Stabilization of native and immobilized urease

The stability of native and immobilized urease isolated from Staphylococcus saprophyticus was studied at 4 degrees and 25 degrees C. The activity yield was 20% and 1.4% on the enzyme immobilization in albumin gel and latex membrane, respectively. Inactivation of native microbial urease proceeded 10 times slower in the solution containing 1 mM EDTA and 30 mM sodium sulfite. This solution contributed to a great extent to stabilization of immobilized urease both during storage in the phosphate buffer solution and in case of lyophilization.     

Properties of urease immobilized on the functional organic silica surface

The paper deals with kinetics of the urea hydrolysis by microbial-origin urease dissolved and immobilized on the organic silica surface. It is shown that hydrolysis kinetics for soluble urease is described by the Michaelis-Menten equation until the concentration of urea reaches 1 M. Two fractions differing in the Michaelis constant are revealed for silochrome immobilized urease. The rate of urea hydrolysis by native and immobilized urease was studied depending on the pH value in presence of the substrate in the 1 M and 5 mM concentration. The hydrolysis rate of 1 M urea in the buffer-free solution by silochrome-immobilized urease is practically independent of pH within 4.5-6.5. Application of a 2.5 mM phosphate-citrate buffer as a solvent causes an increase in the hydrolysis rate within this pH range. For a soluble urease the 1 M urea hydrolysis rate dependence on pH is ordinary at pH 5.8-6.0. If the substrate concentration is 5 mM, the pH-dependences for the rate of the urea hydrolysis by silochrome- and aerosil-immobilized urease are close and at pH above 6.0 coincide with those for a soluble enzyme. The found differences in the properties of soluble and immobilized ureases are explained by the substrate and reaction products diffusion.     

Electric field effects on immobilized urease activity.

The behavior of an enzyme/membrane system containing urease is studied when an external electric field is applied. The device using a potential difference across the enzyme/membrane system is first described. Optimal operating conditions with respect to substrate concentration, ionic strength and pH are studied. Possible mechanisms of the change in membrane activity by electric field are discussed.     

Preparation and characterization of kappa-carrageenan immobilized urease

Urease was encapsulated within kappa-carrageenan beads. Various parameters, such as amount of kappa-carrageenan and enzyme activity, were optimized for the immobilization of urease. Immobilized urease was thoroughly characterized for pH, temperature, and storage stabilities and these properties were compared with the free enzyme. The free urease activity quickly decreased and the half time of the activity decay was about 3 days at 4 degrees C. The immobilized urease remained very active over a long period of time and this enzyme lost about 70.43% of its orginal activity over the period of 26 days for storage at 4 degrees C. The Michaelis constant (Km) and maximum reaction velocity (Vmax) were calculated from Lineweaver-Burk plots for both free and immobilized enzyme systems. Vmax = 227.3 U/mg protein, Km = 65.6 mM for free urease and Vmax = 153.9 U/mg protein, Km = 96.42 mM for immobilized urease showed a moderate decrease of enzyme specific activity and change of substrate affinity.     

Investigation of deactivation thermodynamics of lipase immobilized on polymeric carrier

In the present work, we have investigated biochemical thermo-kinetic stability of lipases immobilized on a biocompatible polymeric material. Immobilization of lipase Candida rugosa (CRL) was carried out on biocompatible blend of poly vinyl alcohol (PVA) and chitosan (CHY) support via entrapment and glutardehyde (Glu) cross-linking method to produce PVA:CHY:CRL and PVA:CHY:Glu:CRL as robust biocatalyst. These immobilized lipases were characterized by various physico-biochemical characterization techniques. Later on, thermal and solvent stability of polymer immobilized lipase was determined in term of half-life time (t _0.5), D values, enthalpy (ΔH°), entropy (ΔS°), and free energy (ΔG°) of deactivation at different temperatures and in various solvents. The thermodynamic deactivation stability trend was found as: cross-linked lipase CRL > entrapped lipase CRL > free lipase CRL. Moreover, kinetic parameters, such as K _m, V _max, and catalytic efficiency, were also determined to understand the kinetic features. The polymer immobilized enzyme was reused to investigate the economic viability of the developed biocatalyst.

     

Properties of urease immobilized on silochrome by means of disulfide bonds

Grafting of SH-groups to the silica surface through the hydrolytically stable Si-C-bond is conducted by gamma-mercaptopropyltrimethoxysilane. After 2,2'-dithiobis-p-nitrobenzoic acid (Ellman's reagent) activation of sulphydryl groups urease of microbial origin was immobilized by these carriers. Certain properties of the preparations obtained were studied. The Km of the enzyme during nonporous silicon aerosil immobilization is shown to remain without considerable changes. The found variations in properties of silochrome-immobilized urease are caused by the diffusion inhibition for the substrate and product of the reaction observed even when the substrate concentration is two orders higher than Km.     

A deactivation model for immobilized and soluble enzymes

Summary A first-order deactivation model which includes a step that does not destroy enzyme activity, but is a compulsory precursor of the step that does, is shown to fit reasonably well the immobilized and soluble enzyme deactivation data presented. The deactivation rate coefficient increases with time, and the model may be generalized to include n such compulsory precursors.     

