Kinetic behaviour of muscle LDH immobilized in nylon tubing

Immobilization was carried out of the lactate dehydrogenase (LDH) from rabbit muscle (EC 1.1.1.27), cross-linked through the bifunctional reactive glutar-aldehyde on to nylon tubing (1 m long, 53cm² internal surface area). Immobilized LDH inactivation kinetics are of first order (t_1/2 = 3·6 years, k = 5·4,e^?4 day^?1 to 5°C). The smaller effect of pH on activity than in the case of LDH in solution can be explained on the basis of limitation to proton diffusion towards the support. A limiting effect to free external diffusion of the substrate towards and products from the support was also observed, an effect which seems to determine the effective kinetic behaviour of immobilized LDH. The apparent optimum temperature is centred around 40°C, observing a clear inactivation (thermal denaturation) above this temperature. In the temperature range studied (10–40°C), the co-existence was seen of a kinetic control accompanied by another control, involving diffusional transport of substrates and products, on the global activity of the immobilized enzyme. This makes the Arrhenius profiles curvilinear. Both graphic and statistical non-linear regression analysis of the kinetic data—rate, v, versus substrate concentration [S]—carried out under conditions in which the diffusional limitations can be considered negligible (high recirculation flow rate), permitted investigation of the intrinsic kinetic behaviour of immobilized LDH. In this sense, it can be deduced that the rate equation to which these data seem to be fitted is of the polynomial quotient type in [S] of minimum degree 2:2. Although the diffusional limitations have a marked effect on the type of global kinetics shown by immobilized LDH, temperature was not found to affect its v[S] behaviour. The experimental evidence obtained thus indicates that the rate equation in the 10-40°C temperature range continues to be a rational equation of at least degree 2:2 in [S].     

Phosphatase active cross-flow microfiltration poly(vinylidene difluoride) bioreactor

Alkaline phosphatase from human placenta has been chemically immobilized on a hydrophilic cross-flow microfiltration membrane made from poly(vinylidene difluoride) (PVDF) derivatized with 1,1′-carbonyldiimidazole. The physicochemical characterization of the immobilized biocatalyst paid special attention to the irreversibility of the bonding of the enzyme to the support, the effects of pH, temperature and ionic strength on this activity, the existence of limitations of internal and external diffusion for H⁺, substrate and/or products, and the kinetic behavior (intrinsic and/or effective) of the immobilized enzyme. With respect to enzyme stability, patterns of hysteresis or memory are proposed, to account for a catalytic activity affected by previous experimental events and situations. The intrinsic kinetic behaviour, rate versus substrate concentration in the absence of diffusional restrictions, was analysed graphically and numerically (by non-linear regression and by utilizing the F statistical test for model discrimination), postulating a minimum rational rate equation of 2:2 degree in substrate concentration. In concordance, a mechanistic kinetic scheme for the catalytic enzyme action has been postulated.     

Immobilization of invertase onto dimer acid‐co‐alkyl polyamine

Invertase was immobilized onto the dimer acid‐co‐alkyl polyamine after activation with 1,2‐diamine ethane and 1,3‐diamine propane. The effects of pH, temperature, substrate concentration, and storage stability on free and immobilized invertase were investigated. Kinetic parameters were calculated as 18.2 mM for K_m and 6.43 × 10^?5 mol dm^?3 min^?1 for V_max of free enzyme and in the range of 23.8–35.3 mM for K_m and 7.97–11.71 × 10^?5 mol dm^?3 min^?1 for V_max of immobilized enzyme. After storage at 4°C for 1 month, the enzyme activities were 21.0 and 60.0–70.0% of the initial activity for free and immobilized enzyme, respectively. The optimum pH values for free and immobilized enzymes were determined as 4.5. The optimum temperatures for free and immobilized enzymes were 45 and 50°C, respectively. After using immobilized enzyme in 3 days for 43 times, it showed 76–80% of its original activity. As a result of immobilization, thermal and storage stabilities were increased. The aim of this study was to increase the storage stability and reuse number of the immobilized enzyme and also to compare this immobilization method with others with respect to storage stability and reuse number. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 93: 1526–1530, 2004     

Immobilization of a nonspecific chitosan hydrolytic enzyme for application in preparation of water‐soluble low‐molecular‐weight chitosan

