Urease composition

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],... 

The composition, visible spectroscopy, and catalytic properties of urease have been reviewed by Blakeley and Zemer [25] and by Hausinger [2]. The urease from jack bean is typical of the enzymes from plants. It has a protein of relative... 

The best-known enzyme electrode is that used to analyze for urea in blood. The enzyme urease is immobilized in a polyacrylamide hydrophilic gel and fixed at the bottom of a glass electrode whose characteristics make it an NH4 ISE. Alternatively, the ISE can be a composite system designed to detect NH3. In the presence of the enzyme, urea is hydrolyzed according to the reaction... 

The first enzyme to be obtained purified and in crystalline form was urease (in 1926, by Sumner in America). Since then many hundreds have been extensively purified and isolated as crystals. All have turned out to be proteins, and it may be taken as a general rule that all enzymes are in fact largely protein in composition, although many also have non-protein material such as metal ions or nucleotides bound to them. 

In addition to nucleic acids, enzymes have also been incorporated into conducting a polymer matrix for biosensor applications. As a transducer, a CP can convert the chemical response into an electric current. To enhance the sensitivity and the response time, fabrication of CPs/enzyme nanocomposites with large surface area is a meaningful objective. Syu and Chang demonstrated the immobilization of urease onto PPy nanotubes over carbon paper substrate by a physical entrapment approach [124]. The composite electrodes exhibited a detection sensitivity for the determination of urea of 53.74 mVdecade and a detection limit on the urea concentration of 1.0 pM. Furthermore, the composite electrode shows rapid response, storage stability and reusability. Lipase can also be covalently immobilized... 

The experiments and calculations have demonstrated that the best pairs of polyelectrolytes for the shells of microcapsules are PAA/DS and PAA/PSS for LDH, and PSS/PAAand PSS/PDADMA for urease in the given sequence of layer deposition. On this basis, we designed and prepared PEMC containing LDH and urease with different polyelectrolyte compositions and different numbers of layers. 

Most biosensors described in the literature for the determination of urea are potentiometric based on NH4 or HCOj" sensitive electrodes [181, 182]. Osaka and co-workers constructed a highly sensitive and rapid flow injection system for urea analysis with a composite film of electropolymerised inactive PPy and a polyion complex [183]. Pandey and co-workers fabricated a urea biosensor based on immobilised urease on the tip of an ammonia gas electrode (diameter 10 pm) made from a PPy film coated onto a platinum wire [170]. The enzymatic response was achieved in the wide range of 0.001-0.05 M with a stability of more than 32 days. Cho and co-workers [184] developed a procedure for urea determination by crosslinking urease onto PANI-Nafion composite electrodes, which could sense the ammonium ions efficiently. Such a urea biosensor has a detection limit of about 0.5 pM and a response time of 40 seconds. 

The basic component of tiie erqimimental system applied to this study was an absorbent material normally used in personal hygiene articles. In principle tiiis would be loose or bonded cellulose flufi possibly blen with cross-linked sodium polyacrylate granules (known as superabsorbent polymer, SAP) for enhanced liquid capacity. Cellulose fibers or whole composites served as carriers of known and potmitial urease inhibitors. These substances were physically attached to the absoibent core by exhaustion fiom their solutions and subsequent drying of the substrates. A few examples of materials treated this... 

Our studies with urease inhibitor-modified composites indicated that the new materials were able to maintain the slightly acid pH on the surface of the absorbent pad containing urine. Healthy skin has its own pH at a level which is somewhat lower than neutral. This condition ensures the stability of natural oil layer present on the skin and reduces the risk of microbial infections and irritations. 

The aim of this paper was to report the results of our studies on the modification of cellulose and cellulose-based composites with urease inhibitors. The obtained data support our suggestion that the new technology is a simple and effective way of controlling ammonia odor caused by the decomposition of urine. Another seemingly important benefit of this approach is the ability of the modified systems to maintain the pH at a level benign to skin. In addition to that, the evidence bas on our lab data suggests that the applied treatment not only retards the emission of ammonia but can also help reduce other odors typically related to menses and sweat. 

M D Sweidloff, J F Kolc, M M Rogic and L L Hmbickstni, Phosphoroamide urease inhibitors and urease inhibited urea based fertilizer compositions, US Patent Office, Pat No... 

The present work is a continuation of our research activity towards the development of chemical and bio-sensors of this type. Two types of composite films were produced with PESA method and studied throughout this work PAA/CuPc, and CTCT/PAA/Urease, Attempts to incorporate some other organic indicators, such as methyl red and thymol blue into PESA films have also been made. 

The enzyme Urease (SIGMA), which was negatively charged in Img/ml solution in the Trisma base/HCl buffer having pH 8.0-8.3, was deposited by alternation with PAA. A few layers of Urease/ PAA were deposited on top of CTCT/PAA films. Composite membranes with the typical structure of glass/PAA/(CTCT /PAA)n/(Urease/PAA)m with n=10-40 and m=l-5 were finally produced (see the scheme in Fig. 3b). 

Optical spectral transformations of composite PESA films having a structure PAA(CTCT/PAA)n(Urease/PAA)ni were recorded by using UV-visible optical spectroscopy in order to study the reaction of urea decomposition. Typical UV/visible absorption spectra of these films are shown in Fig. 10. When the sample was soaked in urea solution, the band at 550 nm was found to be slightly shifted towards higher wavelengths with an additional band appeared at 440 nm. 

Enzyme/indicator optrodes for registration of enzyme reactions and their inhibitors, such as heavy metal ions and pesticides, can be produced by PESA technique. Composite films containing the enzyme urease and cyclo-tetra-chromotropylene as indicator molecules show some characteristic spectral transformations caused by urea decomposition. The reaction of inhibition of urease by heavy metal ions can be also registered with this method. Further development of the enzyme sensors and sensor arrays lies in finding suitable pairs of enzyme/indicator, their deposition by PESA method and studying the enzyme reactions (including inhibition) with UV-vis spectroscopy. 

CaCO, Microspherulites Containing Enzymes were prepared as in Ref [6]. One volume of a 1M CaCl aqueous solution was added to two volumes of enzyme solution (1.0 mg/mL) with vigorous stirring. Then an equal volume of aqueous 0.33 M Na COj was rapidly added. After stirring for 30 s, the suspension of particles was allowed to stand for 15 min at room temperature without stirring. The formation of microspheruhtes was controlled with a light microscope. These composite micro-sphemlites with inclusions of urease or LDH had a narrow size distribution with a mean diameter of 4.5 pm. 

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