Urease immobilized on aminated butyl acrylate-ethylenedimethacrylate copolymer

Urease was covalently immobilized on a new acrylic enzyme carrier: aminated butyl acrylate-ethylenedimethacrylate copolymer. The enzyme retained 56% of its original activity. The properties of the free and immobilized urease were determined and compared. The Michaelis constant was found to be higher for the immobilized urease than for the free one, while maximum reaction rate was lower for the immobilized enzyme. The stability of urease against change in pH was considerably improved by the immobilization. The immobilized urease showed very good storage stability and reusability. Resistance of the enzyme against heat inactivation at 70°C, however, was not found to be improved.     

Preparation of a cellulose acetate/organic montmorillonite composite porous ultrafine fiber membrane for enzyme immobilization

Four types of fibrous membranes based on cellulose acetate (CA)—CA membranes with nonporous fibers, CA/organic montmorillonite (O‐MMT) membranes with nonporous fibers, CA membranes with porous fibers, and CA/O‐MMT membranes with porous fibers—were prepared by electrospinning, and then, they were used for enzyme immobilization. The surface morphologies of the composite fibrous membranes were investigated with scanning electron microscopy and transmission electron microscopy. The optimum pH was 3.5 for all of the immobilized enzymes, and the optimum temperature was 50 °C. Compared with the free enzyme, the immobilized enzyme showed better stability for pH and temperature changes. Moreover, the addition of O‐MMT and the pores on the fibers improved the storage stability and the operational stability. Among the four kinds of fibrous membranes, the CA/O‐MMT membranes with porous fibers showed the best stability for the immobilized enzymes. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 43818.     

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     

Epoxy‐derived pHEMA membrane for use bioactive macromolecules immobilization: Covalently bound urease in a continuous model system

Poly(2‐hydroxyethylmethacrylate) (pHEMA) membranes were prepared by UV‐initiated photopolymerization of HEMA in the presence of an initiator (α‐α′‐azobisisobutyronitrile, AIBN). The epoxy group, i.e., epichlorohydrin, was incorporated covalently, and the urease was immobilized onto pHEMA membranes by covalent bonding through the epoxy group. The retained activity of the immobilized enzyme was found to be 27%. The K_m values were 18 and 34 mM for the free and the immobilized enzymes, respectively, and the V_max values were found to be 59.7 and 16.2 U mg⁻¹ for the free and the immobilized enzyme. The optimum pHs was 7.2 for both forms, and the optimum temperature for the free and the immobilized enzymes were determined to be 45 and 50°C, respectively. The immobilized urease was characterized in a continuous system and during urea degradation the operational stability rate constant for the immobilized enzyme was found to be 5.83 × 10⁻⁵ min⁻¹. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 77: 2000–2008, 2000     

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     

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     

Comparison of β-galactosidase immobilization by entrapment in and adsorption on poly(2-hydroxyethylmethacrylate) membranes

β-Galactosidase was immobilized in/on poly(2-hydroxyethyl methacrylate) (pHEMA) membranes by two different methods: adsorption on Cibacron F3GA derivatized pHEMA membranes (pHEMA-CB), and entrapment in the bulk of the pHEMA membranes. The maximum β-galactosidase adsorption on pHEMA-CB membranes was obtained as 95·6μgcm^-2 in 2·0mgcm^-3 enzyme solution. The adsorption phenomena appeared to follow a typical Langmuir isotherm. In the entrapment, an increase in β-galactosidase loading resulted in a consistent increase in membrane activity from 3·3×10^-2 to 17·8×10^-2Ucm^-2 pHEMA membranes. The K_m values for both immobilized β-galactosidase (adsorbed 0·32mM and entrapped 0·81mM ) were higher than that of the free enzyme (0·26mM ). The optimum reaction temperature of the adsorbed enzyme was 5°C higher than that of both the free and the entrapped enzyme. The optimum reaction pH was 7·5 for free and both immobilized preparations. After 15 successive uses the retained activity of the adsorbed and the entrapped enzymes was 80% and 95%, respectively. The storage stability of the enzyme was found to increase upon immobilization. ©1997 SCI     

明胶膜固定化脲酶的制备及性质

以明胶为载体,戊二醛为交联剂,采用包埋-交联联用法制备了明胶膜固定化脲酶,其酶活力为6 07U/g载体,酶活力收率为66 1%。最优固定化条件是包酶量为10mg酶/g明胶,ρ(明胶)=100g/L,φ(戊二醛)=0 5%。研究了固定化酶的性质,并与游离酶作了比较,游离酶的最适pH=7 0,固定化酶的最适pH=6 5;游离酶的最适温度为60℃,固定化酶的最适温度升至70℃;固定化酶与游离酶的米氏常数Km分别为11 7mM和12 4mM;固定化酶在80℃下180min仍保留初始活力的10%,而游离酶几乎完全失活。固定化酶重复使用20次其活力仅下降15%,4℃下贮存35d后仍保持初始活力的55%。     

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.     

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 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     

酸性脲酶的固定化研究

以明胶为包埋材料,戊二醛为交联剂固定化粪产碱菌所产的酸性脲酶。对酸性脲酶的固定化条件(包括明胶含量、戊二醛浓度、吸附时间、交联时间和粗酶液用量)及酶学性质(温度和pH值)进行了研究。结果表明,固定化酶的最适宜条件为：明胶的质量分数15％,戊二醛质量分数0.3％,吸附时间4 h,交联时间20 min,粗酶液用量4 mL。...     