A nonspecific chitosan hydrolytic enzyme, cellulase, was immobilized onto magnetic chitosan microspheres, which was prepared in a well spherical shape by the suspension crosslinking technique. The morphology characterization of the microspheres was carried out with scanning electron microscope and the homogeneity of the magnetic materials (Fe₃O₄) in the microspheres was determined from optical micrograph. Factors affecting the immobilization, and the properties and stabilities of the immobilized enzyme were studied. The optimum concentration of the crosslinker and cellulase solution for the immobilization was 4% (v/v) and 6 mg/mL, respectively. The immobilized enzyme had a broader pH range of high activity and the loss of the activity of immobilized cellulase was lower than that of the free cellulase at high temperatures. This immobilized cellulase has higher apparent Michaelis–Menten constant K_m (1.28 mg/mL) than that of free cellulase (0.78 mg/mL), and the maximum apparent initial catalytic rate V_max of immobilized cellulase (0.39 mg mL^?1 h^?1) was lower than free enzyme (0.48 mg mL^?1 h^?1). Storage stability was enhanced after immobilization. The residual activity of the immobilized enzyme was 78% of original after 10 batch hydrolytic cycles, and the morphology of carrier was not changed. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 101: 1334–1339, 2006     

Trametes versicolor laccase immobilized poly(glycidyl methacrylate) based cryogels for phenol degradation from aqueous media

This study aims removal of phenols in wastewater by enzymatic oxidation method. In this study, Trametes versicolor laccase was covalently immobilized onto a cryogel matrix by the nucleophilic attack of amino groups of laccase to epoxy groups of matrix. Glycidyl methacrylate was chosen as functional monomer to prepare poly(2‐hydroxyethyl methacrylate‐co‐glycidyl methacrylate) [p(HEMA‐co‐GMA)] cryogels. The enzyme immobilized matrix was characterized by FTIR, SEM, and swelling tests. The effect of pH, reaction time, temperature, substrate concentration, enzyme concentration, and storage period on immobilized enzyme activity was determined and compared with those of free enzyme. The model substrate was 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid (ABTS). Lineweaver‐Burk plots were used to calculate K_m and V_m values. K_m values were 165.1 and 156.0 µM while V_m values were 55.2 µM min^?1 and 1.57 µM min^?1 for free and immobilized laccase, respectively. Immobilized enzyme was determined to retain 82.5% and 72.0% of the original activity, respectively, after 6 consecutive use and storage period of 4 weeks. The free enzyme retained only 24.0% of its original activity following the same storage period. Lastly, decomposition products resulting from enzymatic oxidation of a model phenolic compound (3,5‐dinitrosalicylic acid) in aqueous solution were identified by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 41981.     

Cinnamic ester of D‐sorbitol for immobilization of mushroom tyrosinase

Mushroom tyrosinase was immobilized by adsorption onto the totally cinnamoylated derivative of D ‐sorbitol. The polymerization and cross‐linking of the derivative initially obtained was achieved by irradiation in the ultraviolet region, where this prepolymer shows maximum sensitivity. Immobilization of tyrosinase on this support involves a process of physical adsorption and intense hydrophobic interactions between the cinnamoyl groups of the support and related groups of the enzyme. The pH value, enzyme concentration and immobilization time were all important parameters affecting immobilization efficiency; also, enzyme immobilization efficiency correlated well with the tyrosinase isoelectric point. The immobilized enzyme showed an optimum measuring pH of 3.5 and greater activity at acid and neutral pH values than the soluble enzyme. The optimal reaction temperature was 35 °C and the temperature profile was broader than that of the free enzyme or of the enzyme immobilized on other supports. The apparent Michaelis constant of mushroom tyrosinase immobilized on the SOTCN derivative acting on 4‐tert‐butylcatechol (TBC) was 0.40 ± 0.02 mmol dm^?3, which was lower than for the soluble enzyme, suggesting that the affinity of this enzyme for this substrate was greater when immobilized than when in solution. Immobilization stabilized the enzyme and made it less susceptible to activity loss during storage at pH values in the range 4–5.5, and the suicide inactivation of the immobilized tyrosinase was null or negligible in a reaction medium with 4‐tert‐butylcatechol at a concentration of 0.4 mmol dm^?3. The results show that cinnamic carbohydrate esters of D ‐sorbitol are an appropriate support for tyrosinase immobilization and could be of use for several tyrosinase applications. Copyright © 2005 Society of Chemical Industry     

