醋酸纤维素膜固定化脲酶的研究

酶固定膜反应器兼具有反应和分离两种功能,是酶工程领域中较活跃的研究课题.随着酶固定化技术和水平的提高,各种固定化酶生物反应器不断涌现,其中以采用固定化脲酶技术制作的人工肾最为成功.     

Immobilization of Urease on (HEMA/IA) Hydrogel Prepared by Gamma Radiation

In the present study, the copolymeric hydrogels based on 2-hydroxyethyl methacrylate (HEMA) and itaconic acid (IA) were synthesized by gamma radiation induced radical polymerization, in order to examine the potential use of these hydrogels in immobilization of Citrullus vulgaris urease. Gelation and Swelling properties of PHEMA and copolymeric P (HEMA/IA) hydrogels with different IA contents (96.5/3.5, 94.4/5.6 and 92.5/7.5 mol) were studied in a wide pH range. Initial studies of so-prepared hydrogels show interesting pH sensitivity in swelling and immobilization. C. vulgaris urease was immobilized on HEMA/IA (92.5/7.5) at 6 kGy with 41.3% retention of activity. The properties of free and immobilized urease were compared. Immobilized urease maintained a higher relative activity than free urease at both lower and higher pH levels, indicating that the immobilized urease was less sensitive to pH changes than the free urease. The K_m value of the immobilized urease was approximately 2 times higher than that of the free urease. Temperature stability was improved for immobilized enzyme. The free form exhibited a loss about 80% of activity upon incubation for 15 min at 80°C. The influence of various heavy metal ions at the concentration of l mM was improved after enzyme immobilization. The immobilization of C. vulgaris urease on HEMA/IA (92.5/7.5) at 6 kGy showed a residual activity of 47 % after 4 reuses.     

磁性壳聚糖微球用于酶的固定化研究*Ⅲ磁性壳聚糖微球的活化及其对脲酶的固定化研究

以环氧氯丙烷活化的磁性壳聚糖微球作为载体,对脲酶进行固定化研究。结果表明, 在25℃时, 活化磁性壳聚糖微球对脲酶的固定化在2h时就达到了最大值,固定化酶和自由酶的最适温度都在65℃左右,自由酶和固定化酶的米氏常数Km值分别为0.042mol/L和0.008mol/L,固定化酶的Km降低了5倍。     

磁性壳聚糖微球用于酶的固定化研究：Ⅲ磁性壳聚糖微球的活化及其对脲酶的固化研究

以环氧氯丙烷活化的磁性壳聚糖微球作为载体、对脲酶进行固定化研究,结果表明,在25℃时,活化磁性壳聚糖微球对脲酶的固定化在2h时就达到了最大值,固定化酶和自由酶的最适温度都在65℃左右,自由酶和固定化酶的米氏常数Km值分别为0．042mol/L和0．008mol/L,固定化酶的Km降低了5倍。     

Electrical Control of Urease Activity Immobilized to the Conducting Polymer on the Carbon Felt Electrode

The activity of urease varies by its redox reaction. Active urease has an SH group that is essential to exhibit its activity, however, oxidation agents such as quinone compounds can oxidize the SH group in urease and a S–S bond is produced, resulting in the loss of enzyme activity. The reduction potential of cystine was almost the same as that of the recovery of urease activity. In this work, it has been found that the SH group of urease can be oxidized by not only chemical reaction but also by the direct electrode oxidation of urease and the produced S–S bond can be reduced to SH group by chemical and electrode reactions, and the original enzyme activity is recovered. This research shows that the regulation of urease activity is easily possible by changing the electrode potential of the porous carbon felt immobilized urease. The variation of urease activity was monitored by ammonia or carbon dioxide electrode equipped with the urease immobilized carbon felt, and the ammonia or carbon oxide generated from urea can transfer through the carbon felt to reach the each gas permeable membrane. The combination of gas electrode with porous conducting material such as carbon can supply the novel device for the electrochemical investigation of enzyme activity.     

