Immobilized urease activity

Urease (EC 3.5.1.5 Type IX, Sigma-Aldrich from Jack Beans) was used throughout the experiments. Before immobilizing urease onto the microreactor systems, the enzyme was evaluated for activity in the chosen buffer system (Tris[hydroxymethyl]aminomethane [THAM]). Free enzyme tests of the urease showed an approximate activity of 44,800 U/g of solid. 

Batch studies for evaluating immobilized enzyme activity and properties of the "bioplastic" (urease entrapped in PDMS) material were conducted in 250-mL shake flasks in an environmentally controlled shaker/ incubator. 

The first electrode for urea was prepared by immobilizing urease in a poly-acrylcimide gel on nylon or Dacron nets. The nets were placed onto a Beckman electrode (NH J selective) (59). In a later development, the electrode was improved by covering the enzyme gel layer with a cellophane membrane to prevent leaching of urease into the solution (60). The urease electrode could be used for 21 days with no loss of activity. 

Fig. 26 Cross-sectional view of a bioerosion-regulated hydrocortisone delivery system, a feedback-regulated drug delivery system, showing the drug-dispersed monolithic bioerodible polymer matrix with surface-immobilized ureases. The mechanism of release and time course for the urea-activated release of hydrocortisone are also shown. (From Ref > 1)...

GC mode SECM experiments have been used to detect localized activity of a variety of enzymes (see Table 2). Since microelectrodes are used in the life sciences to detect neurotransmitters (36), metal cations (37), and free radicals (38), a wide variety of probes may be combined with SECM positioning technology to obtain spatially resolved information. The GC mode experiments may be carried out with potentiometric or amperometric tips depending on the species to be sensed. The tip must be chosen to determine a product or reactant consumed by the enzyme reaction, e.g., H+ for immobilized urease, H202 for immobilized glucose oxidase, or 4-aminophen-olate for alkaline phosphatase. A list of enzymes and the tips employed is given in Table 2. 

Evaluation of stability and catalytic properties of the immobilized system must take into account possible pH differences between the inner core of the fiber, where the reaction takes place, and the bulk of the feed solution. The production of compounds which alter the pH, like ammonia produced from urea via immobilized urease,48 and the partition properties in hollow fiber membranes can result in creating such pH gradients. Experimentally, these differences produce more or less pronounced shifts in the optimum pH dependence of enzyme activity relative to its free form dependence and thereby affect the activity of the enzyme at work.2 3 48... 

Immobilization of Urease on Sweetzyme Pellets For co-immobilization of urease on the Sweetzyme pellets, 500 ml of 1 g/1 urease solution and 2 g of Sweetzyme pellets was added to a 1-1 beaker [35]. The beaker was left on the benchtop at room temperature for 24 h. The pellets were separated from the solution by decanting and gravity filtration and dried on a paper towel at room temperature for 24 h or until dry. Co-immobilized pellets were stored at 4 °C until use. Activity of immobilized urease was measured at pH 7.5 and 25°C using a standard assay procedure that measures the rate of ammonia liberation [43]. The urease activities obtained with our immobilization procedure were in the range of 550-577 U/g pellets, where a unit liberates 1 pmol of ammonia per minute under the assay conditions. 

Fig. 5 Effect of Xl/urease activity on the isomerization kinetics and xylose/xylulose production for the co-immobilized enzyme pellets. All pellets were from the same coimmobilization batch and have the same urease and XI activities per gram of pellet at pH 7.5, The initial urea concentration used in all experiments was 0.01 M. The improvement in the xylulose yields with increased enzyme loading can be attributed to the dual role of tetrahydroxyborate ions in our co-immobilized pellet system...

Entrapment in polyacrylamide gel Active immobilized enzyme study of the characteristics and overall reaction rate of unbuffered gel-immobilized urease particles 817... 