Covalent immobilization of trypsin onto thermo‐sensitive poly(N‐isopropylacrylamide‐co‐acrylic acid) microspheres with high activity and stability

Poly(N‐isopropylacrylamide‐co‐acrylic acid) (P(NIPAM‐co‐AA)) microspheres with a high copolymerized AA content were fabricated using rapid membrane emulsification technique. The uniform size, good hydrophilicity, and thermo sensitivity of the microspheres were favorable for trypsin immobilization. Trypsin molecules were immobilized onto the microspheres surfaces by covalent attachment. The effects of various parameters such as immobilization pH value, enzyme concentration, concentration of buffer solution, and immobilization time on protein loading amount and enzyme activity were systematically investigated. Under the optimum conditions, the protein loading was 493 ± 20 mg g^?1 and the activity yield of immobilized trypsin was 155% ± 3%. The maximum activity (V_max) and Michaelis constant (K_m) of immobilized enzyme were found to be 0.74 μM s^?1 and 0.54 mM, respectively. The immobilized trypsin showed better thermal and storage stability than the free trypsin. The enzyme‐immobilized microspheres with high protein loading amount still can show a thermo reversible phase transition behavior. The research could provide a strategy to immobilize enzyme for application in proteomics. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 43343.     

Nostoc calcicola Immobilized in Silica-coated Calcium Alginate and Silica Gel for Applications in Heavy Metal Biosorption

The present study reports the preparation and characterization of silica-based immobilization matrices for the purpose of metal accumulation using immobilized cyanobacterium Nostoc calcicola. Silica gel was prepared using aqueous sodium silicate and colloidal silica. Calcium alginate (CAG) beads were coated with silica using sodium silicate solutions. Microscopy observations and TTC tests confirmed that the immobilized cells were intact and viable. Ultrastructural studies with electron microscopy revealed a membrane thickness of approximately 10 μm around the CAG and the silica gel to be of mesoporous nature. BET surface area of silica gel-immobilized N. calcicola was 160 m² g^?1. The porous volume and average pore diameter were 0.40 cm³ g^?1 and ca. 100 Å, respectively, as calculated using the BJH model. Studies on silica-coated calcium alginate immobilized cells showed that these were superior to the uncoated CAG beads in terms of mechanical strength and metal accumulation. The silica matrices were found to be stable for repeated cycles of metal removal and with commonly used eluants for desorption processes. These matrices have potential applications in immobilization of industrially important biocatalysts.     

Production of L‐methionine by immobilized pellets of Aspergillus oryzae in a packed bed reactor

Production of L ‐methionine by immobilized pellets of Aspergillus oryzae in a packed bed reactor was investigated. Based on the determination of relative enzymatic activity in the immobilized pellets, the optimum pH and temperature for the resolution reaction were 8.0 and 60 °C, respectively. The effects of substrate concentration on the resolution reaction were also investigated and the kinetic constants (K_m and V_m) of immobilized pellets were found to be 7.99 mmol dm^?3 and 1.38 mmol dm^?3 h^?1, respectively. The maximum substrate concentration for the resolution reaction without inhibition was 0.2 mol dm^?3. The L ‐methionine conversion rate reached 94% and 78% when substrate concentrations were 0.2 and 0.4 mol dm^?3, respectively, at a flow rate of 7.5 cm³ h^?1 using the small‐scale packed bed reactor developed. The half‐life of the L ‐aminoacylase in immobilized pellets was 70 days in continuous operation. All the results obtained in this paper exhibit a practical potential of using immobilized pellets of Aspergillus oryzae in the production of L ‐methionine. © 2002 Society of Chemical Industry     

