Co-immobilization of manganese peroxidase from Phlebia radiata and glucose oxidase from Aspergillus niger on porous silica beads

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Biotechnology Letters 22: 641–646, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands.

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Co-immobilization of manganese peroxidase from Phlebia radiata and glucose oxidase from Aspergillus niger on porous silica beads Benoît Van Aken, Philippe Ledent, Henry Naveau & Spiros N. Agathos∗ Unit of Bioengineering, Universit´e Catholique de Louvain, 2/19 Place Croix du Sud, B-1348 Louvain-la-Neuve, Belgium ∗ Author for correspondence (Fax: +32-10-473062; E-mail: [email protected]) Received 31 January 2000; Revisions requested 8 February 2000; Revisions received 22 February 2000; Accepted 24 February 2000

Key words: co-immobilisation, glucose oxidase, manganese peroxidase, Phlebia radiata

Abstract Manganese peroxidase (MnP) from Phlebia radiata and glucose oxidase from Aspergillus niger were coimmobilized on porous silica beads. Immobilization of both enzymes on the same carrier provided an integrated system in which H2 O2 required by MnP was produced by glucose oxidase. The immobilization process resulted in a decrease of both enzymatic activities and substrate affinities. However, immobilization improved the stability of MnP against H2 O2 or high pH, as well as the storage stability of this enzyme.

Introduction White-rot fungi are ligninolytic organisms able to secrete powerful oxidative enzymes (Barr & Aust 1994, Tien & Kirk 1983) including manganesedependent peroxidases (MnP) and lignin peroxidases (LiP) (Kuwahara et al. 1984). Due to its random and complex structure, lignin is usually resistant to the action of enzymes, often specific to a given substrate (Barr & Aust 1994). The non-specificity of ligninolytic peroxidases secreted by white-rot fungi allows them to degrade lignin, but also a wide range of dangerous xenobiotic pollutants (Paszczynski & Crawford 1995, Van Aken et al. 1997). In the presence of H2 O2 as electron acceptor, MnP oxidizes Mn(II) to Mn(III) which is a strong oxidative agent acting as a diffusible mediator in the MnP-catalyzed degrading process (Wariishi et al. 1989). A cell-free enzymatic preparation involving concentrated MnP from Nematoloma frowardii and glucose oxidase (Glox) from Aspergillus niger as a H2 O2 -generating system was shown to be able to mineralize several recalcitrant environmental pollutants (Hofrichter et al. 1998). However, industrial use of free biocatalysts in continuous remediation processes would be expensive because of enzyme loss in the effluent (Hartmeier 1988). Im-

mobilization of enzymes allows their maintenance in a continuous reactor and may improve their stability against denaturing agents (Royer 1975). In this work, we have co-immobilized MnP from the whiterot basidiomycete Phlebia radiata together with Glox from A. niger on porous silica beads activated by aminoalkylethoxysilane (Germain & Crichton 1988). The coupling agent was glutaraldehyde, the most common bifunctional reagent used for protein immobilization (Hartmeier 1988). The kinetic parameters of both soluble and immobilized enzymes were quantified and the ratio Glox/MnP exhibiting optimal MnP activity was determined. Finally, the thermal, pH, and storage stability of immobilized enzymes were compared to those of free enzymes. Although the successful immobilization of MnP from white-rot fungi has been reported previously (Grabski et al. 1996, 1998), this paper presents the first co-immobilization of both MnP and Glox on the same carrier.

