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 catalase in poly(isopropylacrylamide-co-hydroxyethylmethacrylate) thermally reversible hydrogels

Catalase was entrapped in thermally reversible poly(isopropylacrylamide-co-hydroxyethylmethacrylate) (pNIPAM/HEMA) copolymer hydrogels. The thermoresponsive hydrogels, in cylindrical geometry, were prepared in an aqueous buffer by redox polymerization. It was observed that upon entrapment, the activity retention of catalase was decreased between 47 and 14%, and that increasing the catalase loading of hydrogel adversely affected the activity. The kinetic behaviour of the entrapped enzyme was investigated in a batch reactor. The apparent kinetic constant of the entrapped enzyme was determined by the application of Michaelis–Menten model and indicated that the overall reaction rate was controlled by the substrate diffusion rate through the hydrogel matrix. Due to the thermoresponsive character of the hydrogel matrix, the maximum activity was achieved at 25 °C with the immobilized enzyme. The K_m value for immobilized catalase (28.6 mM) was higher than that of free enzyme (16.5 mM). Optimum pH was the same for both free and immobilized enzyme. Operational, thermal and storage stabilities of the enzyme were found to increase with immobilization. © 1999 Society of Chemical Industry     

Enzyme immobilization: Part 1. Modified bentonite as a new and efficient support for immobilization of Candida rugosa lipase

Immobilization of Candida rugosa lipase onto modified and unmodified bentonites is described. The effect of hydrophilic or hydrophobic nature of the support, the reuse efficiency, and kinetic behavior of immobilized lipase were studied. The modified bentonite with monolayer surfactant (BMS), was the best support, for immobilization. The activity of the immobilized enzyme was examined under varying experimental conditions. The effect of various factors such as concentration of enzyme solution, pH and temperature, stirring and various thermodynamic parameters were also evaluated. The activity of lipase on Na-bentonite, on BMS and on bentonite with bilayer surfactant (BBS) at the optimum pH was 7.2%, 56.6% and 3.6%, respectively. The adsorption isotherm was modelled by the Langmuir equation. The amounts of immobilized lipase on Na-bentonite, BMS and BBS at the highest activity were 42.6%, 61.2% and 28.3%, respectively. The effect of substrate concentration on enzymatic activity of the free and immobilized enzymes showed a good fit to the Michaelis–Menten plots. The immobilized enzyme exhibited an activity comparable to the free enzyme after storage at 30 °C. The thermal stability of free and immobilized lipase were also studied.     

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.     

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     

Preparation of glucose oxidase immobilized in different carriers using radiation polymerization

Glucose oxidase (EC 1.1.3.4) was immobilized on different polymeric materials using different immobilization techniques (entrapping by γ‐irradiation, and covalent binding using epichlorohydrin). Studies were carried out to increase the thermal stability of glucose oxidase (GOD) for different applications. The activity and stability of the resulting biopolymers have been compared with those of free GOD. The effect of different polyvinyl alcohol/polyacrylamide (PVA/PAAm) compositions of the copolymer carrier on the enzymatic activity of the immobilized GOD was studied. The maximum enzymatic activity was obtained with the composition ratio of PVA/PAAm of 60:40. The behaviour of the free and immobilized enzyme was analysed as a function of pH. A broadening in the pH profile (5.5–8) was observed for immobilized preparations. The activity and stability of the resulting biopolymers produced by immobilization of GOD onto different carriers have been compared, in both aqueous and organic media, with those of the free GOD. The enzyme's tolerance toward both heat and organic solvent was enhanced by immobilization onto polymers. The addition of different concentrations of organic solvents (10–50%, v/v) to the enzyme at higher temperature (60 °C) was found to stabilize the enzyme molecule. The strongest stabilizing effect on the enzymatic activity was achieved at a concentration of 10%. Copyright © 2005 Society of Chemical Industry     

Covalent immobilization of penicillin G acylase onto amine-functionalized PVC membranes for 6-APA production from penicillin hydrolysis process. II. Enzyme immobilization and characterization

