate beads. The beads were kept for solidification at room temperature for 30 min,and then the beads were filtered and washed thoroughly until there was no detectable protein and copper ion in the wash-out solution. The above condition was optimized for laccase immobilization in the literature [21, 22],and the anion concentrations were various and subjected to study. Finally,the copper solutions and Cualginate laccase beads were characterized using an Agilent 8453 diode array spectrophotometer and a BaySpec Desktop Raman analyzer. 2.2. Spectrophotometric assay

Each assay was monitored for at least 5 min after the addition of the enzyme,using o-tolidine as the substrate [23]. Absorbances were monitored using an Agilent 8453 diode array spectrophotometer in conjunction with UV-visible ChemStation software programs. Assays were carried out in a 1 cm quartz cuvette in a total volume of 2.0 mL at room temperature. Stock solutions ofotolidine were prepared as 3.34 mmol/L in 3 mol/L H+Cl. In a typical assay,a 0.2 mL of 3.34 mmol/Lo-tolidine solution and 5 beads of immobilized laccase (12.5±1.0 mg,wet weight) were added into 1.8 mL 0.15 mmol/L pH 4.6 sodium acetate/acetic acid buffer. Then the UV-vis absorption spectra were recorded every 30 s. Each experiment of kinetic measurement was repeated at least 5 times to secure the statistic meanings. 2.3. Kinetics analysis

Subjected to the kinetic measurement,the final concentrations ofo-tolidine in the reaction system are also set as 0.017,0.034, 0.084,0.167,0.334,and 0.668 mmol/L,respectively,using the same amount of immobilized laccase beads. The resulting spectrophotometric data were then analyzed to determine the enzyme kinetic parameters. Velocities,take from the linear portion of the absorbance at 365,630,and 850 nm,were reported as changes in A/min and averaged from the three repetitive assays. TheKmand Vmax values were determined using the Lineweaver- Burk double-reciprocal plot with classical Michaelis-Menten model [24]. 2.4. Degradation of 2,4-DCP

2,4-Dichlorophenole (DCP),a toxic residual compound in environment from the production of germicides and soil sterilizants,was used to evaluate the immobilized laccase within Cualginate matrices made under the gelating solutions of a 0.05 mol/L cupric concentration and 0.10 mol/L counteranion [25, 26]. 0.2 mmol/L 15 mL solution of 2,4-DCP and 80 beads (~1g) of immobilized laccase were placed in a bubbling reactor at a flow air rate of 1 L/min (saturated with water) at room temperature and pH 4.6 sodium acetate/acetic acid buffer. The reaction samples were periodically withdrawn (0.5 mL each time) and transferred into a prepared solution in which 2 mL of water,0.3 mL of 0.5 mol/L ammonia,0.050 mL of 2% 4-aminoantipyrine,and 0.050 mL of 80 g/L potassium hexacyanoferrate (III) were mixed together. The pH value of the system was adjusted to 7.9 with~0.2 mL of 0.2 mol/L pH 6.7 phosphate buffer. The mixture was incubated for 15 min and then the absorbance at 509 nm was measured [27]. 3. Results and discussion

3.1. Enzyme immobilization

Laccase from T. versicolor was immobilized by physical entrapment in Cu-alginate beads. In this work five different anions such as perchlorate,nitrate,sulfate,chloride,and acetate were used for the preparation of Cu-alginate matrices,respectively. Cupric sulfate is the most commonly used for the preparation of Cu-alginate matrices in laccase immobilization [14]. No significant difference was observed in mechanical and structural properties of the resulting beads with different counteranion-gelating solutions, but with different colors. As shown in Fig. 1c,the acetate beads are in light blue,the chloride green,and the perchlorate,nitrate,and sulfate are in proper order of colors between the green and blue. Compared to the colors of the solid cupric salts,the Cu-alginate beads are much lighter. The bead colors indicated that the counteranions were partially entrapped in the Cu-matrices as expected. Basically the anions diffuse into the soft matters when cupric ion takes the place of sodium ion in the alginate-laccase solution (see Fig. S1 in Supporting information).