Immobilization of proteolytic enzyme on highly porous activated carbon derived from rice bran

Highly porous activated carbon (HPAC) was used as carrier matrix for immobilization of acid protease (AP). Immobilization of acid protease on mesoporous activated carbon (AP-HPAC) performs as best enzyme carrier. At pH 6.0, 250 mg acid protease g⁻¹ HPAC was immobilized. The optimum temperature for both free and immobilized AP activities were 50 °C. After incubation at 50 °C, the immobilized AP maintained about 50% of its initial activity, while the free enzyme was completely inactivated. When testing the reusability of AP-HPAC combination immobilized system, a significant catalytic efficiency was maintained along more than five consecutive reaction cycles. The highly porous nature of the carbon permits significant higher loadings of enzyme, which results in a higher enzyme-support strength and increased stability. The changes in the AP, HPAC and AP-HPAC were confirmed by Fourier Transform Infrared spectroscopy (FT-IR). Furthermore, scanning electron microscopy (SEM) allowed us to observe that the morphology of the surface of HPAC and the AP-HPAC.     

<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.     

Characterization and application of immobilized lipase enzyme on different radiation grafted polymeric films: Assessment of the immobilization process using spectroscopic analysis

Lipase has been immobilized onto different films, polypropylene and poly(tetrafluoroethylene‐perfluroro‐propyl vinyl ether) using glutalaradehyde as a crosslinker. Differential scanning calorimetery, Fourier transform infrared spectroscopic, x‐ray diffraction, and scanning electron microscopy measurements were carried out to confirm the structure of the polymer films as well as the immobilization process of the enzyme onto the polymeric carrier. The activity and stability of the resulting biopolymers produced by lipase have been compared to those for the native lipase. The experimental results showed that the optimum temperature and pH were 40°C and 8.0, respectively. The activity of the immobilized lipases varied with lipase concentration and with the yield of grafting. Subjecting the immobilized enzymes to a dose of γ‐radiation of (0.5–10 Mrad) showed complete loss in the activity of the free enzyme at a dose of 5 Mrad. A leakage of the enzyme from the irradiated membranes was not observed in the repeated batch enzyme reactions. The operational stability of the free and immobilized lipase in n‐hexane showed that the immobilized enzyme was much more stable than the free one. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 90: 155–167, 2003     

Photosynthetic membranes, 21. Immobilization of catalase by photochemically grafted ultrafiltration membranes

Catalase has been immobilized in membranes prepared by photoinduced grafting onto microporous polymeric supports and its catalytic activity on hydrogen peroxide decomposition has been studied under ultrafiltration conditions by means of a recirculation apparatus. The membranes showed a very good catalytic performance and the enzyme reaction took place exclusively within the membrane structure. Initial reaction rates measured in the temperature range 5 – 35°C as a function of both substrate concentration and enzyme amount immobilized per unit membrane surface indicate that the mechanism of action of catalase is not altered after immobilization.     

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 urease on cation-exchange membranes prepared by radiation-initiated graft copolymerization of acrylic acid on polyethene thin films

Summary A covalent immobilization of urease was conducted on carboxylic cation-exchange membranes (CEM) prepared by radiation-initiated graft copolymerization of acrylic acid (AA) on polyethene (PE) thin films. Six types of CEM with different grafting degree (from 26.5 to 95.2%) were used as carry. The carboxyl groups were activated by the carbodiimide method in order to carry out a covalently immobilization. The amount of bound protein and the enzyme activity were determined in each immobilized system. It was established that the urease, immobilized on CEM with 64.2% grafting degree, featured the highest relative activity – 80.32%. The amount of bound protein on this membrane type was 6.01 mg/cm². The basic characteristics of the immobilized and the free enzymes were determined (pH_opt, T_opt and pH_stab). It was found out that the immobilized urease had greater thermal and storage stability in comparison with the free enzyme. It was proven that CEM with a grafting degree of 64.2% would be a suitable carrier for urease immobilization.     

乙基纤维素固定化α-淀粉酶的研究

对乙基纤维素固定化α-淀粉酶进行了研究,优化了α-淀粉酶的固定化工艺条件,并比较了游离酶和固定化酶的特性。结果表明,在α-淀粉酶浓度为4g·L-1、乙基纤维素质量分数为0.50%、4℃的条件下,固定化α-淀粉酶的重复操作稳定性最好;固定化α-淀粉酶的最适反应pH值为7.0、最适反应温度为60℃,具有良好的热稳定性、重复使用性和储存稳定性;该固定化方法操作简便,减少了酶变性的可能,最大程度保留了酶的活力。     

Behavior of immobilized glucose oxidase on membranes from polyacrylonitrile and copolymer of methylmethacrylate‐dichlorophenylmaleimide

Two new ultrafiltration membranes were obtained from a polymer mixture, containing 60% polyacrylonitrile (PAN) and 40% copolymer of methylmethacrylate‐dichlorophenylmaleimide (MMA‐DCPMI). Membrane 1 (MB1) contains 40% DCPMI of the copolymer, and membrane 2 (MB2) contains 15% of the copolymer. The pore size, the specific surface, the water content, the water flux, and the selectivity were determined for the two membranes. The presence of dichlorophenylmaleimide in the copolymer ensures the preparation of membranes suitable for direct covalent enzyme immobilization without further modifications. These membranes were used for immobilization of glucose oxidase (GOD). High amount of bound protein was found on each of the membranes. High relative activities of the immobilized GOD were achieved, 72% for MB1 and 68% for MB2. The properties of the immobilized enzyme (GOD) were determined: optimum pH and temperature and pH, thermal, and storage stability, and then compared with the properties of the native enzyme. The kinetic parameters of the enzyme reaction, Michaelis constant (K_m) and maximum reaction rate (V_max), were also investigated. The results obtained showed that the ultrafiltration membranes prepared from the mixture of PAN and the copolymer MMA‐DCPMI were suitable for use as carriers for the immobilization of GOD. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 101: 4334–4340, 2006