642 Materials and methods Production and preparation of manganese peroxidase (MnP) Partially purified MnP from P. radiata (ATCC 64658) was prepared as previously described (Van Aken et al. 1999). Briefly, the extracellular fluid of liquid cultures of P. radiata grown over two weeks was concentrated 50-fold by ultrafiltration on a 10-kDa cut-off membrane (Pall Filtron Corp., Northborough, MA, USA). In order to remove low-molecular weight compounds, the ultrafiltration was repeated twice more after redilution in acetate buffer (25 mM, pH 5.0). Partially purified MnP exhibited a protein concentration of 2100 ± 110 mg l−1 and an activity of 231 ± 8 nkat mg−1 protein (Paszczynski et al. 1986). Enzymatic assays for LiP (Tien & Kirk 1983) and laccase (Niku-Paavola et al. 1988) did not reveal any measurable activities. Immobilization of MnP and glucose oxidase (Glox) MnP and Glox were immobilized on controlled-pore silica beads (30–45 mesh, pore size 375 Å; CPCSilica Carrier, Fluka, Buchs, Switzerland) according to Robinson et al. (1971) and Germain & Crichton (1988). Briefly, silica beads were treated successively with 3-aminopropyltriethoxysilane and glutaraldehyde before being immersed in the enzymatic solution. The MnP solution contained partially purified MnP (20% v/v) and 2 mM MnSO4 · H2 O in malonate buffer (30 mM, pH 4.5). It exhibited a final protein content of 458 ± 6 mg l−1 and an enzymatic activity of 247 ± 12 nkat mg−1 protein. The Glox solution was prepared by diluting Glox from A. niger, low in catalase (Sigma) in malonate buffer containing 100 mM glucose to a final protein content of 452 ± 4 mg l−1 and an enzymatic activity of 371 ± 5 nkat mg−1 . For the co-immobilization experiments, the above enzymatic solutions were mixed in the following Glox/MnP ratios (v/v): 1:4, 1:2, 1:1, 2:1, and 4:1.

Glox was measured by the oxidation of glucose to gluconic acid and H2 O2 ; H2 O2 produced was determined spectrophotometrically at 450 nm by oxidation of odianisidine in the presence of horseradish peroxidase. The reaction mixture (1 ml) contained 120 µM odianisiline dihydrochloride, 17 nkat ml−1 horseradish peroxidase, 100 mM glucose, and a 5% (v/v) Glox in sodium tartrate buffer (100 mM, pH 4.5). The activity of the composite system MnP/Glox was determined using the MnP assay in which 100 mM glucose instead of H2 O2 were added to start the reaction. To determine the activities of immobilized enzymes, the assays were performed in a stirred reactor containing 4 ml assay mixture and connected in-loop to a circulation cell (Fisher Scientific, Pittsburgh, PA, USA) placed in the spectrophotometer. The enzyme sample consisted of distilled water containing the solid carrier. Enzymatic activities were expressed in nkat mg−1 protein. The protein concentrations were determined using the bicinchoninic acid method (bicinchoninic acid protein assay kit, Sigma). Kinetic studies on free and immobilized enzymes A known amount of free or immobilized enzymes was incubated as in the enzymatic assays described above, but in the presence of different concentrations of substrate (MnSO4 · H2 O and H2 O2 for MnP, and glucose for Glox). The initial transformation rate of the substrate was recorded. The kinetic parameters describing the relationship between this initial velocity and the substrate concentration were computed by fitting the experimental data (SigmaPlot, Jendel Scientific, San Francisco, CA, USA). Chemicals All chemicals were of analytical grade and were purchased from Sigma, Aldrich or Merck. Controlled pore ceramic silica beads were obtained from Fluka (Buchs, Switzerland). Vanillyl acetone was prepared by reaction of vanillin and acetone according to Paszczynski et al. (1986).

Activity of free and immobilized enzymes Activity of free MnP was determined photometrically by the oxidation of vanillyl acetone at 334 nm (Paszczynski et al. 1986). The reaction mixture (1 ml) contained 100 µM vanillyl acetone, 1 mM MnSO4 · H2 O, 100 µM H2 O2 , and a 10% (v/v) MnP in sodium malonate buffer (40 mM, pH 4.5). Activity of free

Results and discussion Immobilization and co-immobilization of MnP and Glox Table 1 compares the enzymatic activities of free and immobilized enzymes. The immobilization process resulted in a loss of enzymatic activity of 40.0% for

