A new enzyme immobilization procedure using copper alginate gel: Application to a fungal phenol oxidase

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A new enzyme immobilization procedure using copper alginate gel: application to a fungal phenol oxidase. Enzyme Microb Technol 16, 151158 ARTICLE in ENZYME AND MICROBIAL TECHNOLOGY · MARCH 1994 Impact Factor: 2.32 · DOI: 10.1016/0141-0229(94)90078-7 · Source: PubMed

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A new enzyme immobilization procedure using copper alginate gel: Application to a fungal phenol oxidase Gianna Palmieri, Paola Giardina, Bianca Desiderio, Liberato Marzullo, Marta Giamberini* and Giovanni Sannia Dipartimento di Chimica Organica e Biologica and *Dipartimento di lngegneria dei Materiali e della Produzione, Universita degli Studi di Napoli "Federico H , " Naples, Italy

A new procedure was developed for enzyme immobilization by entrapment in copper alginate gel. The mechanical properties of the copper alginate gel were characterized and compared with those of the most widely used calcium alginate. The system was applied to the immobilization of a fungal phenol oxidase. Optimal conditions for enzyme immobilization were set up: the system immobilized 85% of the enzyme, and the remaining 15% was recovered in the aqueous immobilization medium. The stability and activity of the immobilized enzyme were studied. After immobilization, the enzyme was active in a widerpH range, the temperature of its optimal activity was shifted to lower values, and the possibility of storage at 4°C was greatly improved. The immobilized enzyme generally increased the rate of oxidation of various substrates. The results indicate a potential use of this system for the construction of bioreactors to be used in the detoxification of polluted waste waters.

Keywords:Enzyme immobilization; phenol oxidase; copper alginate Introduction Recently an increasing trend has been observed in the use of immobilized enzymes as catalysts in several industrial chemical processes. Immobilization is important to maintain constant environmental conditions m order to protect the enzyme against changes in pH, temperature, or ionic strength; this is generally reflected in enhanced stability.l Moreover, immobilized enzymes can be more easily separated from substrates and reaction products and used repeatedly. Many different procedures have been developed for enzyme immobilization; these include adsorption to insoluble materials, entrapment in polymeric gels, encapsulation in membranes, crosslinking with a bifunctional reagent, or covalent linking to an insoluble carrier. 2 Among these, entrapment in calcium alginate gel is one of the simplest methods of immobilization. The success of the calcium alginate gel entrapment technique is due mainly to the gentle environment it provides for the entrapped material. However, there are some limita-

Address reprint requests to Dr. Sannia at the Dipartimentodi Chimica Organicae Biologica,via Mezzocannone,16, 80134Naples, Italy Received 18 May 1993; revised 20 July 1993

© 1994 Butterworth-Heinemann

tions, such as low stability and high porosity of the gel. 1These characteristics could lead to leakage of large molecules like proteins, thus generally limiting its use to whole cells or cell organelles. 3 This paper describes the setting up of a new procedure for enzyme entrapment in alginate gel which makes use of copper ions as the gelifying agent, thus greatly improving the stability and mechanical properties of the gel in comparison with calcium alginate. 4 This method promises to be a general system for immobilization of enzymes which are not inactivated by high concentrations of copper salts. The application of the system to a fungal phenol oxidase was studied. Fungal phenol oxidases are involved, together with peroxidases, in the modification of lignin macromolecules in nature. 5 These enzymes catalyze the oxidation of phenylpropanoid subunits containing phenolic hydroxyl groups using 0 2 as the electron acceptor. 6'7 It has been suggested that lignin-degrading enzymes may be used in the pulp and paper industry, where the removal of residual lignin from pulp and waste waters is a challenging t a s k ) Moreover, it has also been suggested that these enzymes may be used for the detoxification of various water and soil pollutants. 9 The best producers of ligninolytic enzymes are the basidiomycete fungi belonging to the so-called "white-

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Papers The purpose of this study is to explore the improvement of the activity and stability of a phenol oxidase by e n t r a p m e n t in c o p p e r alginate beads and to c o m p a r e the properties of the immobilized e n z y m e with those of the free enzyme. The results obtained might be considered of general interest for the immobilization of c o p p e r - d e p e n d e n t enzymes.

