Detoxification of mercury by immobilized mercuric reductase

Mercuric reductase which originated from a recombinant Escherichia coli PWS1 was purified and immobilized on a chemically modified diatomaceous earth support. The mercury reduction kinetics, pH dependence, storage stability, and reusability of the immobilized enzyme were investigated. Four dyes were examined for their electron transfer efficiency with the soluble and bound mercuric reductase. Continuous mercury detoxification by the immobilized mercuric reductase was also performed in fixed‐bed processes. The effects of bed‐length, mercury loading rate, and electron donor on the performance of the fixed beds were assessed. Immobilized mercuric reductase exhibited substrate‐inhibition‐type kinetics with a maximal activity (1.2 nmol Hg mg⁻¹ protein s⁻¹) occurring at an initial Hg²⁺ concentration of 50 µmol dm⁻³. The optimal pH was 7.0 for the soluble and immobilized mercuric reductase, but the immobilized enzyme maintained higher relative activity for less favorable pH values. Immobilization of the enzyme appeared to significantly enhance its storage stability and reusability. Of four artificial electron donors tested, azure A (5 mmol dm⁻³) demonstrated the highest relative activity (78%) for soluble mercuric reductase. For the immobilized enzyme, neutral red (5 mmol dm⁻³) gave a relative activity of nearly 82%. With a fixed‐bed, the mercury‐reducing efficiency of using neutral red was only 30–40% of that obtained using NADPH. Fixed‐bed operations also showed that increased bed length facilitated mercury reduction rate, and the optimal performance of the beds was achieved at a flow rate of approximately 100–200 cm³ h⁻¹. © 1999 Society of Chemical Industry     

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     

Stability of horseradish peroxidase immobilized on different cinnamic carbohydrate esters

Four types of organic polymer were used to cover 3 mm diameter glass beads for subsequent use in immobilizing horseradish peroxidase (HRP). These supports were the totally cinnamoylated derivatives of D ‐glucosone (GSOCN), D ‐sorbitol (SOTCN), ethyl‐D ‐glucopyranoside (EGSCN) or inuline (INCN). In some assays, a partially cinnamoylated derivative, 2,3,4,6‐tetracinnamoyl‐D ‐glucopyranoside (GPSTCN) (obtained from the EGSCN derivative by hydrolysis in acidic medium) was used. Polymerization and cross‐linking of the derivatives obtained initially was carried out by irradiation in the ultraviolet region, where these polymers show the greatest degree of sensitivity. The enzyme was immobilized by adsorption to the support. Immobilized enzymatic activity varied with the incubation time used (2–21 h), depending on the chemical nature of the immobilization support. The affinity of HRP for H₂O₂ and 2,2′‐azinobis(3‐ethylbenzothiazolinesulfonic acid) (ABTS) was slightly lower in the case of the enzyme immobilized on the GSOCN, SOTCN and EGSCN derivatives than in the case of the soluble enzyme, as can be seen from the corresponding apparent kinetic constants ( and ). However, the affinity of HRP immobilized on the INCN derivative for these substrates was higher than that shown by the enzyme in solution. In turn, the enzyme immobilized on all the supports was more resistant than the soluble form to inactivation by H₂O₂ and by heat at neutral pH. When the stability and durability of the immobilized HRP were analysed, the cinnamoylated derivatives prepared were seen to function as suitable supports for immobilizing peroxidase and to be suitable for industrial application of the enzyme. Copyright © 2004 Society of Chemical Industry     

Stabilized and Immobilized Bacillus subtilis Arginase for the Biobased Production of Nitrogen‐Containing Chemicals

L ‐Ornithine could serve as an intermediate in the biobased production of 1,4‐diaminobutane from L ‐arginine. Using the concept of biorefinery, L ‐arginine could become widely available from biomass waste streams via the nitrogen storage polypeptide cyanophycin. Selective hydrolysis of L ‐arginine to L ‐ornithine is difficult to perform chemically, therefore the stabilization and immobilization of Bacillus subtilis arginase (EC 3.5.3.1) was studied in a continuously stirred membrane reactor system. Initial pH of the substrate solution, addition of L ‐aspartic acid and reducing agents all appeared to have an effect on the operational stability of B. subtilis arginase. A remarkably good operational stability (total turnover number, TTN=1.13⋅10⁸) at the pH of arginine free base (pH 11.0) was observed, which was further improved with the addition of sodium dithionite to the substrate solution (TTN>1⋅10⁹). B. subtilis arginase was successfully immobilized on three commercially available epoxy‐activated supports. Immobilization on Sepabeads EC‐EP was most promising, resulting in a recovered activity of 75% and enhanced thermostability. In conclusion, the stabilization and immobilization of B. subtilis arginase has opened up possibilities for its application in the biobased production of nitrogen‐containing chemicals as an alternative to the petrochemical production.     

