Immobilization of urease using Amberlite MB-1

Amberlite MB-1 was used to immobilize urease (EC.3.5.1.5). The thermal stability of the immobilized urease was better than that of the free urease. Its highest activity was obtained at 75?°C and at pH 6.5 while the optimum temperature for the free urease was found to be 25?°C. Urease immobilized on Amberlite MB-1 retained 65% of the original activity after 5 repeated uses and 62% of the activity after 60 days when stored at 4?°C.     

Immobilization of urease on vermiculite

Urease (EC 3.5.1.5) of high activity was obtained when the enzyme was immobilized on vermiculite crosslinked with 2.5% glutaraldehyde in chilled EDTA-phosphate buffer (pH 5.5). The highest activity of the immobilized enzyme was at 65°C and pH 6.5 while the optimum temperature for free urease was found to be 25°C. The thermal stability of immobilized urease was observed to be much better than that of the free urease. When stored at 4°C, urease immobilized on vermiculite retained 69 to 81% of its activity after 60 days and 61 to 75% of its original activity was retained after 4 repeated uses.     

Stability evaluation of 6-phosphogluconate dehydrogenase immobilized on amino-functionalized magnetic nanoparticles

Abstract

In this study, 6-phosphogluconate dehydrogenase was covalently immobilized onto the N-2-aminoethyl-3-aminopropyltriethoxysilane (APTES) modified core-shell Fe₃O₄@SiO₂ magnetic nanoparticles (ASMNPs) using glutaraldehyde (GA). Immobilization of 6PGDH on ASMNPs was confirmed using fourier transform-infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM) analysis. The NADP⁺ conversion ratio, the reusability, thermal, and storage stability of the immobilized 6PGDH were determined and compared with those of the free enzyme. The maximum retention of enzyme activity reached to 96% when the enzyme was immobilized on ASMNPs activated with monomer form of GA. Although the thermal stability of free and immobilized enzymes was similar, at 30?°C, the immobilized 6PGDH showed the improved thermal stability at 40?°C and 50?°C compared with free 6PGDH. While the free 6PGDH only converted 33% of NADP⁺ in reaction medium upon 480?s, the immobilized 6PGDH performed 56% conversion of NADP⁺ at same time. The immobilized 6PGDH retained 62% of its initial activity up to the fifth cycle and 35% of its initial activity after 22?days of storage at 4?°C.     

Activity and stability of urease entrapped in thermosensitive poly(N-isopropylacrylamide-co-poly(ethyleneglycol)-methacrylate) hydrogel

Urease was entrapped in thermally responsive poly(N-isopropylacrylamide-co-poly(ethyleneglycol)-methacrylate), p[NIPAM-p(PEG)-MA], copolymer hydrogels. The copolymer membrane shows temperature-responsive properties similar to conventional p(NIPAM) hydrogels, which reversibly swell below and de-swell above the lower critical solution temperature of p(NIPAM) hydrogel at around 32 °C. The retained activities of the entrapped urease (in p[NIPAM-p(PEG)-MA]-4 hydrogels) were between 83 and 53 % compared to that of the same quantity of free enzyme. Due to the thermo-responsive character of the hydrogel matrix, the maximum activity was achieved at around 25 °C with the immobilized urease. Optimum pH was the same for both free and entrapped enzyme. Operational, thermal and storage stabilities of the enzyme were found to increase with entrapment of urease in the thermoresponsive hydrogel matrixes. As for reusability, the immobilized urease retained 89 % of its activity after ten repeated uses.     

Immobilization of glucansucrase for the production of gluco-oligosaccharides from Leuconostoc mesenteroides

Glucansucrase from Leuconostoc mesenteroides was immobilized in 1?% (w/v) with sodium alginate to produce oligosaccharides. Glucansucrase gave three activity bands of approx. 240, 178, and 165?kDa after periodic acid-Schiff staining with sucrose. The immobilized enzyme had 40?% activity after ten batch reactions at 30?°C and 75?% activity after a month of storage at 4?°C, which is six times more stable than the free enzyme. Immobilized enzyme was more stable at lower (3.5?4.5) and higher (6.5?7.0) pH ranges and higher temperatures (35?40?°C) compared with the free enzyme. Immobilized and free glucansucrase were employed in the acceptor reaction with maltose and each produced gluco-oligosaccharide ranging from trisaccharides to homologous pentasaccharides.     