Fig. 1. Characterization of immobilized laccase in Cu-alginate matrices,including the visible absorbance of 0.05 mol/L cupric solutions (a),Cu-matrices (b),the bead samples in 2 mL vials (c),and the Raman spectra of the beads (d).

The UV-vis absorbances of the five cupric solutions (0.05 mol/L) and immobilized laccase beads (prepared in 0.1 mol/L counteranion) are presented in Fig. 1a and b. The spectra of the aqueous solutions had absorption bands at the wavelength of 765,813,811, 807,and 812 nm,and the molar absorption coefficient ε of 1.12, 0.62,0.58,0.56,and 0.50 L cm-1mol-1 for the cases of perchlorate, nitrate,sulfate,chloride,and acetate,respectively. These bands corresponded to the characteristic absorption peaks of Cu-anion coordination,suggesting that the coordination intensity to cupric ion is likely in an order of acetate>chloride~sulfate~nitrate> perchlorate. The maximum absorption bands of the Cu-alginate beads,respectively,located at 752,744,728/786,752,and 749 nm, slightly different from those in the aqueous solution and solids. Using the 600 nm absorbance as background,it could be estimated with the absorption band at the wavelength of 720-800 nm that cupric concentration in Cu-alginate matrix is at about 5 mmol/L level. The different color appearances suggested that counteranions were entrapped in the Cu-alginate matrices.

The Raman spectra of the five Cu-alginates are presented in Fig. 1d. The most characteristic bands obtained are a group of COO stretching vibrations in a range of 1400-1700 cm-1.Aweak absorption in a range of 1530-1550 cm-1was detected and assigned as asymmetric COO stretching vibration,consistent with the Raman absorption at 1574 cm-1in a binuclear structure of coumarin-3-carboxylate cupric salt [28]. The observed asymmetric COO stretching vibrations were redshifted in the Cu-alginate matrices,compared with those in the FTIR spectra of solid alginate salts: sodium (1596 cm-1), calcium (1590 cm-1),copper (1585 cm-1),cadmium (1578 cm-1),lead (1563 cm-1),and nickel (1583 cm-1)[29]. As proposed by Fukset al.,the bidentate chelating mode of asymmetric vibration is shifted to shorter wavelengths in relating to sodium alginate,while the bidentate bridging or monodentate mode to longer [30]. This indicated that the bidentate chelating mode is dominant in the Cu-alginate matrices,as shown in Scheme 1. 3.2. Time-resolved spectrophotometric measurement

Laccase catalyzed the formation of a brilliant blue product from colorlesso-tolidine solutions. Like benzidine,o-tolidine has two oxidation products at least. The primary oxidation product corresponds to a blue pigment with a maximum absorption peak at 630 nm,and the secondary oxidation product corresponds to a yellow pigment with a maximum absorption at 435 nm [31, 32]. In this work,wavelength scans of a reaction mixture were taken every 30 s to identify wavelengths at which the products showed the greatest change in absorbance during the treatment with immobilized laccase. The time-resolved UV-vis absorption spectra are presented in Fig. 2. The oxidation showed three major absorbance peaks at 365,630,and 850 nm,consistent with the two absorption bands of 366 and 630 nm in Miller’s paper [23]. A smaller absorbance peak was also present at about 430 nm, corresponding to the secondary oxidation product. The absorption bands increased with time,and those at wavelengths of the 365, 630,and 850 nm were kept in a linear mode for more than 5 min at room temperature,indicating that the immobilized-laccase beads catalyze the primary oxidation ofo-tolidine to the blue product.

