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.     

Studies on the deactivation of immobilized urease

The results of experiments in a fixed-bed reactor and a CSTR containing urease 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 the product ammonia attacked the free form of the enzyme and thereby illustrates the importance of mechanism in determining deactivation kinetics.     

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.     

Stabilization of enzyme immobilized in temperature-sensitive hydrogels

-Galactosidase was immobilized in a crosslinked poly(N-isopropylacrylamide-co-acrylamide) hydrogel which exhibits an LCST(lower critical solution temperature) behavior. The hydrogel collapses above the LCST, and expands below the LCST. The temperature-dependent phase transition was around 37 °C. The stability of immobilized enzyme was investigated at different temperatures which allow different degrees of collapse in the hydrogel matrix. It was hypothesized that the immobilzed enzyme is more stable in the collapsed matrix due to the physical restraint imposed on the enzyme entrapped.     

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     

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.     

Amperometric electrode for determination of urea using electrodeposited rhodium and immobilized urease

An amperometric biosensor was developed for determination of urea using electrodeposited rhodium on a polymer membrane and immobilized urease. The urease catalyzes the hydrolysis of urea to NH₄⁺ and HCO₃⁻ ions and the liberated ammonia is catalytically and electrochemically oxidized by rhodium present in the rhodinized membrane on the Pt working electrode. Three types of rhodinized polymer membranes were prepared by varying the number of electrodeposition cycles: membrane 1 with 10 deposition cycles, membrane 2 with 40 cycles and membrane 3 with 60 cycles. The morphologies of the rhodinized membranes were investigated by scanning electron microscopy and the results showed that the deposition of rhodium was like flowers with cornices-like centers. The influence of the amount of electrodeposited rhodium over the electrode sensitivity to different concentrations of ammonia was examined initially based on the cyclic voltammetric curves using the three rhodium modified electrodes. The obtained results convincingly show that electrode with rhodinized membrane 1, which contain the lowest amount of electrodeposited rhodium is the most active and sensitive regarding ammonia. It was found that the anodic oxidation peak of ammonia to nitrogen occurs at 0.60 V. In order to study the performance of urease amperometric sensor for the determination of urea, experiments at constant potential (0.60 V) were performed. The current–time experiments were carried out with urease rhodinized membrane 1 (10 cycles). The amperometric response increased linearly up to 1.75 mM urea. The detection limit was 0.05 mM. The urea biosensor exhibited a high sensitivity of 1.85 μA mM⁻¹ cm⁻² with a response time 15 s. The Michaelis–Menten constant K_m for the urea biosensor was calculated to be 6.5 mM, indicating that the immobilized enzyme featured a high affinity to urea. The urea sensor showed a good reproducibility and stability. Both components rhodium and urease contribute to the decreasing of the production cost of biosensor by avoiding the use of a second enzyme.     

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.     

Stabilization of immobilized cathepsin L in non-aqueous medium

A lysosomal cysteine protease cathepsin L (3.4.22.15) purified from goat brain has been immobilized in calcium alginate beads in the presence of BSA through entrapment. Most favorable conditions for the entrapment were standardized as 3.0%(w/v) alginate and 1.5%(w/v) calcium chloride. Comparing the properties of free and immobilized enzyme using Z-Phe-Arg-4mβNA as chromogenic substrate, it was found that the immobilized enzyme could retain～70% of the original activity after five successive batch reactions. Vis-à-vis the free enzyme, immobilization conferred high stability to the enzyme both in the acidic and alkaline range, the enzyme lost no activity up to 60°C (Temperature stability for free enzyme is only up to 50°C). The pH optima for the enzyme shifted from 6.2 to 6.6 on entrapment. The increase in activity and stability of the enzyme in immobilized form even in the presence of high concentration of DMSO and ethanol is surprising and may make it useful for catalyzing organic reactions like trans-esterification and trans-amidation.     