Factors affecting ureolytic activities ofPenicillium waksmanii isolated from sewage sludge

Twenty one fungal isolates belonging to 7 genera were screened for ureolytic activity. APenicillium waksmanii isolate was found to be the most potent and was selected for further study. No ammonia-nitrogen was detected inP. waksmanii cultures either urea-free or containing up to 1 g urea per L. The maximum extracellular urease production was recorded at a urea concentration of 15 g/L. It peaked after 6 d of incubation at 25°C when the initial pH of the glucose—peptone broth was adjusted to 6. On the other hand, the highest fungus biomass was detected at a concentration of 2 g urea per L after 4 d of incubation at 35°C when the pH of the medium was 8. The intracellular urease activity (measured in cell-free extract) was the highest at 12 mg urea per mL after 75-min incubation at 25°C at pH 8. Incubation temperature of 25°C favored both urease production and activity.     

High throughput covalent immobilization process for improvement of shelf-life,operational cycles,relative activity in organic media and enzymatic kinetics of urease and its application for urea removal from water samples

High throughput covalent urease immobilization was performed through the amide bond formation between the urease and the amino-functional MNPs. The enzyme’s performances, including shelf-life, reusability, enzymatic kinetics, and the enzyme relative activity in organic media was improved. At optimal conditions, the immobilization efficiency was calculated about 95.0% with keeping 94.7% of the urease initial specific activity. The optimal pH for maximum activity of the free and immobilized urease was calculated as 7.0 at 37.0 °C and 8.0 at 60.0 °C, respectively. The kinetics studies showed the K_m of 26.0 mM and 8.0 mM and the V_max of 5.31 μmol mg⁻¹ min⁻¹ and 3.93 μmol mg⁻¹ min⁻¹ for the free and immobilized urease, respectively. The ratio K_cat/K_m as a measure of catalytic efficiency and enzyme specificity was calculated as 0.09 mg mL⁻¹ min⁻¹ and 0.22 mg mL⁻¹ min⁻¹ for the free and immobilized urease, respectively, indicating an improvement in the enzymatic kinetics. The shelf-life and operational studies of immobilized urease indicated that approximately 97.7% and 88.5% of its initial activity was retained after 40 days and 17 operational cycles, respectively. The immobilized urease was utilized to urea removal from water samples with an efficiency between 91.5–95.0%.     

Simultaneous purification and immobilization of soybean hull peroxidase with a dye attached to chitosan mini-spheres

Soybean hull peroxidase (EC 1.11.1.7, SBP) was simultaneously purified and immobilized by dye affinity chromatography with Reactive Blue 4 attached to chitosan mini-spheres. Under optimized conditions, 96% of SBP was adsorbed to the matrix. Under the most stringent condition, only 49% was desorbed, whereas 2 M NaCl failed to desorb a significant amount of SBP. This behaviour allowed proposing the dye matrix as a support to immobilize SBP from a crude extract. The pH of maximum activity shifted from 7 to 3–5. SBP gained thermostability after immobilization: after 5?h at 85?°C, the remaining activity was 54%, whereas that of the free enzyme was 31%. The optimum temperature for the immobilized SBP was 75?°C, whereas that of the free enzyme was 55?°C. After two months at 4?°C, the activity loss of the immobilized SBP was only 3%. Immobilized SBP removed 80% of 2-bromophenol from wastewater in 180?min and, after five cycles of use, the activity loss was only 12.8%.     

Synthesis of urease hybrid nanoflowers and their enhanced catalytic properties

Increasing numbers of materials have been extensively used as platforms for enzyme immobilization to enhance catalytic activity and stability. Although stability of enzyme was accomplished with immobilization approaches, activity of the most of the enzymes was declined after immobilization. Herein, we synthesize the flower shaped-hybrid nanomaterials called hybrid nanoflower (HNF) consisting of urease enzyme and copper ions (Cu²⁺) and report a mechanistic elucidation of enhancement in both activity and stability of the HNF. We demonstrated how experimental factors influence morphology of the HNF. We proved that the HNF (synthesized from 0.02 mg mL⁻¹ urease in 10 mM PBS (pH 7.4) at +4 °C) exhibited the highest catalytic activity of ∼2000% and ∼4000% when stored at +4 °C and RT, respectively compared to free urease. The highest stability was also achieved by this HNF by maintaining 96.3% and 90.28% of its initial activity within storage of 30 days at +4 °C and RT, respectively. This dramatically enhanced activity is attributed to high surface area, nanoscale-entrapped urease and favorable urease conformation of the HNF. The exceptional catalytic activity and stability properties of HNF can be taken advantage of to use it in fields of biomedicine and chemistry.     