This article describes the covalent immobilization of penicillin G acylase (PGA) onto glutaraldehyde-activated NH₂-PVC membranes. The immobilized enzyme was used for 6-aminopenicillanic acid production from penicillin hydrolysis. Parameters affecting the immobilization process, which affecting the catalytic activity of the immobilized enzyme, such as enzyme concentration, immobilization's time and temperature were investigated. Enzyme concentration and immobilization's time were found of determine effect. Higher activity was obtained through performing enzyme immobilization at room temperature. Both optimum temperature (35°C) and pH (8.0) of immobilized enzyme have not been altered upon immobilization. However, immobilized enzyme acquires stability against changes in the substrate's pH and temperature values especially in the higher temperature region and lower pH region. The residual relative activities after incubation at 60°C were more than 75% compared to 45% for free enzyme and above 50% compared to 20% for free enzyme after incubation at pH 4.5. The apparent kinetic parameters K_M and V_M were determined. K_M of the immobilized PGA (125.8 mM) was higher than that of the free enzyme (5.4 mM), indicating a lower substrate affinity of the immobilized PGA. Operational stability for immobilized PGA was monitored over 21 repeated cycles. The catalytic membranes were retained up to 40% of its initial activity after 10.5 working h. © 2012 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

醇脱氢酶在聚乙烯膜表面的固定化研究

以聚丙烯酸（PAA）改性的聚乙烯（PE）膜为载体,研究了醇脱氢酶（ADH）的两种固定化路线,并以甲醛为底物考察了固定化酶的催化性能。路线1用聚乙烯亚胺（PEI）进一步改性,使用戊二醛（GA）固定化ADH。最优固定化pH为6.0,温度为5～15℃,酶浓度为1.0 mg/ml,GA浓度为0.01%(质量);固定化酶的最适反应pH为6.5,温度为15～30℃,反应速率最高为9.6 μmol/(L·min);重复利用10次后可保持47.3%的活性。路线2以PAA-PE为载体,用1-(3-二甲氨基丙基)-2-乙基碳二亚胺盐酸盐（EDC）和N-羟基琥珀酰亚胺（NHS）为活化剂,固定化ADH。EDC和NHS最优摩尔比为1∶0.5,固定化时间为24 h;固定化酶的最适反应pH为6.5,温度为20～37℃,反应速率为15.58 μmol/(L·min);重复利用10次后可保持53.8%的活性。     

Immobilization of alcohol dehydrogenase on the surface of polyethylene membrane

Using polyacrylic acid (PAA) modified polyethylene (PE) membrane as a carrier, two immobilization routes of alcohol dehydrogenase (ADH) were studied, and the catalytic performance of immobilized enzyme was investigated using formaldehyde as a substrate. In the first route, PAA-PE membrane was further modified by polyethyleneimine (PEI) and then ADH was covalently linked by glutaraldehyde (GA) to the surface of PEI/PAA-PE. The results show that the optimal immobilization pH was 6.0, immobilization temperature was 5—15℃, ADH and GA concentrations were 1.0mg/ml and 0.01%(mass). For immobilized enzyme, the optimal reaction pH was 6.5, temperature was 15—30℃, and the highest reaction rate was 9.6 μmol/(L·min), the remaining activity was 47.3% after 10 use cycles. In the second route, ADH was immobilized on PAA-PE membrane with 1-(3-dimethylaminopropyl)-2-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) as activators. The results show that the optimal molar ratio of EDC and NHS was 1∶0.5, and the immobilization time was 24 h. For immobilized enzyme, the optimal reaction pH was 6.5, temperature was 20—37℃, and the highest reaction rate was 15.58 μmol/(L·min), 53.8% activity was remained after 10 cycles.     

A new approach for describing and solving the reversible Briggs-Haldane mechanism using immobilized enzyme

Recently immobilized enzymes have been widely used in industrial processes due to their outstanding advantages, such as high stability and recyclability; however, their kinetic behaviour is generally controlled by mass diffusion effects. Thus, in order to improve these enzymatic processes, a clear discernment between the kinetic and diffusion mechanisms that control the production of the metabolite require investigation. In practice, it is typical to establish apparent kinetics for immobilized enzyme operations, and the validity of the apparent kinetics is restricted to the studied cases. In this work, a new approach for mathematically describing the kinetic and diffusion mechanics in an immobilized biocatalyst bead is established, in which the fraction of residual enzymatic activity is included, and is defined as a measure of the active and available enzymes in the bead porous network. In addition, the diffusion and kinetic mechanisms are described by the effective diffusion coefficient and the free enzyme kinetics, since the porous network of the bead is assumed as the bioreaction volume. Therefore, free enzyme kinetics were determined from glucose to fructose bioconversion using a stirred tank reactor with free glucose-isomerase, in which substrate and enzyme concentrations and temperature were varied. The fraction of residual enzymatic activity () and the effective diffusion coefficient () were obtained from the isomerization of glucose to fructose using a stirred tank reactor with immobilized glucose-isomerase in calcium alginate beads at different substrate and enzyme concentrations. Finally, simulations were carried out to establish the bioreaction solid-phase characteristics that most significantly influence productivity.     