Fig. 2. The time-resolved UV-vis spectra ofo-tolidine oxidation catalyzed by immobilized laccases,prepared with different counteranions such as acetate (a), chloride (b),sulfate (c),nitrate (d),and perchlorate (e).
3.3. Michaelis-Menten kinetics

The formation of the blue pigment from the oxidation of otolidine,as measured by the change in A/min,was directly proportional to laccase activity. Using a constant amount of immobilized laccase and varying theo-tolidine concentration up to 0.67 mmol/L,a plot of reaction velocityvmeasured at 850 nm versusincreasing substrate concentration [S] was constructed as shown in Fig. 3a. The Lineweaver-Burk plots are presented in Fig. 3b-f. The apparentKmand Vmax at 365,630,and 850 nm with Michaelis-Menten kinetic model are presented in Table 1,and some detailed data are available in Tables S1-S7 in Supporting information.

Fig. 3. The classic Michaelis-Menten curves (a),and the Lineweaver-Burk plots for the cases of acetate (b),chloride (c),sulfate (d),nitrate (e),and perchlorate (f),using the absorption peaks at 850 nm.

Table 1
The apparent Km(mmol/L) and Vmax (A/min) for immobilized laccase beads prepared in different anion solutions ([X] = 0.1 mol/L),where 1/ Vmax (min/A) and Km/ Vmax (mmol min L-1A-1) were determined by the Lineweaver-Burk doublereciprocal plots.

Reaction rates monitored at 365,630,or 850 nm showed similar profiles in the kinetic measurement. Compared to the data with the absorption bands at 365 nm and 630 nm,those at 850 nm were in the smallest errors and used for the following discussion in the report. Using the absorption band at 850 nm,maximum activity Vmax was observed as 0.014,0.014,0.019,0.0078,and 0.044 A/min, and Michaelis constant (Km) was determined as 57,46,46,24,and 35umol/L foro-tolidine to the immobilized laccase beads prepared in perchlorate,nitrate,sulfate,chloride,and acetate solution,respectively. If the cases of perchlorate,nitrate,and sulfate provided a general reference for immobilized laccase, chloride would be an inhibitor and acetate an activator. Indeed, both chloride and acetate reduced the Michaelis constant by about a half amount compared to nitrate and sulfate,while perchlorate increased slightly. The Michaelis constants are in good agreement with the coordination ability of the five anions,that is, ClO4->NO3-~SlO42->Ac->Cl-,and is opposite to the order of molar absorption coefficients (ClO4--~SlO42-~ Cl-<Ac-,Fig. 1a). It indicated that the counteranions were cooperative with the substrate-binding to the multicopper center in laccase. The stronger the coordination of counteranion is,the smaller the Michaelis constants are,and the higher the affinity ofotolidine is. For some reasons,the two strongest coordinating anions (chloride and acetate) had opposite influences on the enzyme activity - one inhibition and another activation,even though both anions enhanced the binding affinity ofo-tolidine to laccase. 3.4. Counteranion concentration

The relationship of enzyme activity and counteranion concentration is presented in Fig. 4. The inhibition of chloride seemed saturated after 0.2 mol/L,and the promotion of acetate declined drastically as its concentration was increased to 0.5 mol/L. In the cases of sulfate and nitrate,the enzyme activity decreased in the range of 0.1-0.5 mol/L as well. Only perchlorate helped the oxidation at higher concentrations. It indicated that the good coordinating anions were competitive in binding to the multicopper center of laccase,though the ion intensity is able to help the laccase oxidation in the case of perchlorate. It also suggested that the laccase immobilization benefited from the relatively low concentration of counteranions. In the following evaluation,the immobilized laccase beads were prepared in a 0.1 mol/L counteranion solution (initial data are available in Table S8 in Supporting information).

Fig. 4. The absorption increasement at the wavelength of 850 nm in the typical laccase-oxidation by Cu-alginate beads,prepared in the different concentrations of counteranion solutions.
3.5. Biodegradation of 2,4-DCP

The concentrations of 2,4-dichlorophenol were determined colorimetrically by 4-aminoantipyrine,as described in Standard Methods. The biodegradation of 2,4-DCP by immobilized laccase is depicted in Fig. 5. After 5 h,the 2,4-DCP removal efficiency reached 65-75% in the cases of acetate,perchlorate,nitrate,and sulfate,but only 50% in the case of chloride. No significant decrease was observed afterwards.