Immobilization of thermostable exo-inulinase from mutant thermophilic Aspergillus tamarii-U4 using kaolin clay and its application in inulin hydrolysis

In this study, attempts were made to immobilize purified exo-inulinase from mutant thermophic Aspergillus tamarii-U4 onto Kaolinite clay by covalent bonding cross-linked with glutaraldehyde with an immobilization yield of 66% achieved. The free and immobilized inulinases were then characterized and characterization of the enzymes revealed that temperature and pH optima for the activity of the free and immobilized enzymes were both 65?°C and pH 4.5 respectively. The free inulinase completely lost its activity after incubation at 65?°C for 6 h while the immobilized inulinase retained 16.4% of its activity under the same condition of temperature and incubation time. The estimated kinetic parameters K_m and V_max for the free inulinase as estimated from Lineweaver-Burk plots were 0.39?mM and 4.21?µmol/min for the free inulinase and 0.37?mM and 4.01?µmol/min for the immobilized inulinase respectively. Inulin at 2.5% (w/v) and a flow rate of 0.1?mL was completely hydrolysed for 10?days at 60?°C in a continuous packed bed column and the operational stability of the system revealed that the half-life of the immobilized inulinase was 51?days. These properties make the immobilized exo-inulinase from Aspergillus tamarii-U4 a potential candidate for the production of fructose from inulin hydrolysis.     

Immobilization and characterization of lipase from an indigenous Bacillus aryabhattai SE3-PB isolated from lipid-rich wastewater

Abstract

Extracellular lipase from an indigenous Bacillus aryabhattai SE3-PB was immobilized in alginate beads by entrapment method. After optimization of immobilization conditions, maximum immobilization efficiencies of 77%?±?1.53% and 75.99%?±?3.49% were recorded at optimum concentrations of 2% (w/v) sodium alginate and 0.2?M calcium chloride, respectively, for the entrapped enzyme. Biochemical properties of both free and immobilized lipase revealed no change in the optimum temperature and pH of both enzyme preparations, with maximum activity attained at 60?°C and 9.5, respectively. In comparison to free lipase, the immobilized enzyme exhibited improved stability over the studied pH range (8.5–9.5) and temperature (55–65?°C) when incubated for 3?h. Furthermore, the immobilized lipase showed enhanced enzyme-substrate affinity and higher catalytic efficiency when compared to soluble enzyme. The entrapped enzyme was also found to be more stable, retaining 61.51% and 49.44% of its original activity after being stored for 30 days at 4?°C and 25?°C, respectively. In addition, the insolubilized enzyme exhibited good reusability with 18.46% relative activity after being repeatedly used for six times. These findings suggest the efficient and sustainable use of the developed immobilized lipase for various biotechnological applications.     

Immobilization of urease on vapour phase stain etched porous silicon

Porous silicon layers fabricated by the reaction-induced vapor phase stain etch method were coated with 5% polyethylenimine. Urease from Canavalia brasiliensis beans was immobilized on this support through covalent linking with 2.5% glutaraldehyde. The pH and temperature profile of the immobilized and free urease exhibited higher activity at pH 6.5 and 37 °C. After being stored for 30 days at 4 °C, the immobilized enzyme had 75% of the initial activity. The maximum apparent Michaelis constant for free urease (K_m) was 94.33 mM whereas for immobilized urease was 53.04 mM. The maximum reaction velocity (V_max) for free urease was 3.51 mmol/min and for immobilized urease was 1.57 mmol/min.     

Study on the potential of cold-active lipases from psychrotrophic fungi for detergent formulation

Lipases from psychrotrophic fungal isolates BPF4 and BPF6 identified as Penicilium canesense and Pseudogymnoascus roseus respectively were characterized for their compatibility towards laundry detergent. BPF4 and BPF6 lipases showed maximum activity at pH 11 and 9 respectively and at 40?°C. The residual activities at 20?°C and 4?°C of BPF4 lipase were 35% and 20% and of BPF6 lipase were 70% and 20?°C respectively. Both the enzymes were stable at 4?°C, 20?°C and 40?°C for 2?h losing at the most 20% of activities. Both the enzymes were metalloenzymes with activity enhancement by nearly threefold by Ca²⁺. Contrary to BPF6 lipase, BPF4 enzyme was not stimulated by EDTA nor inhibited, rather stimulated by SDS and Triton X-100 by 125% and 330% respectively. Both the lipases showed minor to moderate inhibition by NaClO₃ and H₂O₂, and exhibited nearly 90% residual activity after 1?h of incubation in selected detergent brands thus indicating potential for their inclusion in detergent formulation thereby facilitating cold-washing as a step towards mitigation of climate change.     