Diffusion with simultaneous reaction of reactive dyes in cellulose

The theoretical equations which described the concentration profile of immobilized species of reactive dyes in the substrate were derived from the diffusion equation with the chemical reaction of first-order or pseudofirst-order. The theoretical profiles in the substrate described by the equations were discussed. The larger the diffusion coefficient D of active species and the smaller the reaction rate constant k, the deeper is the penetration of the immobilized and active species of reactive dyes into the substrate. The method of estimating D and k from the diffusion profiles of both species obtained by means of the method of the cylindrical cellophane film roll was described. The diffusion coefficients of the hydrolyzed species of C.I. Reactive Orange 1 and Red 1 were nearly constant in all the pH values examined. The concentration profiles of both the species of Orange 1 at pH 8.8 were identical with the theoretical ones; while the profiles of immobilized species of Red 1 at pH 10 and of Orange 1 at pH 12 agreed with the theoretical ones and those of active species did not because of the hydrolysis. The diffusion coefficients of active species of these dyes at these pH ranges were smaller than those of the hydrolyzed species.     

Immobilization strategy of accessible transmission for trypsin to catalyze synthesis of dipeptide in mesoporous support

Immobilized trypsin in mesoporous silica foams was used to catalyze dipeptide synthesis in hydrophilic organic solvent instead of soluble form. The area surface of nano support was measured. The catalytic activity, coupled yield and kinetic characterization of immobilized trypsin were examined. Bz-Arg-OEt was chosen as the acyl donor with Lys-OH as the nucleophile. The trypsin-catalyzed synthesis condition was optimized, such as catalytic temperature, pH, reaction time, physical properties and content of organic solvents, together with the added enzyme amount. The immobilized trypsin showed 112.8% of residual activity with 91.9% of coupled yield, and the kinetic parameters exhibited accessibility for transmission. The product yield of 5.8% was reached at the optimum conditions for enzymatic synthesis of dipeptide: 800 mg of wet immobilized trypsin (200 mg/g support) was used in Tris-HCl buffer (0.1 mol/L, pH 8.0) containing 80% (v/v) ethanol solvents for 6 h of reaction time at 35 °C. This attempt of immobilized strategy for trypsin in nanopores renders the possibility of wide application of inorganic nano-sized support in catalytic synthesis process, which can avoid usage of large amounts of organic solvents in washing steps by chemical methods and reduce the tedious purification process of its soluble form.     

pH-stat methodology in continuous monitoring of the kinetics of hydrolysis of phosphate esters catalysed by alkaline phosphatase from human placenta: II. Kinetic aspects

The adaptation of the pH-stat to continuous monitoring of the in-vitro hydrolase activity of alkaline phosphatase in solution, an activity which until now has only been analysed by means of spectrophotometric methods in a continuous or static state, is described. To control the reliability of the method in the monitoring of these enzymic reactions, a series of kinetics of hydrolysis of chromogenic (p-nitrophenyl and o-carboxyphenyl phosphates) and non-chromogenic (ATP) substrates was carried out, comparing the results obtained via continuous spectrophotometric analysis of the corresponding phenol released or via a discontinuous technique by the phosphate produced with those results obtained via pH-stat analysis of the H⁺ released or utilized by the enzymic reaction of hydrolysis. It can be concluded that kinetic studies carried out on the pH-stat of in-vitro alkaline phosphatase activity offer results analogous to those obtained for the same system through classic spectrophotometric methods, offering as well notable advantages of speed and simplicity in the kinetic assays since it is always possible to monitor the enzymic activity continuously, with chromogenic or non-chromogenic substrates. The pH-stat methodology affords additional information on the kinetics of action of alkaline phosphatase from placenta acting on non-chromogenic biological substrates. In this sense, kinetic studies with the pH-stat on the enzymic hydrolysis of ATP were begun, determining their rate versus pH profile (optimum pH at 9.1) and proposing possible ‘non-Michaelian’ type kinetic behaviour of the enzyme in solution as deduced from the graphical analysis of the data v([ATP]).     

