Dioxane enhanced immobilization of urease on modified macro-porous silica
Various methods of immobilization of urease already have been reported including covalent bonding, entrapment, physical adsorption and micro-encapsulation.In this study, efficient immobilization of urease was achieved upon dioxane-induced denaturation on alkylated macro porous silica with an average pore size 60 nm. Structural exploration of the urease to find the optimum condition which provides maximum accessible hydrophobic surfaces of the enzyme molecule was carried out using spectroscopic methods. Also the effect of dioxin on the activity of enzyme was considered by Berthelot reaction. According to these results, the optimum immobilization was achieved at 30 % (v/v) dioxane. Results indicate that enzyme will be immobilized on the alkylated macro porous silica with high efficiency.
Urease is enzyme widely occurring in nature and is found in numerous organisms, including plants, bacteria, yeast, algae, fungi and invertebrates. Urease is used for monitoring of urea which is very important in biomedical and clinical approaches . Also in blood dialysis , agro-food chemistry  and environmental monitoring  urease have been considered in applied view of the interest. Recently, determination of urea has been developed through urease based biosensors, as an analytical device .
Numerous methods have reported for urease immobilization . Among them, conventional adsorptions of urease have been associated with the disadvantage of low loading and leaching of the enzyme which result in low sufficiency in various applications . However, we used a novel dioxane-induced high loading urease immobilization on octadecyl substituted porous-silica upon improved hydrophobic interactions in organo-aqueous media.
The present article addresses the in efficiencies of adsorption immobilization methods of urease through reversible dioxane enhanced surface hydrophobicity of the enzyme on octadecyl-porous silica.
Macro porous silica with an average pore size 60 nm, octadecyltrichlorosilane (OTS), potassium dihydrogen phosphate, di-potassium hydrogen phosphate, phenol, sodium hydroxide, sodium hypochlorite solution (5% available chlorine), toluene, dioxane, urea and ammonia were purchased from Merck. 8-anilino-1-naphthalen sulfonic acid (ANS), sodium nitroprusside (sodium pentacyanonitrosyloferrate III) and urease (EC 184.108.40.206) from Jack beans were purchased from Sigma. All chemicals were of analytical reagent grade and distillated water was used in all experiment.
Urease assay activity
Enzyme assay was carried out on Berthelot (phenol-hydrochlorite) reaction . This method is based on the amount of ammonia which is released by hydrolysis of urea. Resulted ammonia (NH3) reacts with hypochlorite (OCl-) to form a monochloramine which can be read out at λmax=625 nm.
In brief, solutions A and B prepared for urease activity assay in which solution (A) contained of 5.0 gr phenol and 25 mg of sodium nitroprusside and solution (B) contained of 2.5 gr sodium hydroxide and 4.2 ml of sodium hypochlorite in 500 ml of distillated water. These solutions can be stored for one month in amber bottle and 4°C. Potassium phosphate buffer 0.1 M, pH 7.6 that was prepared by distillated water and dioxane in a different ratios 0-50% (v/v).
The enzyme activity was measured by addition of 1 ml buffer phosphate solution 0.1 M, and urea substrate solution with a final concentration of 25 mM. The mixture was incubated at 37°C for 30 min. Then 20 µl of the obtained incubated solution was removed and added to a vial containing 500 µl of solution A. Then equal volume of solution B(500 µl) was added, mixed well and incubated at 37 °C for 30 min. The resulted blue solution absorbance was measured photometrically at 625 nm against control solution.
The samples of urease solution for dioxane-induced unfolding studies were prepared using potassium phosphate buffer at different ratios of dioxane: water at 0-50% (v/v).
A structural study of the enzyme was carried out at 280 nm on Camspac (Modle-550) UV spectrophotometer. The final concentration of enzyme was 0.2 mg.ml-1
Fluorescence analyses of the urease at different ratios of dioxane were carried out in Hitachi (Model MPF-4) spectrofluorimeter in quartz cuvette with 1 cm path length. All the measurements were made at 25°C using the 0.1 mM enzyme, for each experimenting appropriate blank was used for base line correction of fluorescence intensity. The intrinsic fluorescence was recorded in the emission wavelength ranging from 300 to 500 nm after excitation of the enzyme at 280 nm. The slit width for both excitation and emission was set at 5 nm. For binding studies of ANS, urease samples at different ratios of dioxane with final concentration of 5 mM ANS were prepared. The fluorescence of ANS was excited at 350 nm and emission was collected between 450 to 600 nm. Assays in absence of the urease were performed in order to correct the measurement against the unbound ANS emission fluorescence intensities.
