<Emphasis Type="Italic">Penicillium</Emphasis> sp. ZD-Z1 chitosanase immobilized on DEAE cellulose by cross-linking reaction

Chitosanase obtained fromPenicillium sp.ZD-Z1 was immobilized on DEAE cellulose with glutaraldehyde by cross-linking reaction. The optimal conditions of immobilization were as follows: 0.1 g DEAE cellulose was treated with 5 ml 5% glutaraldehyde solution; then 2.3 mg chitosanase was immobilized on the carrier. The optimal temperature and pH was 60 °C and 4.0, and the K _m value was 18.87 g/L. Under optimal conditions, the activity of immobilized enzyme is 1.5 U/g, and the recovery of enzyme activity is 81.3%. After immobilization, the optimal temperature and K _m value increased (from 50 °C to 60 °C, from 2.49 g/L to 18.87 g/L), whereas the optimal pH was reduced (from 5.0 to 4.0). The enzyme activity loss was less than 20% after 10 times batch reaction; the immobilized enzyme showed good operation stability.     

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     

Urease immobilized on chitosan membrane: Preparation and properties

Urease was covalently immobilized on glutaraldehyde-pretreated chitosan membranes. The optimum immobilization conditions were determined with respect to glutaraldehyde pretreatment of membranes and to reaction of glutaraldehyde-pretreated membranes with urease. The immobilized enzyme retained 94% of its original activity. The properties of free and immobilized urease were compared. The Michaelis constant was about five times higher for immobilized urease than for the free enzyme, while the maximum reaction rate was lower for the immobilized enzyme. The stability of urease at low pH values was improved by immobilization; temperature stability was also improved. The optimum temperature was determined to be 65°C for the free urease and 75°C for the immobilized form. The immobilized enzyme had good storage and operational stability and good reusability, properties that offer potential for practical application.     

Stabilization of penicillin V acylase from Streptomyces lavendulae by covalent immobilization

Penicillin V acylase from the actinomycete Streptomyces lavendulae ATCC 13664 has been immobilized to epoxy‐activated acrylic beads (Eupergit C®) by covalent binding. Further linkage of bovine serum albumin after enzyme immobilization was carried out in order to remove the remaining oxirane groups of the support. The obtained immobilized biocatalyst displayed double exponential deactivation kinetics at temperatures below 55 °C, while the native enzyme followed single exponential decay at the same temperatures. We concluded that soluble penicillin acylase was deactivated in one step, whereas the immobilized enzyme showed an enzymatic intermediate state which is highly thermostable. As a consequence of the immobilization process, the enzyme displayed a 10‐fold increase in its half‐life at 40 °C. At this temperature, the enzymatic intermediate state was progressively destabilized as the pH of the medium was increased. Thus, the optimum pH range for the immobilized enzyme preparation was established as being from 7.0 to 8.0. Higher pH values led to quicker enzyme deactivation. © 2001 Society of Chemical Industry     

Immobilization of β‐amylase using polyacrylamide polymer derivatives

Barley β‐amlyase was immobilized on two polymeric materials; poly(acrylamide–acrylic acid) resin [P(AM‐AAc)] and poly(acrylamide–acrylic acid–diallylamine–HCl) resin [P(P(AM‐AAc‐DAA‐HCl) using two different methods: covalent and cross‐linking immobilization. Thionyl chloride, used to activate the polymers for covalent immobilization, has the advantage that it is able to react with a number of surface groups of protein under very mild conditions. Cross‐linking with glutaraldehyde gave a higher coupling yield (approximately 70%) than covalent immobilization (approximately 20%). The activity and stability of the resulting biopolymers have been compared with those of free β‐amylase. The specific activity of the immobilized enzyme was significantly influenced by the amount of enzyme loaded onto the polymers, the optimal level being 3.5 mg g^?1 polymer. It was found that the immobilized β‐amylase stored at 4°C retained approximately 90% of its original activity after 30 days, whereas free β‐amylase stored in solution at 4°C retained only 47% of its activity after same period. The difference in long term stability was more significant when the enzyme was stored at room temperature; the immobilized enzyme maintained 40% of its activity after 30 days, whereas the residual activity of free enzyme was only 10%. Copyright © 2003 Society of Chemical Industry     