Xylitol production by liquid emulsion membrane encapsulated yeast cells

BACKGROUND: Liquid emulsion membrane (LEM)‐encapsulated live cells can be used to produce various products. This work reports on LEM‐encapsulated cells for producing xylitol and models the production process. RESULTS: Encapsulated cells of Candida mogii ATCC 18364 were used to produce xylitol from xylose. Soybean oil LEM consisting of 5% (w/v) lanolin and microwaxes was found most suitable for this process. The LEM‐encapsulated cells were immobilized in a tubular biocatalytic loop. Xylitol was produced under oxygen‐limited and aerobic conditions. Xylitol productivity and yield were 0.005 g L^?1 h^?1 and 0.52 g g^?1, respectively, for oxygen‐limited operation. Under aerobic conditions, xylitol productivity increased greatly to 0.022 g L^?1 h^?1, but yield on xylose declined to 0.49 g g^?1. A mathematical model successfully described substrate consumption and product formation in the LEM‐immobilized cell system. CONCLUSION: Potentially, immobilized cell LEM systems are useful for certain fermentations and they can be successfully modeled, as shown by the example of xylitol from xylose process. Copyright © 2009 Society of Chemical Industry     

Production of New Functionalized Polymer Nanoparticles and Use for Manufacture of Novel Nanobiocatalysts

New nanoparticles are synthesized through emulsion polymerization, using distinct comonomers (styrene, divinylbenzene, glycidyl methacrylate and pentafluorostyrene). Then, for the first time, two strategies are adopted to functionalize such nanoparticles using benzylamine and thiophenol: (i) after the manufacture of the nanoparticles; and (ii) in situ during the polymerization reaction. Afterwards, the functionalized nanoparticles are used as nanosupports for immobilization of lipase B from Candida antarctica and the performance of the novel nanobiocatalysts are evaluated. It is shown that the nanoparticles exhibit different properties (specific areas ranging from 34 m² g^?1 to 324 m² g^?1; and contact angles ranging from 29° to 126°), indicating that both procedures can be used to adjust the properties of the polymer supports. Moreover, the nanobiocatalysts are applied successfully in hydrolysis and esterification reactions, exhibiting higher activities than the non‐functionalized biocatalysts. It is also observed that more hydrophilic supports result in more active biocatalysts in hydrolysis (27 ± 1 U g^?1) and intermediate hydrophobic matrices conduct to more active biocatalysts in esterification reactions (1564 ± 50 U g^?1). It is shown that highly hydrophobic surfaces may cause a significant decrease in the activity of such biocatalysts, probably due to distortions on the enzyme active center and to more intense chemical partitioning effects.     

Application of chitosan‐entrapped β‐galactosidase in a packed‐bed reactor system

The model enzyme β‐galactosidase was entrapped in chitosan gel beads and tested for hydrolytic activity and its potential for application in a packed‐bed reactor. The chitosan beads had an enzyme entrapment efficiency of 59% and retained 56% of the enzyme activity of the free enzyme. The Michaelis constant (K_m) was 0.0086 and 0.011 μmol/mL for the free and immobilized enzymes, respectively. The maximum velocity of the reaction (V_max) was 285.7 and 55.25 μmol mL^?1 min^?1 for the free and immobilized enzymes, respectively. In pH stability tests, the immobilized enzyme exhibited a greater range of pH stability and shifted to include a more acidic pH optimum, compared to that of the free enzyme. A 2.54 × 16.51‐cm tubular reactor was constructed to hold 300 mL of chitosan‐immobilized enzyme. A full‐factorial test design was implemented to test the effect of substrate flow (20 and 100 mL/min), concentration (0.0015 and 0.003M), and repeated use of the test bed on efficiency of the system. Parameters were analyzed using repeated‐measures analysis of variance. Flow (p < 0.05) and concentration (p < 0.05) significantly affected substrate conversion, as did the interaction progressing from Run 1 to Run 2 on a bed (p < 0.05). Reactor stability tests indicated that the packed‐bed reactor continued to convert substrate for more than 12 h with a minimal reduction in conversion efficiency. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 91: 1294–1299, 2004     

Immobilization of glucoamylase on the plain and on the spacer arm‐attached poly(HEMA‐EGDMA) microspheres