Immimodiagnostics and enzyme biosensors are two of the leading technologies that have a greatest impact on the food industry. The use of these two systems has reduced the time for detection of pathogens such as Salmonella to 24 hr and has provided detection of biological compounds such as cholesterol or chymotrypsin [15]. Biosensors analyses Beta lactams in milk and presence of urea in milk that lead to production of synthetic milk, the biocomponent part of the urea biosensor is an immobilized urease yielding bacterial cell biomass isolated from soil and is coupled to the ammonium ions selective electrodes of a potentiometric transducer. The membrane potential of all types of potentiometric cell based probes is related to the activity of electrochemically-detected product [16]. 

The carrier nature is clearly evidenced to affect directly the activity of the immobilized urease, the maximum being directly proportional to the matrix hydrophilicity 4-aminobenzylcellulose (DS 0.6)=7.5% 4-aminobenzoylcellulose (DS 1.2) =1.2% poly(vinyl-4-aminobenzoate)= 0.7%. ... 

Reaction of an immobilized, glutaraldehyde-activated form with urease (see p. 510)... 

Fig. 9. Experimental shift of the activity-external pH curve due to the feed-back action of pH-active products, (a) The product is a base immobilized urease (0-0) urease in stirred solution (x-x) substrate urea, product (NH4)2 CO3 (From Sel gny et al. [40].) (b) papain immobilized in a collodion membrane (J-J) papain in stirred solution ( ) substrate benzoyl-L-arginine ethyl ester / iC00R2->i iC00"+H++i 20H (From Goldman et al. [53] and Katchalski et al. In both cases concentrated buffer or grinding to powder of membranes strongly decreases the differences observed between soluble and insoluble enzymes. 

U. of immobilized urease (Boehringer-Enzygel-Urease-650 units/g Coming glass-bound urease-300 units/g) is coated onto the teflon stirrer of an air-gap electrode, and a nylon net is placed over it and tied at the end. Thanks to the favorable hydrodynamics of this constmction, an analysis requires only about two minutes, even though the 2-amino-2-hydroxymethyl-1,3-propandiol buffer used (0.5 M, pH 8.5) results in a pH value at which the enzyme cannot display its optimal activity. 

Several enzymes have been immobilized in sol-gel matrices effectively and employed in diverse applications. Urease, catalase, and adenylic acid deaminase were first encapsulated in sol-gel matrices [72], The encapsulated urease and catalase retained partial activity but adenylic acid deaminase completely lost its activity. After three decades considerable attention has been paid again towards the bioencapsulation using sol-gel glasses. Braun et al. [73] successfully encapsulated alkaline phosphatase in silica gel, which retained its activity up to 2 months (30% of initial) with improved thermal stability. Further Shtelzer et al. [58] sequestered trypsin within a binary sol-gel-derived composite using TEOS and PEG. Ellerby et al. [74] entrapped other proteins such as cytochrome c and Mb in TEOS sol-gel. Later several proteins such as Mb [8], hemoglobin (Hb) [56], cyt c [55, 75], bacteriorhodopsin (bR) [76], lactate oxidase [77], alkaline phosphatase (AP) [78], GOD [51], HRP [79], urease [80], superoxide dismutase [8], tyrosinase [81], acetylcholinesterase [82], etc. have been immobilized into different sol-gel matrices. Hitherto some reports have described the various aspects of sol-gel entrapped biomolecules such as conformation [50, 60], dynamics [12, 83], accessibility [46], reaction kinetics [50, 54], activity [7, 84], and stability [1, 80],... 

In another kinetics study, Huang and Chen immobilized jack bean urease in the form of a thin film on the surface of a reticulated polyurethane foam. The residual apparent activity of the urease after immobilization was about 50%. The good hydrodynamic properties and flexibility of the support were retained in solution after immobilization. Urea hydrolysis was examined in both a batch squeezer and circulated flow reactor. The results suggest potential for practical applications in various reactors. 