Towards the design of non-amyloidogenic proteins

Glucose oxidase was immobilized onto poly(2-hydroxyethyl methacrylate) (pHEMA) membranes by two methods: by covalent bonding through epichlorohydrin and by entrapment between pHEMA membranes. The highest immobilization efficiency was found to be 17.4% and 93.7% for the covalent bonding and entrapment, respectively. The K_m values were 5.9 mmol dm^?3, 8.8 mmol dm^?3 and 12.4 mmol dm^?3 for free, bound and entrapped enzyme, respectively. The V_max values were 0.071 mmol dm^?3 min^?1, 0.067 mmol dm^?3 min^?1 and 0.056 mmol dm^?3 min^?1 for free, bound and entrapped enzyme. When the medium was saturated with oxygen, K_m was not significantly altered but V_max was. The optimum pH values for the free, covalently-bound and entrapped enzyme were determined to be 5, 6, and 7, respectively. The optimum temperature was 30°C for free or covalently-bound enzyme but 35°C for entrapped enzyme. The deactivation constant for bound enzyme was determined as 1.7 × 10^?4 min^?1 and 6.9 × 10^?4 min^?1 for the entrapped enzyme.     

Effect of water‐miscible solvents on the organic solvent‐resistant tyrosinase from Streptomyces sp. REN‐21

The effect of various water‐miscible organic solvents (ethanol, methanol, acetone, acetonitrile, N,N‐dimethylformamide (DMF) and dimethylsulfoxide (DMSO)) on the kinetics of 4‐tert‐butylcatechol (tBC) oxidation in the presence of different samples of organic solvent‐resistant tyrosinase (OSRT) has been studied. In contrast to mushroom tyrosinase the enzyme shows a high relative stability in solutions of organic solvents and increased activity toward the bulky and hydrophobic substrate, tBC, in respect to catechol. Rates of the studied OSRT‐catalyzed reactions are however reduced by the presence of organic solvents and for all studied samples of OSRT decrease exponentially with the content of an organic solvent. The effect has been satisfactorily described by the effect of organic solvents on the thermodynamic activity of tBC. The correlation of the inhibition parameters with the hydrophobicity of a particular solvent (log P), its intrinsic molar volume, V_i, and the Dimroth–Reichardt parameter, E_T(30), are shown. The results allow also the prediction of OSRT activity in aqueous solutions of water‐miscible organic solvents. Copyright © 2003 Society of Chemical Industry     

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 Pycnoporus sanguineus laccase by metal affinity adsorption on magnetic chelator particles

BACKGROUND: Immobilized enzymes provide many advantages over free enzymes including repeated or continuous reuse, easy separation of the product from reaction media, easy recovery of the enzyme, and improvement in enzyme stability. In order to improve catalytic activity of laccase and increase its industrial application, there is great interest in developing novel technologies on laccase immobilization. RESULTS: Magnetic Cu²⁺‐chelated particles, prepared by cerium‐initiated graft polymerization of tentacle‐type polymer chains with iminodiacetic acid (IDA) as chelating ligand, were employed for Pycnoporus sanguineus laccase immobilization. The particles showed an obvious high adsorption capacity of laccase (94.1 mg g^?1 support) with an activity recovery of 68.0% after immobilization. The laccase exhibited improved stability in reaction conditions over a broad temperature range between 45 °C and 70 °C and an optimal pH value of 3.0 after being adsorbed on the magnetic metal‐chelated particles. The value of the Michaelis constant (K_m) of the immobilized laccase (1.597 mmol L^?1) was higher than that of the free one (0.761 mmol L^?1), whereas the maximum velocity (V_max) was lower for the adsorbed laccase. Storage stability and temperature endurance of the immobilized laccase were found to increase greatly, and the immobilized laccase retained 87.8% of its initial activity after 10 successive batch reactions. CONCLUSION: The immobilized laccase not only can be operated magnetically, but also exhibits remarkably improved catalytic capacity and stability properties for various parameters, such as pH, temperature, reuse, and storage time, which can provide economic advantages for large‐scale biotechnological applications of laccase. Copyright © 2007 Society of Chemical Industry     

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     

Immobilization of laccase on itaconic acid grafted and Cu(II) ion chelated chitosan membrane for bioremediation of hazardous materials