Study of the covalently immobilized amyloglucosidase on macroporous polymer by mathematical modeling of the pH optima

BACKGROUND: A totally new approach has been applied for mathematical modeling of the enzyme activity/pH relationship, for quantification and distribution of enzyme activity in and out of carrier pores. This is a very simple and elegant method for determination of the distribution of enzyme molecules on the surface and inside the particles, simply through measurement of enzyme activity at different pH values. RESULTS: Amyloglucosidase (AG) from Aspergillus niger was covalently immobilized onto poly(GMA‐co‐EGDMA) by the glutaraldehyde and periodate methods. Mathematical modeling of the pH optima for two types of covalently immobilized AG resulted in higher enzyme amounts on the surface within periodate immobilizate (67%) in comparison with glutaraldehyde immobilizate (53%). These values are modified to 64.25% for periodate immobilizate and 49.95% for glutaraldehyde immobilizate when diffusion effects are taken into account. CONCLUSION: The mathematical model applied enabled observation of the difference between the two types of coupling agents and different immobilization procedures throughout quantification of the immobilized enzyme on the matrix surface and inside pores. Copyright © 2012 Society of Chemical Industry     

Covalent immobilization of cytochrome P450 BM3 (R966D/W1046S) on glutaraldehyde activated SPIONs

Selective hydroxylation of the C‐H bond of saturated hydrocarbon chains at room temperature is the signature of an invaluable biocatalyst, cytochrome P450 BM3 from Bacillus megaterium. Despite this remarkable ability, because of the enzyme's inherent low stability and dependence on electron supply by expensive NADPH, developing stable and economic BM3 systems is a challenging subject. To improve BM3 stability, facilitate its reuse, and reduce the process cost, this study suggests covalent immobilization of R966D/W1046S P450 BM3 on glutaraldehyde pre‐activated super paramagnetic iron oxide nanoparticles (SPIONs). This double mutant consumes less expensive cofactors like NADH and BNAH and its immobilization on magnetic support facilitates its separation and reuse. Free and immobilized enzyme performances were evaluated by 10‐pNCA hydroxylation and BM3 selectivity (hydroxylation at ω (1–3) positions of a fatty acid) was confirmed in a reaction involving myristic acid. The enzyme activity recovery was up to 60 % with 100 % enzyme binding efficiency. BM3‐SPIONs were easily separated from the reaction medium by applying a magnet, and recycled for 5 times, after which they could still present half of their initial activity. The enzyme storage stability was significantly improved: after one month of storage at 4 °C, the immobilized enzyme showed 80 % residual activity toward NADH while the soluble enzyme was inactive after a week. Binding an enzyme to fabricated SPIONs is a promising technique to increase enzyme stability and prevent downstream contamination in biocatalytic processes. In this context, BM3‐SPIONs can be a practical model system in cost‐effective large‐scale applications of such enzymes.

     

Thermostability and esterification of a polyethylene‐immobilized lipase from Bacillus coagulans BTS‐3

