Urease derivatives

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],... 

U. Narang, P.N. Prasad, and F.V. Bright, A novel protocol to entrap active urease in a tetraethoxysi-lane-derived sol-gel thin-film architecture. Chem. Mater. 6, 1596—1598 (1994). 

Acids likewise inhibit the formation of trimethylsilyl derivatives, and thus the acid in hydrolyzates must be carefully neutralized. In cases where deproteinization is necessary, acidic conditions should be avoided.137,138 High concentrations of urea, as in urine samples, also interfere with the trimethylsilylation reaction, and should be eliminated by treatment with urease,138 although some authors have reported successful results without use of this step,118 Various reagents other than hexamethyldisilazane and chlorotri-methylsilane have been recommended for trimethylsilylation. These differ in their effectiveness as trimethylsilylating agents, their stability to water, or both. 

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). 

A partially purified enzyme from P. mirabilis (72) was found to have a molecular weight of 151,000. The urease of P. rettgeri is an inducible enzyme that appears only when urea, but not its analogs, are present in the media (73). Proteus vulgaris urease was found to be inhibited in vitro by thiourea and two derivatives (74), and by hydroxamic acids (93). 

The copolymei(L-Cys, L-Glu) hydrolyzed benzoylarginine derivatives(ethyl ester and amides, 19) with an optimum pH of 6.1 and optimum temperature of 40 C. This pH value corre onds to one of the two optimum pH in the hydrolysis of PNPA. The urease activity was also noted for this copolymer, and increased witii air oxidation of some SH groups (i2<5). 

Infection, gastrointestinal bleeding or injudicious use of sedatives and diuretics can precipitate hepatic encephalopathy in cirrhotic patients. The pathophysiology is complex but ammonia appears to hold a central role. Derived mainly from the action of colonic urease-containing bacteria, ammonia is... 

Life as we know it would be impossible without the astonishing characteristics of enzymic catalysis. This catalysis is not only highly efficient, so that reactions may proceed at low temperature and at neutral pH with the speed required by living cells, but it exhibits also a remarkable specificity. Let us cite two typical examples First, the enzyme urease catalyzes the hydrolysis of urea but of no other compound (1). Second, the catalytic action is frequently restricted to one of the antipodes of optically active substrates. Thus, chymotrypsin will catalyze the hydrolysis of acylated L-tyrosinamides, but will not catalyze the reaction of the corresponding derivatives of D-tyrosine (2). 

Mattiasson B, Rieke E, Munnecke D, Mosbach K (1979) Enzyme analysis of organophosphate insecticides using an enzyme thermistor. J Solid-Phase Biochem 4 263-270 Mattiasson B, Danielsson B, Hermansson C, Mosbach K (1977b) Enzyme thermistor analysis of heavy metal ions with use of immobilized urease. FEBS Lett 85 203-206 Mattiasson B, Danielsson B (1982) Calorimetric analysis of sugars and sugar derivatives with aid of an enzyme thermistor. Carbohydr Res 102 273-283 Mattiasson B, Danielsson B,Winquist F, Nilsson H, Mosbach K (1981) Enzyme thermistor analysis of penicillin in standard solutions and fermentation broth. Appl Environm Microbiol 41(4) pp 903-908... 

Because the measurement of BUN by nesslerization subsequent to hydrolysis by urease is cumbersome, the colorimetric method most widely used for the measurement of BUN is the diacetyl monoxime procedure (24). In this procedure diacetyl released from diacetyl monoxime under acidic conditions is reacted with urea to yield an yellow diazine derivative that is measured spectrophotometrically at 520 or 550 nm. The color intensity of the reaction is increased by use of either pentavalent arsenic or other polyvalent ions, such as ferric ions, or by the use of thiosemicarbazide. Although compounds such as citrulline, allantoin, and alloxan interfere, in practice the level of these constituents in serum is so low as to be of no significance. Even so, the hazard posed by the reagents used in this reaction has... 

Coordinated cyanamide does not add OH" to form the N-bound urea complex since it de-protonates 5.2) to ve the unreactive [Co(NCNH)(NH3)5p ion, but in acid it readily loses NH to form N-coordinated cyanate (equation 27). The analogous dimethylcyanamide complex cannot deprotonate and adds OH" to form urea (Scheme 21), but this does not proceed further to coordinated carbamic add and NHMCj in a process analogous to that suggested for the function of Ni in the metalloenzyme jack bean urease (which catalyzes the convemioo of urea to these products), nor does it lose NHMc2 to form the cyanato complex as suggested by Balahura and Jordan for the similar urea derivatives (equation 28). Alternatively, in acid solution the protonated urea rapidly isomerizes to the O-coordinated complex (r,/2 a 40 s, 25 °C Scheme 21). This isomer undergoes OH -catalyzed hydrolysis at the metal rather than at acyl carbon, so the function of the metalloenzyme has not been duplicated in this system. A similar property is found for 0-bound urea (equation 29). ... 

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