BACKGROUND: Chitosan membranes were formed through a phase inversion technique and then cross‐linked with epichlorohydrin (CHX). Heterogeneous graft copolymerization of itaconic acid (IA) onto membrane was carried out with different monomer concentrations (CHX‐g‐p(IA)). The membrane properties such as equilibrium swelling ratio, porosity, and contact angle were measured, together with analysis by scanning electron microscopy (SEM), energy dispersive analysis of X‐rays (EDAX), atomic force microscopy (AFM), and Fourier transform infrared (FTIR) spectroscopy. RESULTS: The Cu(II) ion incorporated membranes (i.e. CHX‐g‐p(IA)‐Cu(II)) were used for reversible immobilization of laccase using CHX‐g‐p(IA) membrane as a control system. Maximum laccase adsorption capacities of the CHX‐g‐p(IA) and CHX‐g‐p(IA)‐Cu(II) membranes (with 9.7% grafting yield) were found to be 6.3 and 17.6 mg mL^?1 membrane at pH 4.0 and 6.0, respectively. The K_m value for immobilized laccase on CHX‐g‐p(IA)‐Cu(II) (4.16 × 10^?2 mmol L^?1) was 2.11‐fold higher than that of free enzyme (1.97 × 10^?2 mmol L^?1). Finally, the immobilized laccase was used in a batch system for degradation of three different dyes (Reactive Black 5, RB5; Cibacron Blue F3GA, CB; and Methyl Orange, MO). The immobilized laccase on CHX‐g‐p(IA)‐Cu(II) membrane was more effective for removal of MO dye than removal of CB and RB5 dyes. CONCLUSION: Flexibility of the enzyme immobilized grafted polymer chains is expected to provide easy reaction conditions without diffusion limitation for substrate dye molecules and their products. The support described, prepared from green chemicals, can be used for the immobilization of industrially important enzymes. Copyright © 2012 Society of Chemical Industry     

Tyrosinase-glucose dehydrogenase substrate-recycling biosensor: A highly-sensitive measurement of phenolic compounds

Mushroom tyrosinase and glucose dehydrogenase from Acinetobacter calcoaceticus were immobilized in poly(vinyl)alcohol membranes and coupled with a Clark-type oxygen electrode to give a substrate (analyte) regenerating cycle for monitoring of nanomolar concentrations of phenolic compounds. In this way the response for catechol, phenol, p-cresol, p-chlorophenol and p-acetamidophenol was amplified by a factor of 450, 300, 240, 150, and 140, respectively. The resulting detection limit for catechol and phenol is 0·6 nmol dm⁻³ and 0·9 nmol dm⁻³, respectively. The measuring linear range for phenol obtained by the amplified electrode extends from 1 to 400 nmol dm⁻³. The comparison with the chemical (ascorbic acid) regeneration of the phenolic compounds demonstrates the efficiency of the enzymatic procedure. The biosensor can be used for monitoring of phenolic compounds in environmental or industrial samples.     

Immobilization of α-amylase on zirconia: A heterogeneous biocatalyst for starch hydrolysis

α-Amylase was immobilized on zirconia via adsorption. The support and the immobilized enzymes were characterized using XRD, IR spectra and N₂ adsorption studies. The efficiency of immobilized enzymes for starch hydrolysis was tested in a batch reactor. The effect of calcination temperatures on properties of the support as well as upon immobilization was studied. From XRD, IR and N₂ adsorption studies it was confirmed that the enzyme was adsorbed on the external surface of the support. pH, buffer concentration and substrate concentration had a significant influence on the activity of immobilized enzyme. Immobilization improved the pH stability of the enzyme. The Michaelis–Menten kinetic constants were calculated from Hanes–Woolf plot. K_m for immobilized systems was higher than the free enzyme indicating a decreased affinity by the enzyme for its substrate, which may be due to interparticle diffusional mass transfer restrictions.     