固定化酶在生物医学上的应用(Ⅰ)——聚丙烯酰胺固定化尿酶的制备及性能

本文以交联聚丙烯酰胺对尿酶包埋进行了研究,制备出了球形固定化尿酶,其含酶量高(1份酶对1份聚合物),相对活性为50%左右,对尿素的分解活性:120m/ml(固定化尿酶,37℃,2小时),提高了稳定性,在4℃下,66天后活性降低8%,106天后活性降低22%,37℃下11天后活性降低18%,并且不泄漏酶。基于以上特点,该固定化尿酶为吸附型人工肾提供了一个较合适的活性材料。     

Byssus Thread: A Novel Support Material for Urease Immobilization

Byssus threads are tough biopolymer produced by mussels (Mytilus viridis) to attach themselves to rocks. These were collected from mussels in their natural habitat (N) and from animals maintained in laboratory condition (L) as a novel support. Byssus thread surfaces were characterized by SEM analysis, chemically modified and used for adsorption of urease. The efficiency of the immobilization was calculated by examining the relative enzyme activity of free and the immobilized urease. The pH stabilities of immobilized urease were higher (0.5 unit) than free enzyme. Immobilized enzymes on byssus (both N and L) when stored at 6 °C retained 50% of its activity after 30 days, but they were more stable in dry condition. The optimum temperature of immobilized enzymes was found to increase (25 °C). A Michaelis-Menten constant (K (m)) value for immobilized urease was also elevated (2.08 mol).     

Pigeonpea (Cajanus cajan L.) urease immobilized on glutaraldehyde-activated chitosan beads and its analytical applications

Urease from pigeonpea (Cajanus cajan L.) was covalently linked to crab shell chitosan beads using glutaraldehyde. The optimum immobilization (64% activity) was observed at 4°C, with a protein concentration of 0.24 mg/bead and 3% glutaraldehyde. The immobilized enzyme stored in 0.05 M Trisacetate buffer, pH 7.3, at 4°C had a t _1/2 of 110 d. There was practically no leaching of enzyme (<3%) from the immobilized beads in 30 d. The immobilized urease was used 10 times at an interval of 24 h between each use with 80% residual activity at the end of the period. The chitosan-immobilized urease showed a significantly higher Michaelis constant (8.3 mM) compared to that of the soluble urease (3.0 mM). Its apparent optimum pH also shifted from 7.3 to 8.5. Immobilized urease showed an optimal temperature of 77°C, compared with 47°C for the soluble urease. Time-dependent kinetics of the thermal denaturation of immobilized urease was studied and found to be monophasic in nature compared to biphasic in nature for soluble enzyme. This immobilized urease was used to analyze blood urea of some of the clinical samples from the clinical pathology laboratories. The results compared favorably with those obtained by the various chemical/biochemical methods employed in the clinical pathology laboratories. A column packed with immobilized urease beads was also prepared in a syringe for the regular and continuous monitoring of serum urea concentrations.     

复合纤维素固定化脲酶对铜离子的吸附作用

脲酶对重金属离子极为敏感。由复合纤维素（CC）固定化脲酸对铜离子的吸附现象得出的结论是：脲酶吸附的Cu~(2+)总量与其活性没有定量关系，而是与固酶重量成比例。从而可以认为，重金属离子不是直接作用于脲酶活性部位使其中毒的，很可能是作用于脲酶大分子其他部位，使分子构象变形后导致脲酶失活的。由于CC固酶对Cu~(2+)有吸附作用，且又不污染系统，所以可用其除去食品、药物及水中的Cu~(2+)离子。     

Immobilization of urease onto membranes of modified acrylonitrile copolymer

Urease was immobilized onto membranes prepared from acrylonitrile (AN) copolymer (powder) modified preliminarily with 2-dimethylaminoethyl methacrylate (DMAEM) and diacrylamido-2-methylpropanesulfonic acid (AMPSA). The results obtained were compared with those from commercial acrylonitrile copolymer membranes surface modified with DMAEM and AMPSA under the same conditions. The preliminary treatment was found to give higher amount of active groups and higher modification degree compared to surface modified acrylonitrile copolymer membranes. The modified membranes were used as carriers for immobilization of urease. The basic characteristics of the immobilized urease (amount of bound protein and relative activity) were studied. Temperature and pH optima and thermal stability of the immobilized urease were also studied. The membranes prepared from AN copolymer (powder) modified with AMPSA and DMAEM were used to manufacture diagnostic test-strips for analysis of urea in blood.     