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Fig. 1. Relative MnP activity for different MnSO4 (A) and H2 O2 (B) concentrations and relative Glox activity for different glucose concentrations (C). 100% corresponds to an enzymatic activity of 248 nkat mg−1 for free MnP, 170 nkat mg−1 for immobilized MnP, 1928 nkat mg−1 for free Glox, and 400 nkat mg−1 for immobilized Glox. Activities of free (open circles) and immobilized enzymes (closed circles) are presented. Solid lines represent the data fitting according to Michaelis–Menten (A, C) or substrate inhibition kinetics (B). Table 1. Individual immobilization and co-immobilization of manganese peroxidase (MnP) from Phlebia radiata and glucose oxidase (Glox) from Aspergillus niger on controlled pore silica beads: activities of free and immobilized enzymes. Enzyme

MnP Glox Mixture Glox/MnP 1:1 (v/v)

Free

Immobilized

nkat mg−1

%

nkat mg−1

%

247 ± 12 1722 ± 89 254 ± 13

100 100 100

168 ± 4 371 ± 5 79 ± 6

68 22 31

MnP, 87.8% for Glox, and of 69.1% (measured on the basis of the MnP activity) for the Glox/MnP system when Glox and MnP were mixed in equal quantities (ratio 1:1, v/v). The immobilization process is known to bring about a diminution of the enzyme activity, which results either in an increase of the Km (alteration of the binding site of the enzyme or steric hindrance) or in a decrease of the Vmax (partial destruction of the enzyme or reduction of its activity) (Royer 1975). The proportion of proteins immobilized on the solid carrier after a 24-h loading varied from 68% to 89% and increased with the Glox/MnP ratio (Table 2). The immobilization procedure involves covalent bindings between aminoalkylsilane derivatized beads and lysine residues of the enzymes, using glutaraldehyde as a bifunctional coupling agent (Robinson et al. 1971). Even though MnP and Glox have a comparable lysine content, the accessibility of those residues could explain the differences.

No trace of protein release was detected after stirring overnight the solid carrier in liquid phase. Despite considerable losses of enzymatic activities, the successful co-immobilization of MnP from P. radiata together with Glox form A. niger on porous silica beads proved possible, thus providing an integrated system in which the oxidation of glucose produced continuously – and biologically – H2 O2 required by MnP. The immobilized biocatalysts (Glox/MnP) could be used in continuous bioreactors for the treatment of effluents contaminated by xenobiotic environmental pollutants. Considering the loss of enzymatic activities observed, the very common immobilization procedure used may not have been the most appropriate. However, the aim of this paper was not to optimize an immobilization method, but to present the first embodiment of an innovative idea which could be developed further and applied in bioremediation processes.

644 Table 2. Individual immobilization and co-immobilization of manganese peroxidase (MnP) from Phlebia radiata and glucose oxidase (Glox) from Aspergillus niger on controlled pore silica beads: protein content of the enzymatic solution (before and after loading) and of the solid carrier. Enzymes mg Immobilization MnP Glox Co-immobilization Glox/MnP 1:4 1:2 1:1 2:1 4:1

Enzymatic solution (5 ml) Initial After loading (24 h) % mg %

Carrier (1 g) mg

%

2.29 ± 0.03 2.26 ± 0.02

100 100

0.81± 0.05 0.51± 0.02

35 23

1.59 ± 0.19 2.04 ± 0.11

70 89

2.36 ± 0.02 2.33 ± 0.02 2.35 ± 0.14 2.25 ± 0.01 2.31 ± 0.05

100 100 100 100 100

0.86 ± 0.03 0.73 ± 0.03 0.70 ± 0.03 0.54 ± 0.03 0.50 ± 0.03

37 31 30 24 22

1.60 ± 0.09 1.63 ± 0.06 1.71 ± 0.08 1.81 ± 0.12 1.90 ± 0.12

68 70 73 80 82

Fig. 2. Relative MnP activity of the MnP/Glox system for different Glox/MnP ratios. 100% corresponds to an enzymatic activity of 254 nkat mg−1 for free enzymes and 90 nkat mg−1 for co-immobilized enzymes. Activities of free (open circles) and co-immobilized enzymes (closed circles) are presented.