Extracellular lipase from Bacillus coagulans BTS‐3 was immobilized on activated (alkylated, 2.5% glutaraldehyde) and native (nonactivated) polyethylene powder, and its thermostability and esterification efficiency were studied. Immobilization on activated support was found to enhance thermostability as well as esterification efficiency. The optimum time for immobilization on activated (AS) and nonactivated (NS) polyethylene support was found to be 10 min, and the binding of the lipase was markedly higher on AS. Lipase was more efficiently bound to AS (64%) than to NS (30%) at an optimum temperature of 37°C. The pH and temperature optima for AS‐ and NS‐bound lipase were 9.0 and 55°C and 8.5 and 55°C respectively. At 55°C the free lipase, which had a half‐life of 2 h, lost most of its activity at elevated temperatures. In contrast, AS‐bound lipase retained 60%–80% of its original activity at 55°C, 60°C, 65°C, and 70°C for 2 h. Exposure to organic solvents resulted in enhanced lipase activity in n‐hexane (45%) and ethanol (30%). Both AS‐ and NS‐bound biocatalysts were recyclable and retained more than 85% of their initial activity up to the fourth cycle of hydrolysis of p‐nitrophenyl palmitate. The AS‐bound lipase efficiently performed maximum esterification (98%) of ethanol and propionic acid (300 mM each, 1 : 1) in n‐hexane at 55°C. With free or NS‐bound lipase in similar conditions, the conversion of reactants into ester was relatively low (40%). © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 102: 3986–3993, 2006     

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     

A simple method for biocatalyst immobilization using PVA‐based hydrogel particles

BACKGROUND: The aim of this study was to evaluate the feasibility of enzyme immobilization in PVA particles through extrusion of LentiKat^®Liquid in polyethylene glycol. Inulinase, with invertase activity for sucrose hydrolysis, was used as model system. RESULTS: Inulinase was effectively immobilized in PVA particles. The pH optimum of the enzyme activity was broadened for lower pH values. Mechanical instability of the PVA under prolonged incubation above 55 °C was observed. A 1.8‐fold increase in the apparent K_M (Michaelis constant) suggests diffusion limitations as a result of immobilization. The immobilized biocatalyst exhibited considerable operational stability, since a decrease of roughly 10% in the product yield after 24 h biotransformation runs was observed in trials performed at 50 °C, following 20 repeated, consecutive batches. CONCLUSION: The results obtained highlight the potential of PVA‐based particles obtained through extrusion into PEG for the production of suitable biocatalysts for application in large‐scale processes. Copyright © 2008 Society of Chemical Industry     

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.     

Immobilization of invertase onto crosslinked poly(p‐chloromethylstyrene) beads

Invertase was immobilized onto poly(p‐chloromethylstyrene) (PCMS) beads that were produced by a suspension polymerization with an average size of 186 μm. The beads had a nonporous but reasonably rough surface. Because of this, a reasonably large external surface area (i.e., 14.1 m²/g) could be achieved with the proposed carrier. A two‐step functionalization protocol was followed for the covalent attachment of invertase onto the bead surface. For this purpose, a polymeric ligand that carried amine groups, polyethylenimine (PEI), was covalently attached onto the bead surface by a direct chemical reaction. Next, the free amine groups of PEI were activated by glutaraldehyde. Invertase was covalently attached onto the bead surface via the direct chemical reaction between aldehyde and amine groups. The appropriate enzyme binding conditions and the batch‐reactor performance of the immobilized enzyme system were investigated. Under optimum immobilization conditions, 19 mg of invertase was immobilized onto each gram of beads with 80% retained activity after immobilization. The effects of pH and temperature on the immobilized invertase activity were determined and compared with the free enzyme. The kinetic parameters K_M and V_M were determined with the Michealis–Menten model. K_M of immobilized invertase was 1.75 folds higher than that of the free invertase. The immobilization caused a significant improvement in the thermal stability of invertase, especially in the range of 55–65°C. No significant internal diffusion limitation was detected in the immobilized enzyme system, probably due to the surface morphology of the selected carrier. This result was confirmed by the determination of the activation energies of both free and immobilized invertases. The activity half‐life of the immobilized invertase was approximately 5 times longer than that of the free enzyme. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 83: 1268–1279, 2002     

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.     