Preparation and characterization of urease immobilized onto porous chitosan beads for urea hydrolysis

Urease was covalently immobilized onto porous chitosan beads via primary amine groups connected to the backbone via a six-carbon linear alkyl spacer. The optimum conditions for enzyme immobilization are activating the beads with 1%(w/w) glutaraldehyde, reacting the activated beads in pH 7 buffer with the enzyme, using an enzyme to bead weight ratio of 25, and without lyophilization. Chitosan-bound urease was found to fully retain its specific activity. Properties of the immobilized urease were characterized under batch and flow conditions. Increased optimum reaction temperature, enhanced thermal stability and storage stability, and excellent reusability were found after enzyme immobilization. Continuous hydrolysis of urea solution was studied in a column packed with the enzyme-containing beads for its possible application in regenerating dialysate solution during hemodialysis.     

Stabilization of immobilized lectin columns by crosslinking with glutaraldehyde

Procedures to purify membrane proteins usually require the use of detergents and often include affinity chromatography on lectin columns. Some detergents, especially denaturing detergents such as sodium dodecyl sulfate (SDS), can interfere with affinity chromatography by inactivating the bound lectin or by eluting it from the column together with the material of interest. We have developed a procedure that stabilizes lectin-column matrices by crosslinking with glutaraldehyde. This procedure does not impair the binding capacity of the immobilized lectin. It permits subsequent elution by SDS of bound glycoproteins without coelution of lectin subunits.     

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.     

Stabilization of native protein fold by intein-mediated covalent cyclization

A mutant version of the N-terminal domain of Escherichia coli DnaB helicase was used as a model system to assess the stabilization against unfolding gained by covalent cyclization. Cyclization was achieved in vivo by formation of an amide bond between the N and C termini with the help of a split mini-intein. Linear and circular proteins were constructed to be identical in amino acid sequence. Mutagenesis of Phe102 to Glu rendered the protein monomeric even at high concentration. A difference in free energy of unfolding, DeltaDeltaG, between circular and linear protein of 2.3(+/-0.5) kcal mol(-1) was measured at 10 degrees C by circular dichroism. A theoretical estimate of the difference in conformational entropy of linear and circular random chains in a three-dimensional cubic lattice model predicted DeltaDeltaG=2.3 kcal mol(-1), suggesting that stabilization by protein cyclization is driven by the reduced conformational entropy of the unfolded state. Amide-proton exchange rates measured by NMR spectroscopy and mass spectrometry showed a uniform, approximately tenfold decrease of the exchange rates of the most slowly exchanging amide protons, demonstrating that cyclization globally decreases the unfolding rate of the protein. The amide proton exchange was found to follow EX1 kinetics at near-neutral pH, in agreement with an unusually slow refolding rate of less than 4 min(-1) measured by stopped-flow circular dichroism. The linear and circular proteins differed more in their unfolding than in their folding rates. Global unfolding of the N-terminal domain of E.coli DnaB is thus promoted strongly by spatial separation of the N and C termini, whereas their proximity is much less important for folding.     

Reduction of nitro-substituted compounds by native and immobilized Escherichia coli cells

Reduction of nitro-substituted compounds, 1,4-benzodiazepine-2-ones, dibenzo[b,f]-1,4-diazepines, quinolones, and quinoxalinones, by Escherichia coli cells was studied. Physicochemical methods demonstrated the formation of corresponding amines. 4-(p-Nitrophenyl)-1H-6-R-quinolones-2 were nor reduced by Escherichia coli cells. Regiospecific reduction of 2,4-dinitro-5H-11-(p-R-phenyl)-dibenzo[b,f]-1,4-diazepines and 4-(2'-R-3',5'-dinitro)-benzoyl-3,4-dihydroquinoxalinones-2 was shown to result in the formation of 2-nitro-4-amino-5H-11-(p-R-phenyl)-dibenzo[b,f]-1,4-diazepines and 4-(2'-R-3'-nitro-5'-amino)-benzoyl-3,4-dihydroquinoxalinones-2, respectively. Methods for microbiological reduction of nitro compounds and immobilization of Escherichia coli cells into carrageenan and its modified forms were elaborated.     

Stabilization of fly head cholinesterase immobilized on a gelatin membrane

Immobilization of meat fly head cholinesterase in gelatin membrane essentially increases the enzyme stability and changes its kinetic characteristics: the Michaelis constant and maximum velocity of acetylthiocholine hydrolysis and also biomolecular velocity constant of phosphorylation by DDVP.     

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.     

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.