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E n z y m e production, purification, a n d assay Pleurotus ostreatus (strain Florida) was maintained, through 0.01

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Figure 1 Activity retention of different amounts of phenol oxidase by entrapment in alginate beads

periodic transfer, at 4°C on agar-potato dextrose (Difco Laboratories, Detroit, MI) plates containing 0.5% yeast extract, and grown as described previously by Palmieri G. et al. 1~The extracellular phenol oxidase was isolated from the filtered culture broth at the maximum of enzyme production, as described earlier. 11The purified enzyme was stored at -80°C.

E n z y m e immobilization

r o t " group, which play a predominant role in the complete decomposition oflignin. 10One of these organisms is Pleurotus ostreatus, which excretes large amounts of a phenol oxidase previously identified, purified, and fully characterized by the authors. J~

The enzyme was immobilized by entrapment in copper-alginate beads: 100 ~1 of a 10 mg ml -j bovine serum albumin (BSA) solution, containing different amounts of purified phenol oxidase (from 7 × 10 - 2 to 21 units), was mixed with 10 ml 3% sodium alginate solution (alginic acid from Macrocystis pyrifera, low viscosity, Sigma, St. Louis, MO). The resulting homogeneous mixture was centrifuged at 4,000 rev rain- ~for

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5 min, to remove air bubbles, and extruded drop by drop using a syringe needle (gauge 21) into a 0.15 M CuSO4 aqueous solution (pH 4.0). The same procedure was used to immobilize the enzyme in calcium alginate beads using 0.15 M CaCI2 in 10 mM acetate buffer pH 5.0 instead of the CuSO4 solution. In a typical experiment, 0.75 txg (0.3 U) enzyme and 1 mg BSA were added to 10 ml sodium alginate solution. The resulting spherical blue beads were washed exhaustively with distilled water until pH 5.0-5.5 was reached. The diameter of the beads ranged from 3 to 4 ram, and the total wet weight obtained from 10 ml of Na + alginate solution was about 7.0 g. Entrapment yields were determined measuring the phenol oxidase activity lost in the 0.15 M CuSO4 aqueous medium and the enzymatic activity entrapped in the formed beads.

Properties of alginate gel In order to test the resistance of the alginate gel to the presence of a chelating compound, 0.06 g of copper or calcium alginate beads was immersed in 2 ml 0.1 M Na phosphate buffer pH 6.0. The incubation was carried out under continuous stirring at room temperature. Cylindrical alginate gels were prepared by pouring the alginate solution into a dialysis tubing of relevant diameter and dialyzing against 0.15 M CaCI z or CuSO4 overnight at room temperature. Gel samples were cut into 0.2-cm-thick disks, 1.6 cm diameter, and storage modulus (G') and loss modulus (G") were measured at 25°C and 1 Hz by means of

a Bohlin VOR Rheometer (Bohlin Reology, Lund, Sweden) using the parallel-plate configuration (15 mm plate diameter), a 1.59-g cm torsion bar, and the PPI5 measuring system.

Soluble and immobilized enzyme assay Phenol oxidase activity was assayed at 25°C with 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) as the substrate using two different procedures: 1. 0.3 g of beads was incubated in 1 ml reaction mixture (2 mM ABTS, 10 mM Na-citrate, 4 mM CuSO4, pH 3.0) under continuous stirring. The absorbance increase due to oxidation of the substrate was followed discontinuously by withdrawing the liquid phase at different times and measuring its absorbance at 420 n m (8420 = 3.6 10 - 4 M - 1c m - 1 ) . 1 2 After each measurement, the solution was immediately remixed with the immobilized enzyme to follow the enzymatic reaction further. 2. 1.5 g of beads was incubated in 5 ml reaction mixture (2 mM ABTS, 10 mM Na-citrate, 4 mM CuSO4, pH 3.0) under continuous stirring. Aliquots (100 p.1)were withdrawn at fixed time intervals and the oxidation of ABTS was followed by measuring the absorbance increase at 420 nm. The same values of enzymatic activity were obtained using the two procedures described above. The activity of the free and immobilized enzymes towards different substrates was assayed at 25°C by polarographic

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analysis using an oxygen monitor (Orion SA520) with an oxygen electrode (Orion 97-08-00, Orion, Boston, MA) connected to a 2210 LKB recorder (Bromma, Sweden); 1.9 U of free or immobilized enzyme in 20 ml of reaction mixture (20 mM substrates, 10 mM Na phosphate, pH 6.0) were used in each assay. The activity of the entrapped enzyme was measured as a function of pH using a universal buffer solution (Britton and Robinson type) 13adjusted to different pH values in the range 2.5-5.0.