Invertase immobilized on spacer‐arm attached poly(hydroxyethyl methacrylate) membrane: Preparation and properties

Microporous poly(2‐hydroxyethyl methacrylate) (pHEMA) membrane was prepared by UV‐initiated photopolymerization. The spacer arm (i.e., hexamethylene diamine) was attached covalently and then invertase was immobilized by the condensation reaction of the amino groups of the spacer arm with carboxyl groups of the enzyme in the presence of carbodiimides. The values of the Michael's constant K_m of invertase were significantly larger (ca. 2.5 times) upon immobilization, indicating decreased affinity by the enzyme for its substrate, whereas V_max was smaller for the immobilized invertase. Immobilization improved the pH stability of the enzyme as well as its temperature stability. Thermal stability was found to increase with immobilization and at 70°C the half times for the activity decay were 12 min for the free enzyme and 41 min for the immobilized enzyme. The immobilized enzyme activity was found to be quite stable in repeated experiments. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 75: 1685–1692, 2000     

Efficient Immobilization of Yeast Transketolase on Layered Double Hydroxides and Application for Ketose Synthesis

Transketolase (TK) from S. cerevisiae was successfully immobilized on layered double hydroxides (LDH) using simple, affordable and efficient adsorption and coprecipitation based immobilization procedures. Optimization of the preparation was performed using zinc aluminium nitrate (Zn₂Al‐NO₃) and magnesium aluminium nitrate (Mg₂Al‐NO₃) LDH as immobilization supports, and the protein‐to‐LDH weight ratio (Q) was varied. The highest immobilization yields (98–99%) and highest relative specific activities (4.2–4.4 U⋅mg⁻¹ for the immobilized enzyme compared to 4.5 U⋅mg⁻¹ for the free enzyme) were both achieved when using a protein‐to‐LDH weight ratio (Q) of 0.38. Efficient lyophilization of the LDH‐TK bionanocomposites thus synthesized was proven to allow easy use and storage of the supported TK with no significant loss of activity over a three‐month period. The kinetic parameters of the LDH‐TK enzyme were comparable to those of the free TK. The LDH‐TK enzyme was finally tested for the synthesis of L ‐erythrulose starting from hydroxypyruvate lithium salt (Li‐HPA) and glycolaldehyde (GA) as substrates. L ‐erythrulose was characterized and obtained with an isolated yield of 56% similar to that obtained with free TK. The reusability of the LDH‐TK biohybrid material was then investigated, and we found no loss of enzymatic activity over six cycles.     

Chitosaneous hydrogel beads for immobilizing neutral protease for application in the preparation of low molecular weight chitosan and chito‐oligomers

Enzyme hydrolysis with immobilized neutral protease was carried out to produce low molecular weight chitosan (LMWC) and chito‐oligomers. Neutral protease was immobilized on (CS), carboxymethyl chitosan (CMCS), and N‐succinyl chitosan (NSCS) hydrogel beads. The properties of free and immobilized neutral proteases on chitosaneous hydrogel beads were investigated and compared. Immobilization enhanced enzyme stability against changes in pH and temperature. When the three different enzyme supports were compared, the neutral protease immobilized on CS hydrogel beads had the highest thermal stability and storage stability, and the enzyme immobilized on NSCS hydrogel beads had the highest activity compared to those immobilized on the other supports, despite its lower protein loading. Immobilized neutral protease on all the three supports had a higher K_m (Michaelis‐Menten constant) than free enzyme. The V_max (maximum reaction velocity) value of neutral protease immobilized on CS hydrogel beads was lower than the free enzyme, whereas the V_max values of enzyme immobilized on CMCS and NSCS hydrogel beads were higher than that of the free enzyme. Immobilized neutral protease on CS, CMCS, and NSCS hydrogel beads retained 70.4, 78.2, and 82.5% of its initial activity after 10 batch hydrolytic cycles. The activation energy decreased for the immobilization of neutral protease on chitosaneous hydrogel beads. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 101: 3743–3750, 2006     

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.     

Synthesis and characterization of PMMA composites activated with starch for immobilization of L‐asparaginase

Poly (methyl methacrylate) (PMMA)–starch composites were prepared by emulsion polymerization technique for L‐asparaginase (L‐ASNase) immobilization as highly activated support. The hydroxide groups on the prepared composites offer a very simple, mild and firm combination for enzyme immobilization. The pure PMMA and PMMA‐starch composites were characterized as structural, thermal and morphological. PMMA‐starch composites were found to have better thermal stability and more hydrophilic character than pure PMMA. L‐ASNase was immobilized onto PMMA‐starch composites contained the different ratio of starch (1, 3, 5, and 10 wt %). Immobilized L‐ASNase showed better performance as compared to the native enzyme in terms of thermal stability and pH. K_m value of immobilized enzyme decreased approximately eightfold compared with the native enzyme. In addition to, immobilized L‐ASNase was found to retain 60% of activity after 1‐month storage period at 4 °C. Therefore, PMMA‐starch composites can be provided more advantageous in terms of enzymatic affinity, thermal, pH and storage stability as L‐ASNase immobilization matrix. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 43421.     

