Urease Reactors

A urease packed bed reactor combined with an ammonia electrode is being used in a blood urea nitrogen (BUN) analyzer marketed since 1976 by the Kimble Division of Owens-Illinois (USA). Urease is bound to aluminum oxide particles. The reaction is conducted at pH 7.5. In order to obtain a high sensitivity for the ammonia electrode, the product stream leaving the reactor is mixed with NaOH until a pH of 11 is attained (Watson and Keyes, 1976). 

Besides these conventional reactors with spherical immobilizates, urease has also been immobilized inside nylon tubing and pipette tips ( enzyme pipette , Sundaram and Jayonne, 1979), on nylon fibers ( enzyme brush , Raghavan et al., 1986), and on the surface of a magnetic stirrer (Guilbault and Starklov, 1975). The urease reaction was in each case carried out at optimal pH after removal of the immobilized enzyme NH3 was assayed electrochemically or photometrically according to Berthelot s method. 

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The conditions under which chemical indicator reactions are used often necessitates the use of postcolumn addition, however. Figure 4.11 shows an experimental setup for urea assays using an immobilized urease reactor.30 The postcolumn addition of sodium hydroxide allows the NH produced by the reactor to be detected as NH3 at an ammonia gas-sensing electrode placed in a flow cell. 

Na+, NHJ (urea), K+ Dilution, treatment with immobilized urease reactor Ion exchange 5 itiM nitric acid Indirect conductivity 26... 

Capsules with high enzyme loading and activity prepared by templating BMS spheres can also be used as biomimetic reactors [89]. For example, PGA/ PLL capsules with pre-loaded urease (prepared via a BMS sacrificial template) are capable of catalyzing the hydrolysis of urea and have been shown to induce the exclusive formation of CaC03 particles inside the capsules [89]. 

Lvov, Y. and Caruso F. (2001) Biocolloids with ordered urease multilayer shells as enzymatic reactors. Analytical Chemistry, TS, 4212-4217. 

Huang, T.C. and Chen, D.H., Kinetic studies on urea hydrolysis by immobilized urease in a batch squeezer and flow reactor, Biotechnol. Bioeng., (1992) 40,10,1203-09. Storey, K.B., Duncan, J.A., and Chakrabarti, A., Immobilization of amyloglucosidase using two forms of polyurethane polymer, Appl. Biochem. Biotechnol., (1990) 23, 3, 221-36. 

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. 

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. 

Novel microreactors with immobilized enzymes were fabricated using both silicon and polymer-based microfabrication techniques. The effectiveness of these reactors was examined along with their behavior over time. Urease enzyme was successfully incorporated into microchannels of a polymeric matrix of polydimethylsiloxane and through layer-bylayer self-assembly techniques onto silicon. The fabricated microchannels had cross-sectional dimensions ranging from tens to hundreds of micrometers in width and height. The experimental results for continuous-flow microreactors are reported for the conversion of urea to ammonia by urease enzyme. Urea conversions of >90% were observed. 

Calculate the time needed to convert 80% of the urea to ammonia and carbon dioxide in a O.S-dm batch reactor. The initial concentration of urea is 0.1 mol/dm , and the urease concentration is 0.001 g/dm. The reaction is to be carried out isother-mally at the same temperature at which the data in Table E7-7.1 were obtained. 

Figure 4.11. Enzyme reactor system for urea based on immobilized urease and potentio-metric detection.30...

In contrast to reactor arrangements, the intimate physical contact of urease with the signal transducer in enzyme membrane sensors does not permit shifting the sample pH in favor of optimal transducer sensi-... 

Urease, uricase,48 alcohol dehydrogenase,50 and alkaline phosphatase51 have been used in such reactors. 

Similar reactor configurations using flat membranes in place of hollow fibers have even been used with urease, uricase, glucose oxidase and creatinine kinase.49 An enzyme solution is, in this case, introduced into one of the membrane-separated chambers of a flat-membrane dialyzer. Operating the reactor at high feed flow rates leads to a reduction of mass transfer resistances, which can also be achieved using suitable turbulence promoters. The overall behavior of such systems appears to be similar to that of the hollow fiber enzyme reactors, except for an apparent higher efficiency.49... 

Cheetham PSJ (1994) Case studies in applied biocatalysis-from ideas to products. In Cabral IMS, Boros DBL, Tramper J (eds). Applied biocatalysis. Harwood Acad Publ, Chur, pp 47-108 Chen DH, Leu JC, Huang TC (1994) Transport and hydrolysis of urea in a reactor-separator combining an anion-exchange membrane and immobilized urease. J Chem Technol Biotechnol 61(4) 351-357... 

Yu AM, Gentle 1, Lu GQ, et td. (2006) Nanoassembly of biocompatible microcapsules for urease encapsulation and their use as biomimetic reactors. Chem Common 2150-2152... 

The immobilization can be performed with a modality different from that of the reactor filled with the immobilized enzyme. The spectrophotometric control of hemodialysis treatment is based on an optical flow-through biosensor based on Prussian Blue film with chemically linked urease forming a monomo-lecular layer of the enzyme. This pH-enzyme opt-rode-FIA system has been used for the selective determination of postdialyzate urea in real clinical samples. 

Ni/Ni(OH)2 [19]. To our knowledge, no study on direct electrochemical ammonia oxidation in urine has been reported so far. Nicolau et al. [20] proposed an electrolyzer for urine treatment in space missions, which consists of a first reactor with immobilized urease to produce ammonia, which is later oxidized on a platinized BDD to hydrogen and nitrogen. However, all experiments were conducted with urea solutions and not urine. 

Huang, T.-C., and D.-H. Chen. 1992. Kinetic Studies on Urea Hydrolysis by Immobilized Urease in a Batch Squeezer and Flow Reactor. Biotechnology and Bioengineering 40 (10) 1203-1209. 

The indirect method was applied to FIA technique for the simultaneous determination of urea and NH4 in agricultural irrigation waters [204] and natural waters [205]. The final concentrations of NH4 were determined by spectrophotometric and fluorimetric methods, respectively. The continuous-flow technique was proposed for the determination of urea in river and lake waters by Kara et al. [191] in the concentration range of 0.4-8.0 jxmol N L. This automated procedure included the removal of the NH4 initially present in the samples, the decomposition of urea by means of an immobilized urease enzyme reactor, and the final determination of NH4 by a gas-sensing membrane electrode detector system. 

Hara H., Kitagawa T., and Okabe Y. 1993. Continuous-flow determination of low concentrations of urea in natural water using an immobilized urease enzyme reactor and an ammonia gas-sensing membrane electrode detector system. Analyst 118 1317-1320. 

Thus, the first task was to obtain a pH switch from acid to base in the bulk urea-urease reaction in the presence of lipids and to ensure that no significant side reactions took place. Figured A and B shows the beginning and the end of the reaction in a stirred batch reactor, respectively. Panels C and D show the reaction in the presence of [POPC] = 0.5 x 10 M, the colour change indicates that the reaction took place and enough NH3 was produced to raise the pH. 

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