磁性壳聚糖微球用于酶的固定化研究Ⅱ磁性壳聚糖微球对脲酶的吸附及其酶学性质的研究

本文用磁性壳聚糖作为载体用吸附法对脲酶进行固定化研究。结果表明,磁性壳聚糖对脲酶的固载量与磁性壳聚糖微球的粒径、交联度及酶溶液的离子强度成反比;固定化脲酶和自由酶的最适温度分别为80℃和70℃,固定化脲的最适合pH值变化不大,固定化脲酶和自由酶的米氏常数km分别为0.00546mol/L和0.19mol/L。     

Acrylamide grafted poly(ethylene terephthalate) fibers activated by glutaraldehyde as support for urease

Urease was covalently immobilized on acrylamide-grafted poly (ethylene terephthalate) fibers after glutaraldehyde activation. Ureasecontaining fibers showed a very high operational stability and reusability, with about 85% of the initial activity after 90 d. The thermostability of the bound urease was positively influenced, and a slight change in optimum temperature was observed after immobilization, when compared with the free enzyme. The pH optimum of both types of urease was found to be the same, but immobilized urease showed an increased stability in a broader range of pH. The kinetic studies exhibited a slightly higherK _m value for the bound enzyme, with a value of 4.50 mmol dm^-3, when compared with the free enzyme (2.82 mmol dm^-3), which demonstrated that the immobilization procedure did not cause an unfavorable conformation for the substrate-product formation and a hindered diffusion. The graft yield was also found effective on maximum activity of immobilized urease. Twenty-five percent of the acrylamide-grafted fibers exhibited the highest enzymatic activity together with the highest water uptake. Higher graft yields were not suitable for the immobilization of the enzyme molecules as a result of crosslinks formed between the poly(acrylamide) chains and glutaraldehyde.     

Immobilization of Urease from Pigeonpea (<Emphasis Type="Italic">Cajanus cajan</Emphasis>) on Agar Tablets and Its Application in Urea Assay

The pigeonpea urease was immobilized on agar, a common gelling substance. The tablet strips were used as moulds to cast agar tablets of uniform shape and size. The time and temperature of solidification of agar was 6 min and 44 degrees C, respectively. The 5 % agar (w/v) and 0.019 mg protein/agar tablet yielded an optimum immobilization of 51.7%. The optimum pH was shifted through 0.2 U (from 7.3 to 7.5) towards basic side upon immobilization. The optimum temperature of soluble and immobilized urease was 30 degrees C and 60 degrees C, respectively, showing the improvement in thermal stability of urease. There was an increase in K m from 3.23 to 5.07 mM after immobilization. The half-lives of soluble and immobilized urease were 21 and 53 days, respectively, at pH 7.3 and 4 degrees C. The urea was estimated in different blood samples with the help of immobilized urease and the results were consistent with those from clinical pathology laboratory through an autoanalyzer (Zydus Co., Rome, Italy).     

Effect of thermosensitive matrix-phase transition on urease-catalyzed urea hydrolysis

Temperature dependencies of kinetic and equilibrium parameters of urea hydrolysis catalyzed by native urease and the urease immobilized in a thermosensitive poly-N-isopropylacrylamide gel have been studied. The swelling ratio of the collapsed urease-containing gel is shown to increase in the presence of urea. Below a lower critical solution temperature (LCST) of the polymer, the immobilized u reaseactually has thesame catalytic properties as the native enzyme. At temperatures above LCST, the observed catalytic activity of the immobilized enzyme depends chiefly not only on the thermoreversible matrix state, but also on gel water content.     

Modified Polymer for Enzyme Immobilization and Characterization of Immobilized Urease

Abstract

Poly-γ-methyl-L-glutamate was modified by the introduction of -CC1₃ and /or -NH₂ groups. Two kinds of polymer were obtained, and applied to urease immobilization. The enzyme immobilized on polymer containing -CCl₃ (PCCl₃) pendant groups retained more activity than that containing -NH₂ (PNH₂). Stability of urease immobilized on PCCl₃ and PNH₂ was 17 days and 19 days respectively.     

Elaboration of Urease Adsorption on Silicalite for Biosensor Creation

A possibility of efficient urease adsorption on silicalite for the purpose of biosensor creation was investigated. The procedure of urease adsorption on silicalite is notable for such advantages as simple and fast performance and non‐use of toxic or auxiliary compounds. Optimal conditions for modifying transducer surfaces with silicalite and subsequent urease adsorption on these surfaces were selected. The working parameters of the created biosensor were optimized. The developed biosensor with adsorbed urease was characterized by good intra‐reproducibility (RSD – 4.5 %), improved inter‐reproducibility (RSD of urea determination is 9 %) and operational stability (less than 10 % loss of activity after 10 days). Besides, the developed method for enzyme adsorption on silicalite was compared with the traditional methods of urease immobilization in biosensorics. Working conditions of the produced biosensor (pH and ionic strength) were shown to be close to those of the biosensor based on urease immobilized in GA vapor. For these reasons, it was concluded that the method of enzyme adsorption on silicalite is well‐suited for biosensor standardization aimed at its further manufacture.     