Circular dichroism measurements
Circular dichroism spectral measurements were carried out on AVIV (Model 215) spectropolarimeter. All the measurements were performed at 25°C. Far-UV measurements were recorded in the range of 200 to 300 nm using concentration of 0.2 mg.ml-1 with 1 mm path length cell. The results were expressed as ellipticity [θ (deg cm2 / d mol -1)]. The data were analyzed by CDNN software.
Preparation of alkylated porous silica
150 ml toluene was dried by agitating with 30 g anhydrous calcium chloride overnight, followed by distillation. Macro- porous silica with an average pore size of 60 nm was dried under vacuum. OTS was dissolved in dried toluene and added to dry matrix. Alkylation was resulted by heating to reflux overnight. The attachment of the octadecyl moieties on macro- porous silica was approved by using FTIR spectrophotometery (Equinox 55, Bruker Company). IR spectra were recorded in the range 4000- 400 cm-1, using KBr pellets. Spectra were calculated from a total of 18 scans. The mole number of alkyls per gram of macro-porous silica was calculated by thermogravimetric analysis (TGA) using a TAinst Q50, thermal analysis instrument.
For enzyme immobilization, the solution of enzyme at 0.3 mg.ml-1 was prepared in 50 mM phosphate buffer pH 7.6 consisted with different concentration of dioxane in the range of 0-50% (v/v).100 mg of matrix was hydrated thorough successive washing steps with a serial solvent/water solutions . Then enzyme solution in desired polarity condition due to the appropriate concentration of dioxane was added on matrix and incubated for 2 hours at 4ºC with rotary mixing.
Urease establishes adsorptive interaction with alkyl groups in synthesized matrices according to the enhanced accessible surface hydrophobicity of the enzyme molecules. Then samples were centrifuged for 5 min at 13000 rpm in 4ºC (Heraeus Labofuge 400R) and washed with distilled water (dioxane free) to restore protein folded state but immobilized state.
Results and discussion
Unfolding/refolding of plenty of globular proteins has been well described in terms of two state (native↔denature) models. However, many studies have showed that the protein folding involves a discrete pathway with distinct intermediate states between native and denatured states . These intermediate states have been observed under different conditions which do not appear to be native or completely unfolded states. The analysis of these unfolding and refolding intermediates gives an insight to the folding pathways of proteins. Therefore it is important and necessary to identify and define the partially folded conformations of proteins in the protein folding process.
The partially folded intermediates can be made to accumulate in equilibrium either by low concentrations of chemical denaturants  or low pH  or organic solvents .
We use organic solvent to turn-unfold of urease enzyme. Dioxane is an organic solvent which is miscible (without phase forming behavior) with water. Therefore does not disrupt enzyme activity . The organic solvent induced structural changes of urease enzyme were monitored by intrinsic fluorescence, ANS binding and circular dichroism spectroscopic studies.
Intrinsic fluorescence spectra provide a sensitive means to characterize proteins and their conformations. The spectrum is determined mainly by the polarity of the protein environment and by their specific interactions. The emission maximum (λmax) is an excellent parameter commonly used to monitor polarity of Trp, and is sensitive to conformational changes. Intrinsic fluorescence spectra of urease enzyme under native state and unfolded states are shown in figure 1A and the maximum emissions (λmax) of urease at different percent of dioxane are shown in figure 1B.
Circular dichroism spectra of proteins in the far-UV region are particularly sensitive to protein secondary structure and essentially used to monitor the conformation of the polypeptide backbone. Figure 2 shows the effect of dioxane in different percents on the mean residue ellipticity (MRE) of the urease in far-UV regions. The spectrum for native state has a minimum at 220 nm which is characteristic feature of α-helical structure. Up to 20% dioxane the helicity was increased due to stabilization of the hydrogen bands in the first phase of treatment process. However at higher percent concentration of dioxane the percent of α-helix conformational was decreased. Which means protein exposes its hydrophobic portion to the interface.