Fig. 5. Comparison of 2,4-DCP removal by immobilized laccases prepared in different counteranion solutions (immobilized laccase,0.185 g/L,1.0 g; starting solution volume,15 mL; pH 4.6).

Indeed,during the initial 2 h of reaction of the catalytic degradation,the catalytic ability for the immobilized laccase was in the order of acetate>nitrate>perchlorate>chloride> sulfate. After 3 h,the differences among sulfate,nitrate,perchlorate,and acetate became smaller and smaller in catalytic ability, indicating that the counteranions in the Cu-matrices exchanged with the anions in the reaction solution such as acetate and reached an equilibrium. However,chloride seems to be a strong inhibitor even after 5 h of reaction,implying that it may strongly bind to the multicopper site in the protein and is hardly replaced by acetate. This result demonstrated that the immobilized laccase with the other four anions showed a clear catalytic capacity for 2,4-DCP degradation and counteranion was crucial for the catalytic ability. Wanget al. reported that the removal efficiency of 2,4-DCP was approximate 60% in the first 2 h by using magnetic Cu2+-chelating silica particles for laccase immobilization [25]. In comparison with their results,the immobilized laccase in this study showed a good catalytic capacity for 2,4-DCP biodegradation. It should be noted that chloride was generated in the laccasecatalyzed biodegradation of 2,4-DCP and probably was inhibitory when it reached a high concentration.

It is also interesting to observe that the immobilized laccase prepared in sulfate solutions (the most commonly used cupric solution) was inefficient in the biodegradation of 2,4-DCP,even worse than that in chloride solutions in the initial stage,though the catalytic activity was recovered after 3 h for anion exchange within the Cu-alginate matrices. However,the catalytic ability in the case of sulfate was found much higher in theo-tolidine spectrophotometric assay compared to that in the case of chloride. This indicated that the counteranion effect was accompanied with the substrate selectivity and the anions were likely involved in the catalytic sites in laccase. 4. Conclusion

In this work the anion effect on laccase activity was investigated with enzyme immobilization,by which the studied anions were absorbed in the Cu-alginate matrices and temporarily isolated from the pH buffer solution that is also composed of anions. With the UV-vis and Raman spectroscopic characterization,counteranions were found to be entrapped in the immobilized laccase beads and cupric ions were coordinated with the carboxylate group in alginate in the bidentate chelating mode. Using theotolidine spectrophotometric assay,a new absorption band at the wavelength of 850 nm was observed and applied for the kinetic analysis. It was concluded that chloride and acetate enhanced the binding affinity of theo-tolidine substrate to laccase,consistent with their properties of the strong coordination. It appears that acetate acted as an activator that doubled the catalytic activity and chloride as an inhibitor that reduced by half,compared with the other three anions. The coordinating ability was proposed to rationalize the experimental observations about the counteranions: Stronger coordination to cupric ions leads to better binding affinity of the substrate and is more competitive at the higher concentration. Finally,these immobilized laccases were employed in the degradation of an environmental pollutant,2,4-dichlorophenol,showing the fact that anion effect is applicable and chloride should be avoided in the laccase immobilization. The discovery of the new regulation by counteranion species may help to develop a better laccase-mediator-system (LMSs) in future. Acknowledgments

The first author gave special thanks to Mr. Yanbing Qi,[M+H]+r. Lanxuan Liu for discussions. This work is supported in part by the National Migh-Tech R&D Program of China ‘‘863’’ (No. 2012AA020403) and the National Basic Research Program of China ‘‘973’’ (Nos. 2012CB721005,2013CB966802),National Natural Science Foundation of China (Nos. 21377085,21303101, 31121064,J1210047),MOE New Century Excellent Talents in University (No. NCET-12-0354). Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version,at

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