A potentiometric sensor designed on the basis of urease immobilized in polyelectrolyte microcapsules

A potentiometric biosensor has been designed on the basis of glass pH-electrode with a sensing device of the microcellular polyelectrolytic coating containing urease. The polymeric walls of the coating are readily permeable for low-molecular weight compounds, including urea, but are impermeable for macromolecules. The main characteristics of the biosensor in various experimental solutions containing urea, low-molecular-weight salt, and buffer have been obtained. The sensor has been shown to be stable for at least three weeks. The standard curves of the sensor are linear in the range of urea concentrations from 0.2 to 20 mM.     

On the mechanism of immobilized glucose oxidase deactivation by hydrogen peroxide

The result of experiments in a fixed-bed reactor containing glucose oxidase immobilized on a nonporous support and conducted in the absence of diffusional limitations are reported. Kinetic parameters were established by separate batch experiments. The key observation was that, in every case, poisoning by product hydrogen peroxide resulted in a minimum in enzyme activity in the interior of the bed, well away from the ends. The deactivation data were interpreted by fitting the rate constant for poisoning, the only free parameter, to a previously reported theory. The theory postulates several deactivation mechanisms each of which leads to self-consistent kinetics, but the only mechanism which fitted the data assumes that peroxide attack the enzyme when it is the from complexed with glucose. Theory and experiment agreed to within the accuracy (+/- 2%) of the activity measurements.     

Studies on immobilized papain

Papain was covalently coupled to ZrO_2?coated porous glass by several different methods. These derivatives were characterized and their operational half-lives determined using casein substrate. Papain covalently coupled to the porous glass, previously converted to a carboxylic acid derivative, through amide linkage gave a 35 day operational half-life.     

A deactivation protection model for immobilized and soluble enzymes

A model is proposed in which a deactivated enzyme protects an active enzyme from deactivation. Enzyme modifiers, when present, may also appear to induce or enhance enzyme protection by providing intra-molecular covalent cross-links to ‘lock-in’ protein conformation and facilitate enzyme protection by the deactivated enzyme. In both cases a first-order deactivation protection model is shown to fit the immobilized and soluble enzyme deactivation data presented reasonably well. Steric hindrances and the immobile nature of the enzyme-support link of immobilized enzymes would appear to lessen the extent of this protection.     

Effect of immobilized enzyme deactivation in fixed- and fluid-bed reactors

A two-parameter theoretical model is developed to evaluate the effect of immobilized enzyme deactivation on substrate conversion in fixed- and fluid-bed reactors under diffusion-free conditions. The method describes a simple reaction in which three different immobilized enzyme deactivation forms are considered, and an expression is developed to evaluate the effect of immobilized enzyme deactivation on yield in a consecutive reaction. Comparison of reactor performances for the two reactor types reduces to a comparison of the appropriate dimensionless parameters. The practical implications of the development are illustrated through an example.     

Flow rate dependent kinetics of urease immobilized onto diverse matrices

Gas holdup and liquid circulation velocity meassurements were made for a range of liquid viscosities in a 22 l external loop airlift column and 250 l pilot-scale concentric cylinder airlift bioreactor. The results showed that for a fixed superficial gas velocity, liquid circulation velocity decreased monotonically with increasing liquid viscosity. The gas holdup for a fixed gas flow rate showed an initial increase with liquid viscosity followed by a decrease when liquid viscosity increased beyond a critical value. These observations could not be described satisfactorily using the available models of gas holdup and liquid circulation.List of Symbols U _sg m/s Superficial gas velocity - U _sl m/s Superficial liquid velocity in the riser Greek Letters Pas Liquid viscosity - _g Gas holdup in the riser     

Urea potentiometric biosensor based on modified electrodes with urease immobilized on polyethylenimine films

The development of a new electrochemical sensor consisting in a glass-sealed metal microelectrode coated by a polyethylenimine film is described. The use of polymers as the entrapping matrix for enzymes fulfils all the requirements expected for these materials without damaging the biological material. Since enzyme immobilization plays a fundamental role in the performance characteristics of enzymatic biosensors, we have tested four different protocols for enzyme immobilization to determine the most reliable one. Thus the characteristics of the potentiometric biosensors assembled were studied and compared and it appeared that the immobilization method leading to the most efficient biosensors was the one consisting in a physical adsorption followed by reticulation with dilute aqueous glutaraldehyde solutions. Indeed, the glutaraldehyde immobilized urease sensor provides many advantages, compared to the other types of sensors, since this type of urea biosensor exhibits short response times (15–30 s), sigmoidal responses for the urea concentration working range from 1×10^−2.5 to 1×10^−1.5 M and a lifetime of 4 weeks.     

A new process for the treatment of fertilizer effluent using immobilized urease

A method is reported for the treatment of industrial fertilizer effluent rich in urea by a new coupling method to immobilize crude urease onto polyester which is having high flow through property in columns. Kinetics of the immobilized enzyme is established in small column. A typical treatment process with two larger columns in packed bed mode is discussed with and without recycling the treated effluent.     

A deactivation model involving pH for immobilized and soluble enzymes

A pH-dependent deactivation model is developed for immobilized and soluble enzymes in which the rate of enzyme decay is assumed proportional to the concentration of theactive enzyme. The model developed applies reasonably well to pH-stability data of Johnson and coworkers (1977 a,b,c; 1978 a,b,c).