Amperometric Urea Biosensor Using Aminated Glassy Carbon Electrode Covered with Urease Immobilized Carbon Sheet,Based on the Electrode Oxidation of Carbamic Acid

A large oxidation current can be observed when ammonium carbamate aqueous solution is electrolyzed using a glassy carbon electrode (GCE) at a potential exceeding 1.0 V vs. Ag/AgCl and amino groups are introduced at the surface of the GCE. Aminated GCE exhibits the electrocatalytic activity of the oxidation of ammonium carbamate that is produced from urea as an intermediate product of urease reaction, and a distinct oxidation current is observed when the aminated GCE is used to oxidize the urea in the urease solution. A novel amperometric determination method to detect urea has been developed. This method is based on the electrooxidation of carbamic acid produced during urease reactions. Urease is immobilized to polymaleimidostyrene (PMS) coated on the insulated amorphous carbon sheet set on the aminated GCE surface. A good linear relationship is observed between urea concentration and the electrolytic current of the urease‐immobilized electrode in the concentration range from 0.5 mM to 21.0 mM. The proposed urea biosensor has an effective merit in that the interference resulting from ammonia and pH change caused by the urease reaction can be eliminated, differing from conventional urea biosensors.     

Immobilization of urease in poly(1-vinyl imidazole)/poly(acrylic acid) network

In this study, urease was immobilized in a polymer network obtained by complexation of poly(1-vinyl imidazole) (PVI) with poly(acrylic acid) (PAA). Preparation of the polymer network was monitored by FT-IR spectroscopy. Scanning electron microscopy (SEM) revealed that enzyme immobilization had a strong effect on film morphology. Proton conductivity of the PVI/PAA network was measured via impedance spectroscopy under humidified conditions. Values of the Michaelis-Menten constant (K _M) for immobilized urease were higher than for the free enzyme, indicating a decreased affinity of the enzyme to its substrate. The basic characteristics (pH_opt, pH_stability, T _opt, T _stability, reusability, and storage stability) of immobilized urease were determined. The results show that the PAA/PVI polymer network is suitable for enzyme immobilization.     

Immobilized enzymes - A simple calorimetric method for the determination of the catalytic activity

The results communicated in this paper show that rapid and reliable information about the activity of immobilized enzymes follows from calorimetric measurements. The study was done using spherical and plain carriers as well as different enzymes (urease, glucose-oxidase, invertase). The enzyme thermistor developed by Danielsson et al. was used as a measuring system. This measuring system was applied to investigate the activity of enzyme carrier complexes produced by the sol-gel technique. The influence of processing parameters could be pointed out at complexes of different forms (xerogel, gel on ceramic carrier, thin gel layers on foil, etc.). With the described calorimetric method, a fast and reliable technique for comparative determination of the activity of immobilized enzymes is available. A special advantage of this method is its variability in carriers and the generally applicable thermal measuring principle. Therefore, it seems useful for the development of new immobilization techniques.     

Activity and lifetime of urease immobilized using layer-by-layer nano self-assembly on silicon microchannels

Urease has been immobilized and layered onto the walls of manufactured silicon microchannels. Enzyme immobilization was performed using layer-by-layer nano self-assembly. Alternating layers of oppositely charged polyelectrolytes, with enzyme layers “encased” between them, were deposited onto the walls of the silicon microchannels. The polycations used were polyethylenimine (PEI), polydiallyldimethylammonium (PDDA), and polyallylamine (PAH). The polyanions used were polystyrenesulfonate (PSS) and polyvinylsulfate (PVS). The activity of the immobilized enzyme was tested by pumping a 1 g/L urea solution through the microchannels at various flow rates. Effluent concentration was measured using an ultraviolet/visible spectrometer by monitoring the absorbance of a pH sensitive dye. The architecture of PEI/PSS/PEI/urease/PEI with single and multiple layers of enzyme demonstrated superior performance over the PDDA and PAH architectures. The precursor layer of PEI/PSS demonstrably improved the performance of the reactor. Conversion rates of 70% were achieved at a residence time of 26 s, on d 1 of operation, and >50% at 51 s, on d 15 with a six-layer PEI/urease architecture.