UV-curable methacrylated/fumaric acid modified epoxy as a potential support for enzyme immobilization

The immobilization procedure of UV-curing coating is simple and causes less loss of enzymatic activity. UV-curable methacrylated/fumaric acid modified cycloaliphatic epoxide is here proposed as a rigid support material for covalent immobilization of α-amylase. The immobilized enzyme is analyzed in terms of bioactivity retention as a function of repeated use ability, pH, storage, as well as stability under various experimental conditions, taking starch as a substrate. The properties of immobilized enzyme were also compared with those of the free enzyme. The highest activity of free enzyme was obtained at pH 7.0 while this value was shifted to pH 7.5 for immobilized system. Optimum catalytic activity was observed at 30 °C, for both free and immobilized enzyme; however, the immobilized enzyme had a higher activity than the free one. The immobilized enzyme that was used 35 times in 8 h in repeated batch experiments demonstrated that about 73% of the enzyme activity was retained. The free enzyme lost all its activity with in 15 days. The retained activity of immobilized enzyme was found to be around 80%. The amount of bound α-amylase was found 94 mg per gram polymeric support material.     

Immobilization of <Emphasis Type="Italic">α</Emphasis>-amylase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> on developed support using microbial transglutaminase

α-amylase from Bacillus licheniformis was successfully immobilized on developed support, which was prepared by coating a chitosan-casein film on silica, at 20 °C, pH 6.0 for 5 hr with microbial transglutaminase (MTG) as the cross-linking factor. The optimal support was obtained when 1% chitosan and 1% casein were used in the coating mixture. The optimal condition for immobilization catalyzed by MTG was confined to be at MTG concentration of 15 U/mL, pH 6.0, reacting for 6 hr at 20 °C. The highest specific activity of immobilized α-amylase was achieved as 236 U/g. After immobilization, the obtained enzyme showed broader pH profile and maintained more than 70% of the original activity after 20 reuses.     

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 α-Amylase to Temperature-Responsive Polymers by Single or Multiple Point Attachments

Temperature-responsive N-isopropylacrylamide (NIPAAm) polymer (PNIPAAm) with a free carboxyl functional end group and a copolymer (NIPNAS) of NIPAAm and N-acryloxysuccinimide (NAS) were synthesized and used for immobilization of α-amylase. The enzyme forms covalent bonds with the former polymer by single point attachment and with the latter polymer by multiple point attachment. Such a difference influences the enzyme activity and properties of the immobilized enzymes. The polymers are temperature-sensitive with lower critical solution temperatures (LCST) of 34·7 and 36·0°C for NIPNAS and PNIPAAm, respectively. The immobilized enzyme exhibited an LCST of 35·5°C for NIPNAS-amylase and 37·1°C for PNIPAAm-amylase. They precipitated and flocculated in aqueous solution above the LCST and redissolved when cooled below that temperature. The activity of the immobilized enzyme depended on the pH of the coupling buffer, with 8·0 being the optimum value. The specific activities of the immobilized enzymes were 87% and 108% compared with that of free enzyme with soluble starch as the substrate for NIPNAS-amylase and PNIPAAm-amylase, respectively. By characterizing the properties of the immobilized enzymes and comparing with those of free enzyme, no diffusion limitation of substrate was found for the immobilized enzymes and they are more thermal stable than the free enzyme. Within the two immobilized enzymes, NIPNAS-amylase showed better thermal stability and reusability. Repeated batch hydrolysis of soluble starch can be carried out efficiently with the immobilized enzymes by intermittent thermal precipitation and recycle of the enzyme. © 1997 SCI.     

A polyacrylic acid as a carrier for immobilization of penicillin acylase

A copolymer of acrylic acid with divinylbenzene was synthesized by suspension polymerization. This polymer is an effective carrier. Penicillin acylase was immobilized on this carrier to convert benzylpenicillin to 6‐aminopenicillanic acid, which may be employed in the manufacture of semisynthetic penicillins. Factors that affect the activity of immobilized penicillin acrylase, such as temperature, pH, and amount of native enzyme, were studied. Under suitable conditions, the activity and activity recovery of the immobilized enzyme were 3100 U/g (dry carrier, p‐dimethylaminobenzaldehyde method) and 59.7%, respectively. The immobilized penicillin acylase shows a remarkable increase in stability. At 40°C and pH 8.0 the value of the kinetic Michaelis–Menten constant (K_m) of the immobilized enzyme is 2.8 × 10^?3 mol/L, and the value of activation energy of enzyme catalysis is 71.5 kJ/mol. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 86: 2067–2069, 2002     

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