Development of urea biosensor using non-covalent complexes of urease with aldehyde derivative of PEG and analysis on serum samples

Abstract

Non-covalent complexes of urease/polyethylene glycol (PEG)-aldehyde were synthesized using regular molar ratios of urease and PEG-aldehyde at room temperature. The physical properties of the non-covalent complexes were analyzed in order to investigate the impact of coupling ratio, temperature, pH, storage stability, and thermal stability. Urease activity was analyzed by UV–Vis spectrophotometer at 630?nm. The results showed that the strongest thermal resistance was obtained using n_U/n_PEG:1/1 (mg/mL) complex within all molar ratios tested. The enzymatic activity of n_U/n_PEG:1/1 complex doubled the activity of the free enzyme. Therefore, this complex was chosen to be used in the analyses. When coupled with PEG-aldehyde, urease exhibited improved activity between pH 4.0–9.0 and the optimum pH was found to be 7.0. The thermal inactivation results of the complex demonstrated that higher activity remained (40%) when compared with the free enzyme (10%) at 60?°C. The storage stability of the non-covalent complex was 4 weeks which was greater than the storage stability of the free enzyme. A kinetic model was suggested in order to reveal the mechanism of enzymatic conversion. Potentiometric urea biosensor was prepared using two different membranes: carboxylated poly vinyl chloride (PVC) and palmitic acid containing PVC. The potentiometric responses of both sensors were tested against pH and temperature and the best results were obtained at pH 7.0 and 20–30?°C. Also, selectivity of the suggested biosensors toward Na⁺, Li⁺ Ca²⁺, and K⁺ ions was evaluated and the reproducibility responses of the urea biosensors were measured with acceptable results.     

Immobilization of glucose oxidase on Fe<Subscript>3</Subscript>O<Subscript>4</Subscript>/SiO<Subscript>2</Subscript> magnetic nanoparticles

Glucose oxidase (GOD) was covalently immobilized onto Fe₃O₄/SiO₂ magnetic nanoparticles (FSMNs) using glutaraldehyde (GA). Optimal immobilization was at pH 6 with 3-aminopropyltriethoxysilane at 2% (v/v), GA at 3% (v/v) and 0.143 g GOD per g carrier. The activity of immobilized GOD was 4,570 U/g at pH 7 and 50°C. The immobilized GOD retained 80% of its initial activity after 6 h at 45°C while free enzyme retained only 20% activity. The immobilized GOD maintained 60% of its initial activity after 6 cycles of repeated use and retained 75% of its initial activity after 1 month at 4°C whereas free enzymes retained 62% of its activity.     

Physico-Chemical Characterization of Watermelon Urease upon Immobilization in Alginate Beads

Watermelon (Citrullus vulgaris) urease was immobilized in 3.5% alginate leading to 72% immobilization. There was no leaching of the enzyme over a period of 15 days at 4°C. It continued to hydrolyse urea at a faster rate upto 90 min of incubation. The immobilized urease exhibited a shift of apparent pH optimum by one unit towards acidic side (from pH 8.0 to 7.0). The K_m was found to be 13.3 mM; 1.17 times higher than the soluble enzyme (11.4 mM). The beads were fairly stable upto 50°C and exhibited activity even at ?10°C. The enzyme was significantly activated by ME and it exhibited two peaks of activation; one at lower concentration and another at higher concentration. Time-dependent ureolysis in presence of ME progressed at a much elevated rate. Unlike soluble enzyme, which was inhibited at 200 mM urea, the immobilized enzyme was inhibited at 600 mM of urea and above, and about 47% activity was retained at 2000 mM urea. Moreover, the inhibition caused by high urea concentration was partially abolished by ME. The significance of the observations is discussed.     