Immobilized enzyme stability The stability of the immobilized enzyme in organic solvents (methanol, dioxane, and acetonitrile) and in buffer at different pH values (0.1 M glycine, pH 3.0; 0.05 M succinate, pH 4.0 and pH 5.0) was measured by incubating 0.3 g of beads in 0.4 ml of the different solutions at 25°C. The effects of storage time on the activity of the immobilized phenol oxidase were verified by incubating the wet beads at 4°C. The stability to temperature (from 20 to 70°C) was monitored under the same conditions.

Results and discussion A standard immobilization procedure in calcium alginate gel 3 was used to entrap a fungal phenol oxidase purified from P. ostreatus.l~ Under these conditions,

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the immobilized e n z y m e completely lost its activity in about 24 h at 4°C. Studies on the stability of phenol oxidase in the presence of different divalent cations such as Ca 2+ , Zn 2+ , Ba 2+ , Cd 2+ , and Cu 2+ indicate that all the ions used, except copper, cause a progressive inactivation of the enzyme. This observation, together with the data on the higher affinity of alginate 3 for Cu 2+ with respect to Ca 2+ and the fact that the hardness and the mechanical strength of alginate gel increased with the increased affinity for the divalent cation, 4 led the authors to use copper as the crosslinking agent for the immobilization by entrapment of the enzyme under study. Better mechanical properties were indeed found for copper with respect to calcium alginate gel. In fact, strain sweep experiments in the range of linear viscoelasticity gave values of the storage modulus and the loss modulus respectively of 1.6 x 105 and 1.8 x 10 4 Pa for the copper alginate gel, and 6.5 x 10 4 and 9.5 x 10 3 Pa for the calcium alginate gel. These data indicate that a better response to mechanical stresses can be expected in the case of the copper alginate gel. Furthermore, the use of copper alginate gels may o v e r c o m e one of the major limitations of this kind of system in the presence of a chelating compound such

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temperature (°C) Figure 5 Activity of free and immobilized phenol oxidase after 30 min incubation at different temperatures. The free enzyme was incubated in buffer containing 0.1 mg m1-1 BSA

as phosphate, which is one of the most common components of biological buffers. 3 In fact, copper alginate beads were dissolved after about 3 h of incubation in 0.1 M phosphate, while under the same conditions, calcium alginate beads were dissolved at least six times faster. In order to investigate the effect of enzyme loading on the activity of the immobilized biocatalyst, various quantities of phenol oxidase were entrapped in alginate gel beads. In all experiments, only 15% of the activity of the soluble enzyme initially added to the system remained in the aqueous immobilization medium. As shown in Figure 1, activity retention in the range 0.01-0.04 U g-i of beads was as much as 85%, but exponentially decreased to 6.8% when 2.9 U g-1 of beads were immobilized. This behavior could be caused by internal diffusional restrictions so that not all the activity of the immobilized enzyme was expressed. This might happen because the enzyme located in the outer portions of the bead consumes most or all of the substrate, and the enzyme molecules located deeper

within the bead have little opportunity to attack the substrate.' To exclude enzyme leakage from the gel matrix, the absorbance of the liquid phase, sampled after an immobilized enzyme assay, was monitored. No increase in the absorbance of the solution due to substrate conversion was observed for at least 1 h. The properties of the immobilized enzyme were then compared with those of the free enzyme. Figure 2 shows the effect of pH on enzyme activity. It can be seen that the immobilized enzyme shows a broader pH range of optimal activity, together with a shift towards less acidic regions. This phenomenon can be explained by an increase in the hydrogen ion concentration in the enzyme microenvironment, due to the negatively charged alginate gel. 2 Changes in the activity of soluble and immobilized phenol oxidase as a function of temperature were also investigated using assay procedure 2 (see Materials and methods). As shown in Figure 3, the optimum temperature for the activity of the immobilized enzyme E n z y m e M i c r o b . T e c h n o l . , 1994, v o l . 16, F e b r u a r y