Kinetic studies Figure 1 presents the relative activities of both free and immobilized enzymes as a function of substrate concentrations. The initial MnP activity as a function of the Mn(II) concentration followed Michaelis– Menten kinetics: V = Vmax [Mn(II)]/(Km + [Mn(II)]) (Figure 1A). However, when the H2 O2 concentration increased, the MnP activity followed substrate inhibition kinetics: V = Vmax [H2 O2 ]/(Km + [H2 O2 ] + [H2 O2 ]2 /Ks ) (Figure 1B). This was previously ob-

served and can be explained by the inactivation of MnP in the presence of high H2 O2 concentrations (Palma et al. 1997). The Glox activity as a function of the glucose concentration followed Michaelis–Menten kinetics (Figure 1C). In all cases, the immobilization resulted in an increase of the Michaelis constant Km . Binding enzymes on a solid carrier usually brings about alteration and/or steric hindrance of the catalytic site (Royer 1975). Diffusional limitations in the porous carrier could also reduce the affinity of the enzymes for the substrate (Hartmeier 1988). As a consequence, immobilization may result in a decrease of the affinity of enzymes for the substrates (increase of Km ). For the substrate inhibition kinetics (observed for MnP activity as a function of H2 O2 concentration), immobilization increased the enzyme-substrate complex dissociation constant Ks (Figure 1B). Immobilized MnP turned out to be more stable than free MnP in the presence of high H2 O2 concentrations. Immobilization usually improves the rigidity of enzymes and, therefore, their stability against denaturing agents (Royer 1975). Figure 2 presents the relative MnP activity of free and co-immobilized Glox/MnP systems as a function of the Glox/MnP ratio. The MnP activity showed a maximum corresponding to a 1:1 ratio for free enzymes and 2:1 for co-immobilized enzymes. When MnP and Glox were mixed together, H2 O2 was provided through the oxidation of glucose by Glox. Therefore, increasing the ratio Glox/MnP had on the MnP activity a comparable effect as increasing H2 O2 concentration (Figure 2).

645 free enzymes (Hartmeier 1988). However, improvement of thermal stability by immobilization is not a general rule. The pH stability of the MnP/Glox system was higher when immobilized (Figure 3B). The surface of silica beads is negatively charged. The pH close to the surface of the carrier is lower than in the bulk solution and immobilized enzymes are therefore less sensitive to higher pH values (Grabski et al. 1998, Hartmeier 1988). The remaining activity after 6 weeks storage at 4 ◦ C was 24% for the free enzymes and 48% for the co-immobilized ones (Figure 3C). Immobilization tends to decrease the water activity and increase hydrophobic interactions between non-polar amino acid residues in the microenvironment of the carrier (Royer 1975). It appears that increasing the local concentration of enzymes, immobilization of MnP and Glox improved their storage stability.

Acknowledgements The support of a FDS fellowship (Scientific Development Fund, Université Catholique de Louvain) to Benoît Van Aken is gratefully acknowledged. We also thank Dr Robert R. Crichton (Unit of Biochemistry, Université Catholique de Louvain) for useful discussions.

Fig. 3. Temperature dependence (A), pH dependence (B), and storage stability (C) of the co-immobilized MnP/Glox system. The thermal inactivation was determined by incubating free and immobilized enzymes for 10 min at temperatures from 20 ◦ C to 90 ◦ C. The pH stability was measured by incubating enzymes for 60 min over a pH range from 2.0 to 11.0. The storage stability was determined by incubating free and immobilized enzymes at 4 ◦ C over 6 weeks. Activities of free (open circles) and co-immobilized enzymes (closed circles) are presented.

Thermal, pH, and storage stability The immobilization process usually brings about a greater enzyme stability resulting from the multiple interactions between the solid support and the protein molecule (Hartmeier 1988, Royer 1975). Figure 3 shows the thermal, pH and storage stability of the immobilized Glox/MnP system (ratio 1:1) compared to those of the free enzymes. Surprisingly, the immobilized system showed a lower thermal stability than the free enzymes (Figure 3A). Due to their greater rigidity, immobilized enzymes are generally more stable against thermal denaturation than the corresponding

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