Urease adsorption

Fig. 15.3 Kinetic study of urease adsorption on Zn2AI and Zn3AI LDH.

Fig. 15.4 (A) Urease adsorption isotherms for (a) Zn2AI-CI, (b) Zn3AI-CI, (c) Zn4AI-CI (B) amount of immobilized urease in Zn3-AI by the coprecipitation method.

Hg2+ Urease Adsorption on gold nanoparticles in presence of pvc-nh2 Potentiometric [Pg.304]

Rhaman and coworkers [112,113] studied the adsorption of lipase on [MgAl] LDH and its biocatalytic activity for butyl oleate synthesis. They demonstrated that up to 277 and 531 mgg-1 of lipase were adsorbed on [MgAl-N03] and [MgAl-Dodecylsulfate] LDH, respectively, showing the highest adsorption capacity of the anionic clays compared to smectite or inorganic phosphate. Recently, we reported the adsorption isotherms of urease on [ZnRAl] LDH under various experimental conditions (pH, buffer) [117]. The kinetic study showed the fast adsorption process (less than 60 min) (Figure 15.3). 

Figure 15.4(A) shows the effect of the R = Zn2+/Al3+ ratio, which determines the charge density of the LDH layer, on the Freundlich adsorption isotherms. K values are far higher than those measured for smectite or other inorganic matrices. The increase in Kf with the charge density (Kf= 215, 228, 325mg/g, respectively, for R = 4, 3 and 2) is supported by a mechanism of adsorption based on an anion exchange reaction. The desorption isotherms confirm that urease is chemically adsorbed by the LDH surface. The aggregation of the LDH platelets can affect noticeably their adsorption capacity for enzymes and the preparation of LDH adsorbant appears to be a determinant step for the immobilization efficiency. [ZnRAl]-urease hybrid LDH was also prepared by coprecipitation with R = 2, 3 and 4 and Q= urease/ZnRAl from 1 /3 up to 2.5. For Q < 1.0,100 % of the urease is retained by the LDH matrix whatever the R value while for higher Q values an increase in the enzyme/LDH weight ratio leads to a decrease in the percentage of the immobilized amount.

Fig. 15.10 SEM images of [Zn3AI]-a-chymotrypsin prepared by (A) adsorption and (B) delamination and Zn3AI-CI-urease (Q 1) prepared by coprecitation (C) air-dried and (D) lyophilized.

Gianfreda L, Rao MA, Violante A (1992) Adsorption, activity, and kinetic properties of urease on montmorillonite, aluminum hydroxides, and Al(OH)x-montmorillonite complexes. Soil Biol Biochem 24 51-58... 

Skujins and McLaren (1967) co-lyophilized urease and [ CJurea. The rate of reaction, determined by the level of C02, was measured as a function of water content. Onset of enzyme reaction occurred at 0.6 relative humidity. The samples contained a 25 1 weight ratio of urea to urease. Sorption isotherms measured separately for enzyme and urea showed that below 0.75 relative humidity the urea adsorbed no water, and thus that the enzyme changes reflected adsorption of water by the urease. From the sorption isotherm for urease, 0.6 relative humidity corresponds to 0.15 h. 

Fig. 10 Adsorption of ammonia with crosslinked poly(acrylic) acid. Conditions urine, 50 ml urease, 10 mg 37°C 2 hr.

Kobos et al. (1988) described the adsorption of urease on a fluorocarbon membrane for the construction of a urea sensor. The spontaneous adsorption was enhanced by a factor of 7 by perfluoroalkylation of the amino groups of the enzyme. The enzyme membrane was attached to an ammonia gas-sensing electrode. The urea sensor thus prepared exhibited a sensitivity of 50 mV per decade of urea concentration and a response time of 3 min. Only a small amount of enzyme could be adsorbed on the limited membrane surface, so the sensor was stable for only 7 days. 

Since NH4 is stable at pH 7, the solution could be strongly buffered during this experiment to control the pH at the specimen surface. More recently GC mode was used to study the effects of adsorption at a metal surface and the applied potential on enzyme activity (30). Jack Bean urease adsorbs... 

Plasma-polymerised HMDSO film was used to produce a biocompatible surface and an enzyme support system [85]. The adsorption of urease onto a well-defined solid support, petroleum-based activated charcoal, has been achieved to provide the enzymatic hydrolysis of urea. The adsorption of urease, and the activity and stability of the enzyme on the support were studied and optimised, improving its availability for clinical applications. 

Tamura M, Sugihara M, Uragami T (1979), Ultrafiltration, hydrolysis, and adsorption characteristics of membranes from cellulose nitrate, urease stylite, activated charcoal , Augeiv Makromol Chem, 79,67-77. 