Phase transition in gels in response to biochemical reactions [27,28]. Polymer gels were synthesized in which an enzyme (urease) or a biologically active protein (lectin) was immobilized. The volume phase transitions were observed in such gels when biochemical reactions took place. Such mechano-biochemical gels will be used in devices such as, sensors, selective absorbers, and biochemically controlled drug release. 

An unusual type of derivative is the complex that forms between urease and bentonite in acid medium (61). The adsorbed form was found catalytically active. Similarly, urease immobilized in a polyacrylamide gel matrix has been used to prepare a urea-specific enzyme electrode (62). Yet another active water-insoluble derivative has been prepared (63) by allowing p-chloromercuribenzoate-treated urease to react with a diazotized copolymer of p-amino-D,L-Phe and L-Leu. Urease has been found to retain about 20% of its original activity when encapsulated in 100 n microcapsules of benzalkonium-heparin in collodion (64). 

Just one example will be given here.195 Evaporation of water from aqueous solutions of MEEP and the enzyme urease yields films that can be cross-linked by exposure to gamma rays. The cross-linked films absorb water to form hydrogels in which the enzyme molecules are trapped within the interstices of the gel network. Some of the enzyme molecules may also be covalently grafted to the polymer side groups. The immobilized enzyme retained approximately 80% of its activity for the conversion of urea to ammonia. This system can, in principle, be used for the immobilization of a wide variety of enzymes, and for their use in biochemical flow reactors, or in sensors. 

Chemically binding enzymes to nylon net is very simple and gives strong mechanically resistant membranes (135). The nylon net is first activated by methylation and then quickly treated with lysine. Finally, the enzyme is chemically bound with GA. The immobilized disks are fixed direcdy to the sensor surface or stored in a phosphate buffer. GOD, ascorbate oxidase, cholesterol oxidase, galactose oxidase, urease, alcohol oxidase (135), and lactate oxidase (142) have been immobilized by this procedure and the respective enzyme electrode performance has been established. 

The activity of the enzyme is also strongly affected by the presence of inhibitors. Fluoride ions inhibit urease (173) and oxalate ions inhibit lactate oxidase (174), but the major inhibitors are heavy-metal ions, such as Ag+, Hg +, Cu " ", organophosphates, and sulfhydryl reagents (/i-chloromercuribenzoate and phenylmercury(II) acetate), which block the free thiol groups of many enzyme active centers, especially oxidase (69). Inhibiting the enzyme electrodes makes it possible to quantify the inhibitors themselves (69), for example, H2S and HCN detection using a cytochrome oxidase immobilized electrode (176). 

Diagnosis of renal problems, xanthinuria, and toxemia of pregnancy via determination of the ratio of hypoxanthine to xanthine in plasma is facilitated by the use of biosensors. Xanthine oxidase immobilized on aminopropyl-CPG (controlled pore glass) activated with glutaraldehyde oxidizes hypoxanthine first to xanthine and then to uric acid. Use of an IMER with biosensors for hypoxanthine, xanthine, and uric acid provides the necessary data. Pre- or postcolumn enzymatic reactions catalyzed by creatinine deiminase, urease, alkaline phosphatase, ATPase, inorganic pyrophosphatase, or arylsufatase facilitate analysis of uremic toxins (simultaneous detection of electrolytes, serum urea, uric acid, creatinine, and methylguanidine). 

Photocontrol of enzyme activity has also been demonstrated with membrane-bound enzymes. Urease was modified with a splropyran and Immobilized In a collagen membrane matrix. The activity of the modified urease decreased with ultraviolet Irradiation and then was restored to the Initial activity with visible light Irradiation (38). A collagen membrane matrix has also been modified with a splropyran compound to be photosensitive. Trypsin was Immobilized in the photosensitive collagen membrane matrix. The activity of the trypsin membrane under visible light decreased by about 20% in the dark (39). The splropyran-modifled membrane was hydrophilic In the dark and turned hydrophobic under visible light, which may have been responsible for the photo change in the apparent diffusion coefficient of the substrate within the membrane matrix. 

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