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is shifted to lower values with respect to that of the free form. To investigate further the catalytic properties of the immobilized e n z y m e , the kinetic p a r a m e t e r s (K M and Vmax) were determined when ABTS was used as the substrate. The values for the immobilized (apparent K M = 0.3 mM; Vmax = 5.7 10 - 3 m M m i n l) a n d f r e e forms (K M = 0.28 raM; Vma~ = 3.6 10 -3 mM min l)J] of phenol oxidase are very similar. These data seem to be interesting because immobilized e n z y m e s often show a K M 10-20 times greater than that of the free enzyme, t4 The rate of oxygen consumption by the free and immobilized e n z y m e in the presence of different substrates is shown in Table 1; the same amounts of the free and immobilized enzymatic activity, determined using ABTS as the reference substrate, were used in all experiments. The free form catalyzed the oxidation of para- more efficiently than ortho-di-substituted benzenes, while meta-diphenols were not oxidized. The data for the immobilized e n z y m e show a general increase in the rate of oxidation of various substrates. When 2 - a m i n o h y d r o x y b e n z e n e and 2,6-dimethoxyphenoi were used as substrates, the instantaneous formation of a cloudy solution and the orange-brown coloration assumed by the beads indicated substrate 156

transformation, although no 0 2 consumption could be detected. This m a y be due to rapid precipitation of the oxidized products inside the beads, preventing the uptake of substrates into the matrix.

Table 1 Reaction rates of free and immobilized phenol oxidase (02 consumption, ppm min -1, in the presence of different substrates (20 mM))

Substrate o-Diaminobenzene p-Diaminobenzene 1,2-Benzenediol 1,4-Benzenediol 1,3-Benzenediol 1,2,3-Benzenetriol 2-Aminohydroxybenzene 2,6-Dimethoxyphenol (DMP) a DOPA b 3,4,5,-Trihydroxybenzoic acid Ascorbic acid

Free enzyme

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0.62 0.89 0.12 0.18 0 0.59 0.62 0.16 0.62 0.17 0

2.30 16.47 0.63 0.34 0 5.51 nd nd 0 1.07 0

a This substrate was used at a lower concentration (1 mM) because of its low solubility b/3(3,4-dihydroxyphenyl)-c~-alanine

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The effects of storage time on the activity of the free and immobilized enzyme were also examined, and the immobilized phenol oxidase was more stable than the free enzyme. In fact, the apparent half-life at 4°C for the entrapped enzyme was 30 days, while for the soluble enzyme it was only 3 days (Figure 4). However, prolonged incubation of copper alginate beads at 37°C led to the continuous release of enzyme from the matrix, probably due to the swelling and relaxing of the gel. The stabilities of the two forms were also compared after incubation for 30 min at various temperatures. It is worth noting that immobilization produces a moderate increase in enzyme stability to temperature in the ranges 20-40°C and 50-60°C (Figure 5) but does not significantly change the apparent melting temperature of the enzyme. The activities of the free and immobilized enzyme were also compared after incubation in buffer at different pH values from 3.0 to 5.0. The stability of phenol oxidase at pH 3.0 was greatly increased by immobilization (Figure 6), whereas no significant differences were found at pH 4.0 and 5.0. The stability of phenol oxidase immobilized in copper alginate beads was greatly increased after more than 5 h incubation in water mixtures (1 : 1, v/v) with

methanol or dioxane (Figure 7), while the two forms showed similar behavior when incubated in an acetonitrile/water mixture. Moreover, the gel beads were dehydrated by acetone or dimethylsulfoxide (DMSO)/ water mixtures, thus indicating they cannot be used under these conditions.

Conclusions Several methods have been described for the immobilization of phenol oxidases.14-19 The aim of the present investigation was to work out an inexpensive, unconventional, and fast procedure for enzyme entrapment which turned out to be of extreme interest for use with fungal phenol oxidase. The method described in this paper for immobilization into copper alginate beads resulted in the development of a support more resistant towards mechanical stresses and in a great increase of enzyme stability. Moreover, the substrate specificity of the immobilized enzyme was significantly changed. Such an immobilized enzyme may be used to develop systems for the detoxification of wastewaters polluted with phenolic derivatives of agricultural or industrial origin. Furthermore, this method may be considered as a powerful and potentially widely used system for the immobilization of copper-dependent enzymes.

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Acknowledgements The authors would like to thank Dr. Mario Mensitieri for his helpful advice and for his precious aid in performing mechanical tests, and Mr. L. Battista for technical assistance. Grants were obtained from Ministero dell'Universit~t e della Ricerca Scientifica and from Consiglio Nazionale delle Ricerche. Ms. M.E. Lisboa's skillful assistance in preparing the manuscript is gratefully acknowledged.

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