Another interesting immobilization technique based on the adsorption of suitability modified biomolecules on to fluorocarbon surfaces has been described by Kobos et al. [179]. This method minimizes nonspecific binding which affects the detection limit of biosensors unfavourably, because the transducer surface with immobilized enzyme can be treated with neutral fluorosurfactant. An enzyme can be immobilized directly onto the gas-permeable membrane of a gas-sensor. An urea electrode where perfluoroalkylated urease was immobilized onto an ammonia gas-sensing electrode may serve as an example [180],... 

P-D-Fructofuranosidase has been immobilized, with retention of activity, by an ionic reaction with a guanidino derivative of cellulose, by adsorption onto ion-exchange resins and various activated mineral and mineralized supports, by entrapment in gels prepared from dimethylacrylamide or 2-hydroxyethyl acrylate,and by inclusion inside hollow-fibre cartridges. P-o-Fructofurano-sidase has also been coupled with succinylated and immobilized concanavalin A this activated form of the enzyme gave an active, soluble, immobilized conjugate with urease. ... 

As far as the protein extraction is concerned, this field has received increased attention in recent years. Dahurun and Cussler [35] studied protein extraction in membrane contactors under various experimental conditions. Solutions of cytochrome-c, myoglobin, a-chymotrypsin, catalase, and urease in phosphate buffer were extracted using an immiscible aqueous phase, polyethylene glycol (PEG). Also, membrane-based adsorption processes have been commercialized for the separation and recovery of proteins as a replacement for a packed column of adsorbent beads. Often such processes are called membrane chromatography [36a] or adsorptive membrane... 

Two methods for the spatially controlled deposition of proteins on microelectrodes are described. The first technique involves the entrapment of glucose oxidase in photopolymerized polyHK. The second uses electrochemically aid adsorption to deposit urease and to co-deposit glucose oxidase with bovine serum albumin. Both techniques were found to lead to active deposits and the properties and optimisation of the deposition procedures will be described. Further, to facilitate glucose measurement in complex medi the depointion of a thin film of polypyrrole following that of the protdns is described. The properties of s film with respect to two model interferents and complex yeast extract medium will be reported. 

Two principle techniques for electrochemical enzyme deposition have been reported, entrapment in an electrochemically grown polymer and electrochemically aided absorption. A wide range of electrochemically grown polymers have been used. The polymer can function as both an entrapment matrix and as an anti-interference layer (7, 12-20), as a matrix for the immobilisation of the protein with an electron transfer mediator (21-23), and as an electron transfer matrix alone (24, 25), Electrochemically aided adsorption has received comparably less attention (26-30), However, in our experience (31) the latter technique results in larger responses and is more appropriate to microelectrodes. Here we will present results on the electrochemically aided adsorption of GOx and BSA, and also of urease. Furthermore to reduce the interferences at the GOx/ BSA electrode we will describe the deposition of an anti-interference layer of polypyrrole, which is grown on the electrode after the deposition of the proteins. 

In order to make a useful biosensor, enzyme has to be properly attached to the transducer with maintained enzyme activity. This process is known as enzyme immobilization. The choice of immobilization method depends on many factors such as the nature of the enzyme, the type of transducer used, the physiochemical properties of analyte, and the operating conditions [73]. The major requirement out of all these is its maximum activity in immobilized microenvironment. Enzyme-based electrodes provide a tool to combine selectivity of enzyme toward particular analyte and the analytical power of electrochemical devices. The amperometric transducers are highly compatible when enzymes such as urease, generating electro-oxidizable ions, are used [74]. The effective fabrication of enzyme biosensor based on how well the enzyme bounds to the transducer surface and remains there during use. The enzyme molecules dispersed in solutions will have a freedom of their movement randomly. Enzyme immobilization is a technique that prohibits this freedom of movement of enzyme molecules. There are four basic methods of immobilizing enzymes on support materials [75] and they are physical adsorption, entrapment, covalent bonding, and cross-linking, as shown in the Fig. 36. 

Direct determinations of the adsorption of ethylene on egg albumin, urease, sodium oleate, and other sols have been made by Nord and his associates (Ola). These show that only in the case of the sodium oleate is tlic adsorption conspicuous, although they do not completely exclude adsorption on egg albumin, or on untested cellular enzymes. The indirect evidence from the increase in surface tension and the decrease in viscosity of colloids produced by ethylene suggests that adsorption occurs. But ethylene is considered not to show resonance (69a), a prerequisite for the presence of the polarized form suggested by Nord and Franke (63a, 63b) as the basis for its adsorption. It seems still undecided whether or not ethylene is adsorbed at strategic positions on any of the cellular fermentation enzymes. The increased carbon dioxide production in the presence of an ethylene film is well established by Nord et al. (62a, 62b, 63, 63a, 63b) but there is less evidence as to the manner of action of ethylene. 

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