Immobilization glucoamylase onto plain and a six‐carbon spacer arm (i.e., hexamethylene diamine, HMDA) attached poly(2‐hydroxyethylmethacrylate‐ethyleneglycol dimethacrylate) [poly(HEMA‐EGDMA] microspheres was studied. The microspheres were prepared by suspension polymerization and the spacer arm was attached covalently by the reaction of carbonyl groups of poly(HEMA‐EGDMA). Glucoamylase was then covalently immobilized either on the plain of microspheres via CNBr activation or on the spacer arm‐attached microspheres via CNBr activation and/or using carbodiimide (CDI) as a coupling agent. Incorporation of the spacer arm resulted an increase in the apparent activity of the immobilized enzyme with respect to enzyme immobilized on the plain of the microspheres. The activity yield of the immobilized glucoamylase on the spacer arm‐attached poly(HEMA‐EGDMA) microspheres was 63% for CDI coupling and 82% for CNBr coupling. This was 44% for the enzyme, which was immobilized on the plain of the unmodified poly(HEMA‐EGDMA) microspheres via CNBr coupling. The K_m values for the immobilized glucoamylase preparations (on the spacer arm‐attached microspheres) via CDI coupling 0.9% dextrin (w/v) and CNBr coupling 0.6% dextrin (w/v) were higher than that of the free enzyme 0.2% dextrin (w/v).The temperature profiles were broader for both immobilized preparations than that of the free enzyme. The operational inactivation rate constants (k_iop) of immobilized enzymes were found to be 1.42 × 10^?5 min^?1 for CNBr coupled and 3.23 × 10^?5 min^?1 for CDI coupled glucoamylase. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 81: 2702–2710, 2001     

Immobilization of catalases on amidoxime polyacrylonitrile nanofibrous membranes

Amidoxime polyacrylonitrile (AOPAN) nanofibrous membranes were generated by the reaction between electrospun polyacrylonitrile nanofibrous membranes and hydroxylamine hydrochloride. AOPAN nanofibrous membranes were further modified by Fe(III) chelation for immobilizing catalases with coordination bonds. The surface morphologies of the nanofibrous membranes and immobilized catalases were observed by field emission scanning electron microscopy. Chelation of Fe(III) onto AOPAN nanofibrous membranes was studied by the Langmuir isothermal adsorption model. It was found that the maximum amount of coordinated Fe(III) (q_m) was 4.5045 mmol g^?1 (dry nanofibrous membranes) and the binding constant (K_l) was 0.0698 L mmol^?1. The amounts of immobilized enzymes were determined by the method of Bradford. Kinetic parameters were analyzed for both immobilized and free catalases. The value of V_max (7122.6 µmol mg^?1 min^?1) for the immobilized catalases was smaller than that for the free catalases (9203.2 µmol mg^?1 min^?1), and the K_m for the immobilized catalases was larger. The immobilized catalases showed better resistance to pH and temperature change than the free catalases, and the storage stability of immobilized catalases was higher than that of free catalases. As for reusability, the immobilized catalases retained 71% of their activity after eight repeated uses. © 2012 Society of Chemical Industry     

Distribution of an enzyme in porous polymer beads

Trypsin has been immobilized by adsorption onto Amberlite XAD-7 beads. The Michaelis constant (K_m) of the enzyme was increased about sevenfold following the immobilization. Its rate of penetration into the porous beads was determined by staining the beads, which had been split, with naphthol blue black. The extent of diffusional rate limitation of immobilized trypsin was related to the penetration depth of the enzyme into the beads. This can be controlled by manipulating the conditions during the preparation of the immobilized enzyme.     

Flat-sheet and hollow fiber membrane bioreactors: A study of the kinetics and active site conformational changes of immobilized papain including sorption studies of reaction constituents

Papain, a sulfhydryl protease has been immobilized on flat-sheet modified polysulfone membranes and hydroxyethyl cellulose coated polyethersulfone hollow fibers. Amidase activity of the enzyme in solution on the membranes has been assayed. Immobilized papain on the modified polysulfone membrane and the hollow fibers retains 12% and 25% of its activity (with 1 mmol dm^?3 substrate) in solution, respectively. Loading experiments revealed decreased activity on the modified polysulfone membrane with increased enzyme loading. Adsorption experiments for the reaction product, p-nitroaniline, have been performed and an attempt has been made to correct for this in activity calculations. Apparent Michaelis–Menten parameters were determined for the modified polysulfone and hollow fibers with both K_m and V_max being lower in the immobilized cases. Electron paramagnetic resonance study of the changes in active site conformation of an enzyme on a hollow fiber membrane are reported for the first time. Experiments using the sulfhydryl-specific (1-oxyl-2,2,5,5-teetrahyl-Δ³-pyrroline-3-methyl)methanethiolsulfonate spin label depicted the presence of two subpopulations of immobilized papain on the hollow fibers, one of them active and one denatured.     