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     

Urease immobilization on an ion‐exchange textile for urea hydrolysis

Ion‐exchange textiles are used as organic supports for urease immobilization with the aim of developing reactive fibrous materials able to promote urea removal. A non‐woven, polypropylene‐based cation‐exchange textile was prepared using UV‐induced graft polymerization. Urease was covalently immobilized onto the cation‐exchange textile using three different coupling agents: N‐(3‐dimethylaminopropyl)‐N′‐ethylcarbodiimide hydrochloride (EDC), N‐cyclohexyl‐N′‐(b‐[N‐methylmorpholino]ethyl)carbodiimide p‐toluenesulfonate (CMC), and glutaraldehyde (GA). The immobilized biocatalyst was characterized by means of FT‐IR spectrometry, SEM micrographs, dependence of the enzyme activity on pH and temperature, and according to the kinetic constants of the free and immobilized ureases. The biotextile prepared with EDC in the presence of N‐hydroxysuccinimide performs best. The optimum pH was 7.2 for the free urease and 7.6 for the immobilized ureases. The reactivity was maximal at 45 °C for free urease, 50 °C for biotextiles prepared using EDC or CMC, and 55 °C for biotextiles prepared with GA. The activation energy for the immobilized ureases was 4.73–5.67 kcal mol^?1, which is somewhat higher than 4.3 kcal mol^?1 for free urease. The urea conversion for a continuous‐flow immobilized urease reactor is nearly as good as a continuously stirred tank reactor having a much longer residence time, suggesting that the packed bed reactor had sufficient diffusive mixing and residence time to reach nearly optimal results. Urease immobilized on a biotextile using EDC has good storage and operational stability. Copyright © 2006 Society of Chemical Industry     

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.     

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     

Immobilization of glucoamylase on magnetic poly(styrene) particles

Magnetic poly(styrene) particles including active groups were prepared for enzyme immobilization without any activation process. Glucoamylase, which is widely used in industry, was immobilized onto these particles. The effects of pH, buffer concentration, and temperature on immobilization were investigated; moreover, the effect of immobilization temperature on immobilized glucoamylase activity was determined for the hydrolysis of maltose. The acetate buffer with the concentration of 6 × 10⁻⁴ M at pH 4 and 20–30°C was found as the most suitable medium for the immobilization of the glucoamylase. The amount of bound protein is 8 mg/g particle with the immobilization yield of 70%. The maximum activity obtained with immobilized glucoamylase is approximately 70% of the free one. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 72: 69–73, 1999     

Immobilization of Candida rugosa lipase on modified natural wool fibers

A method has been developed to immobilize lipase from Candida rugosa on modified natural wool fibers by means of graft copolymerization of poly ethylacrylate in presence of potassium persulphate and Mohr’s salt redox initiator. The activities of free and immobilized lipase have been studied. FTIR spectroscopy, scanning electron microscopy, and the Bradford method were used to characterize lipase immobilization. The efficiency of the immobilization was evaluated by examining the relative enzymatic activity of free enzyme before and after the immobilization of lipase. The results showed that the optimum temperature of immobilized lipase was 40 °C, which was identical to that of the free enzyme, and the immobilized lipase exhibited a higher relative activity than that of free lipase over 40 °C. The optimal pH for immobilized lipase was 8.0, which was higher than that of the free lipase (pH 7.5), and the immobilization resulted in stabilization of enzyme over a broader pH range. The kinetic constant value (km) of immobilized lipase was higher than that of the free lipase. However, the thermal and operational stabilities of immobilized lipase have been improved greatly.     

Production of fructooligosaccharides by immobilized mycelium of Aspergillus japonicus

β-Fructofuranosidase (EC 3.2.1.26) in Aspergillus japonicus mycelium was immobilized by entrapment in calcium alginate gel. After immobilization, the enzyme was active over a wider pH range, and had improved thermostability. The total amount of fructooligosaccharides produced by immobilized enzyme was similar to that produced by a free enzyme system. A packed-bed reactor was employed for production of fructooligosaccharides at 42°C using the immobilized enzyme. The reactions were continued for 35 days and only 17% of enzyme activity was lost during this period.