Removal of alkylphenols by the combined use of tyrosinase immobilized on ion‐exchange resins and chitosan beads

Mushroom tyrosinase was covalently immobilized on a poly(acrylic acid)‐type, weakly acidic cation‐exchange resin (Daiaion WK10, Mitsubishi Chemical Corp., Tokyo, Japan) with 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide hydrochloride salt as a water‐soluble carbodiimide. Ion‐exchange resins immobilized with tyrosinase were packed in one column, and crosslinked chitosan beads prepared with epichlorohydrin were packed in another column. The enzymatic activity was modified by covalent immobilization, and the immobilized tyrosinase had a high activity in the temperature range of 30–45°C and in the pH range of 7–10. When solutions of various alkylphenols were circulated through the two columns packed with tyrosinase‐immobilized ion‐exchange resins and crosslinked chitosan beads at 45°C and pH 7 (the optimum conditions determined for p‐cresol), alkylphenols were effectively removed through quinone oxidation with immobilized tyrosinase and subsequent quinone adsorption on chitosan beads. The use of chemically crosslinked chitosan beads in place of commercially available chitosan beads was effective in removing alkylphenols from aqueous solutions in shorter treatment times. The removal efficiency increased with an increase in the amount of crosslinked chitosan beads packed in the column because the rate of quinone adsorption became higher than the rate of enzymatic quinone generation. The activity of tyrosinase was iteratively used by covalent immobilization on ion‐exchange resins. One of the most important findings obtained in this study is the fact that linear and branched alkylphenols suspected of weak endocrine‐disrupting effects were effectively removed from aqueous solutions. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2010     

Removal of aqueous phenolic compounds from a model system by oxidative polymerization with turnip (Brassica napus L var purple top white globe) peroxidase

Turnip roots, which are readily available in Mexico, are a good source of peroxidase, and because of their kinetic and biochemical properties have a high potential as an economic alternative to horseradish peroxidase (HRP). The efficiency of using turnip peroxidase (TP) to remove several different phenolic compounds as water‐insoluble polymers from synthetic wastewater was investigated. The phenol derivatives studied included phenol, 2‐chlorophenol, 3‐chlorophenol, o‐cresol, m‐cresol, 2,4‐dichlorophenol and bisphenol‐A. The effect of pH, substrate concentration, amount of enzyme activity, reaction time and added polyethylene glycol (PEG) was investigated in order to optimize reaction conditions. A removal efficiency ≥85% was achieved for 0.5 mmol dm^?3 phenol derivatives at pH values between 4 and 8, after a contact time of 3 h at 25 °C with 1.28 U dm^?3 of TP and 0.8 mmol dm^?3 H₂O₂. Addition of PEG (100–200 mg dm^?3) significantly reduced the reaction time required (to 10 min) to obtain >95% removal efficiency and up to 230% increase in remaining TP activity. A relatively low enzyme activity (0.228 U dm^?3) was required to remove >95% of three phenolic solutions in the presence of 100–200 mg dm^?3 PEG. TP showed efficient and fast removal of aromatic compounds from synthetic wastewaters in the presence of hydrogen peroxide and PEG. These results demonstrate that TP has good potential for the treatment of phenolic‐contaminated solutions. © 2002 Society of Chemical Industry     

New generation polymeric nanospheres for catalase immobilization

Monosize poly(2‐hydroxyethyl methacrylate‐co‐N‐methacryloly‐L ‐histidinemethylester) [mon‐poly(HEMA‐MAH)] nanospheres were prepared via surfactant‐free emulsion polymerization method. L ‐Histidine groups of the mon‐poly(HEMA‐MAH) nanospheres were chelated with Fe³⁺ ions. Mon‐poly(HEMA‐MAH) nanospheres were characterized by Fourier transform infrared spectroscopy, proton NMR, and scanning electron microscopy. Particle size of the mon‐poly(HEMA‐MAH) nanospheres was measured by Zeta Sizer. Elemental analysis of MAH for nitrogen was estimated as 0.94 mmol/g polymer. The catalase immobilized onto the mon‐poly(HEMA‐MAH)–Fe³⁺ nanospheres resulted in increasing the enzyme stability with time. Optimum operational temperature for both immobilized preparations was the same, and the temperature profiles of the immobilized preparations were significantly broader. It was observed that enzyme could be repeatedly adsorbed and desorbed on the mon‐poly(HEMA‐MAH)–Fe³⁺ nanospheres without loss of adsorption capacity or enzymic activity. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009