Immobilization of pepsin on an acrylamide/2‐hydroxyethyl methacrylate copolymer and its use in casein hydrolysis

Pepsin was immobilized through covalent bonding on a copolymer of acrylamide and 2‐hydroxyethyl methacrylate via the individual and simultaneous activation of both groups. The extent of enzyme coupling upon the activation of both the amino and hydroxyl groups of the copolymer resulted in a synergistic effect. However, the order of activation of the support was critical. The covalently bound enzyme retained more than 50% of its activity even after six cycles. The storage stability of the covalently bound enzyme was 60% after storage for 1 month, whereas the free enzyme lost all of its activity within 10 days of storage at 35°C. The Michaelis constant (K_m) and maximum reaction velocity (V_max) were 1.1 × 10^?6 and 0.87 for the free enzyme and 1.2 × 10^?6 and 0.98 for the covalently bound enzyme when the enzyme concentration was kept constant and the substrate concentration was varied. Similarly, K_m and V_max were 6.73 × 10^?11 and 0.47 for the free enzyme and 7.59 × 10^?11 and 0.545 for the covalently bound enzyme when the substrate concentration was kept constant and the enzyme concentration was varied; this indicated no conformational change during coupling, but the reaction was concentration‐dependent. The hydrolysis of casein was carried out with a fixed‐bed reactor (17 cm × 1 cm). Maximum hydrolysis (90%) was obtained at a 2 cm³/min flow rate at 35°C with a 1 mM casein solution. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 96: 1544–1549, 2005     

A novel method to prepare chitosan powder and its application in cellulase immobilization

In this study, chitosan microspheres and sponges with uniform spherical and porous morphologies were prepared by coiling the stretched chains of chitosan with addition of salt and choosing different kinds of organic solvents as evaporation solvents. Cellulase was immobilized to the support by a covalent method. The enzyme exhibited a considerable affinity to the support, and the protein loading of 145.5 mg g^?1 support was fairly high. The immobilized cellulase had a higher K_m than free cellulase and had better stability with respect to pH, thermal stability, reuses and storage stability than free cellulase. Copyright © 2005 Society of Chemical Industry     

Protease immobilization onto porous chitosan beads

Water-insoluble proteases were prepared by immobilizing papain, ficin, and bromelain onto the surface of porous chitosan beads with any length of spacer by covalently fixation. The activity of the immobilized proteases was found to be still high toward small ester substrate, N-benzyl-L -arginine ethyl ester (BAEE), but rather low toward casein, a high-molecular-weight substrate. The relative activity of the immobilized proteases with spacer gave an almost constant value for the substrate hydrolysis within the surface concentration region studied. The values of the Michaelis constant K_m and the maximum reaction velocity V_m for free and immobilized proteases on the porous chitosan beads are estimated. The apparent K_m values were larger for immobilized proteases than for the free ones, while V_m values were smaller for the immobilized proteases. The pH, thermal, and storage stability of the immobilized proteases were higher than those of the free ones. The initial enzymatic activity of the immobilized protease maintained almost unchanged without any elimination and inactivation of proteases, when the batch enzyme reaction was performed repeatedly, indicating the excellent durability.     

Immobilization of polyphenol oxidase on carboxymethylcellulose hydrogel beads: preparation and characterization

Carboxymethylcellulose (CMC) beads were prepared by a liquid curing method in the presence of trivalent ferric ions, and epicholorohydrin was covalently attached to the CMC beads. Polyphenol oxidase (PPO) was then covalently immobilized onto CMC beads. The enzyme loading was 603 µg g⁻¹ bead and the retained activity of the immobilized enzyme was found to be 44%. The K_m values were 0.65 and 0.87 mM for the free and the immobilized enzyme, and the V_max values were found to be 1890 and 760 U mg⁻¹ for the free and the immobilized enzyme, respectively. The optimum pH was 6.5 for the free and 7.0 for the immobilized enzyme. The optimum reaction temperature for the free enzyme was 40 °C and for the immobilized enzyme was 45 °C. Immobilization onto CMC hydrogel beads made PPO more stable to heat and storage, implying that the covalent immobilization imparted higher conformational stability to the enzyme. © 2000 Society of Chemical Industry