ANS, a fluorescence hydrophobic probe widely used to detect the hydrophobic regions of proteins. The exposure of hydrophobic surfaces of different states of urease enzyme was monitored by changes in ANS fluorescence. A comparison of ANS fluorescence emission spectra of urease at different percent of dioxane in the 450–600 nm range is shown in Fig.3A. As can be seen from the figure, the binding of ANS from the native state to unfolded state increased in fluorescence intensity.
The ANS fluorescence emission maximum (λmax) of the enzyme at different percent of dioxane is shown in Fig. 3B. The λmax of urease native state was found to be at 530 nm and decreased to 508nm in the 40% dioxane. This blue shift in emission maximum (λmax) indicated the exposure of hydrophobic regions of the enzyme molecule. A low binding of ANS to urease at native state is decreased until 20% dioxane .This amount apparently related to non-accessibility of hydrophobic regions that confirmed with CD spectra. These observations further suggest that 20% dioxane state becomes a compact molecule with the minimum exposure of hydrophobic clusters and maximum percent of α -helix.
Dioxane is a hydrophobic solvent. When the urease-dioxane interactions favor exclusion of the solvent molecules from the enzyme hydration layer, compaction of the enzyme occurs because of the unfavorable surface energy rise, and this stabilizes the enzyme.
The increase levels of ANS binding to urease in higher than 20% dioxane show exposure of hydrophobic clusters and enzyme unfolding.
Preferential binding leads to denaturation via interaction of the dioxane with nonpolar residues and the concomitant disruption of electrostatic interactions (including H-bonds).
Urease enzyme has low tendency for binding to a hydrophobic support in its native form. Urease is unfolded by dioxane and exposes maximum hydrophobic interfaces during with refolding protein in water. Moreover, substituted alkyl chains on macro-porous silica which are in collapsed state in aqueous conditions are raised to present themselves at dioxane treated condition. The later, improves capability of the matrices to involve efficiently in hydrophobic interactions. Therefore, both of adsorbent and adsorbant are improved to involve more efficiently in adsorption process upon applied dioxane-enhanced immobilization strategy.
Adsorptive of urease with hydrophobic support in the absence or presence of and organic solvent were compared. Figure 4 shows the comparison of urease activity that immobilized in different intermediate structures.
As evident from the figure, a slight decrease in enzyme activity value was observed from native state to 20% dioxane so that enzyme activity was minimal for immobilization at 20% dioxane. A maximum enzyme activity was observed for enzyme immobilization at 30% dioxane induced state. These results were confirmed by fluorescence and CD measurements.
Hence, the results obtained in the present study demonstrate that the existence of a transient intermediate state during unfolding of urease is a process that happens in dioxane. Urease unfolded at 30% dioxane exhibits the characteristics of ‘molten globule' MG state. The (MG) state has been known to be an intermediate state between the native and unfolded states. The MG state is characterized by the presence of substantial secondary structure arranged in a native-like overall fold, a compact shape with slightly larger than the native protein, formation of a hydrophobic core exposed to solvent and lacking in detectable tertiary structure. The results indicate that a MG state of urease at 30% dioxane have been observed by fluorescence, CD, ANS binding studies and urease immobilization on the hydrophobic support.
(A) Intrinsic fluorescence emission spectra of urease at 0-50% dioxane. The excited was 280nm and emission was recorded between 300 and 500 nm.
(B) Intrinsic fluorescence emission maxima (λmax) of urease enzyme at 0- 50% dioxane.
Far-UV circular dichroic spectra of urease enzyme.
The spectra were recorded in the wavelength region 200–300nm using protein concentration of 0.2 mg/ml at 0-40% of dioxane.
(A) ANS fluorescence emission spectra of urease at 0- 50% dioxane. Spectra were recorded in the region of 450–600 nm after exciting at 350nm at 25 ◦C.
(B) Fluorescence emission maxima (λmax) of urease enzyme at 0- 50% dioxane urease enzymes activity that immobilized in 0- 40% dioxane environment.