Investigation on the immobilization of Pseudomonas isoamylase onto polysaccharide matrices

This study examined Pseudomonas isoamylase immobilized onto polysaccharide matrices, among which included agarose, cellulose, and raw corn starch. For chemical binding of polysaccharides activated with tosyl chloride, a high specific activity of 23144?U/g-starch was obtained as compared with matrices of cellulose and agarose with 3229?U/g-cellulose and 84?U/g-agarose, respectively. For raw corn starch, isoamylase desorption occurred when the immobilized enzyme by physical adsorption was subjected to 0.05?M acetate buffer with pH?5.2 at 40?°C; this is despite the considerable affinity between the enzyme and the matrix. In contrast, no detectable activity leached from the matrix for chemical binding, regardless of whether maltose, i.e. an affinity species to isoamylase, was added. For immobilized starch-isoamylase, its optimal activity performance was obtained in broader pH?ranges of 3.5–5.5 and 5?°C higher than those of the free enzymes. More specifically, the free enzyme's activity markedly decreased within five hours while the immobilized starch-isoamylase exhibited a fairly stable behavior over a three day incubation period at 40?°C. After 175 days of storage at 4?°C, the residues of relative activity of 75% and 45% were obtained with respect to immobilized and free isoamylases, respectively.     

Immobilization of watermelon (<Emphasis Type="Italic">Citrullus vulgaris</Emphasis>) urease in agarose gel for urea estimation

Urease from dehusked seeds of watermelon was immobilized in 1.5% agarose gel with 53.9% entrapment. There was negligible leaching (<10% at 4°C) and the same gel membrane could repeatedly be used for seven days. The immobilization exhibited no apparent change in the optimum pH but there was a significant decrease in the optimum temperature (50°C as compared to 65°C for soluble urease). The immobilized urease revealed an apparentK _m of 9.3±0.3 mM; 1.2 times lower than the soluble enzyme (11.4±0.2 mM). Unlike soluble enzyme which was inhibited at 200 mM urea, the immobilized urease was inhibited at 600 mM of urea and above, and about 47% activity was retained at 2 M urea. The time-dependent thermal inactivation kinetics at 48 and 52°C was found to be biphasic, in which half of the initial activity was destroyed more rapidly than the remaining half. These gel membranes were also used for estimating the urea content of the blood samples from the University hospital. The results obtained matched well with those obtained by the usual method employed in the clinical pathology laboratory. The significance of these observations is discussed.     

Enzymatic detection of heavy metal ions in aqueous solution from vegetable wastes by immobilizing pumpkin (<Emphasis Type="Italic">Cucumis melo</Emphasis>) urease in calcium alginate beads

Enzyme urease is extracted from the discarded seeds of pumpkin. Urease was purified to apparent homogeneity (5.2 fold) by heat treatment at 48 ± 1°C and gel filtration through Sephadex G-200. Effect of model metal ions on the activity of the homogeneous enzyme preparation (sp. activity 353 U/mg protein, A₂₈₀/A₂₆₀ = 1.12) of soluble as well as immobilized enzyme was investigated. The soluble and immobilized urease has been used for the quantitative estimation of general water pollution with heavy metal ions like Hg²⁺, Cu²⁺, Cd²⁺, and Co²⁺. The measurements of the urease residual activity have been carried out in tris-acetate buffer after pre-incubation of model metal salt. The inhibition was found to be biphasic with an initial rapid loss of activity and remainder in slow phase of 10∼15 min. The immobilization was done in 3.5% alginate beads leading to 86% of entrapment. There was no leaching of the enzyme over a period of 15 days at 4°C. The beads were fairly stable up to 50°C and exhibited activity even at −10°C. The inhibition by these ions was non-competitive and irreversible, hence could not be restored by dialysis. Based on the values of inhibition constant Ki the heavy-metal ions were found to inhibit urease in the following order Hg²⁺ > Cu²⁺ > Cd²⁺ > Co²⁺.     

Viability and Morphology of rat heart cells after freezing and thawing of the whole heart

Intact adult rat hearts were cooled in the presence of 10% DMSO according to an external cooling program which approximated the optimal external three-step cooling program for the isolated adult heart cells: 20 min at ?20 °C, 0.2 °C/min from ?20 to ?25, ?30, or ?50 °C, and rapid cooling to ?196 °C. Following rapid thawing, cells were isolated after perfusion with a 0.1% collagenase solution. Only cells which originated from the free wall of the right ventricle could be isolated, even after cooling to ?20 °C. Most cells from hearts cooled to ?196 °C did not survive. When the third cooling step was omitted and the end temperature of the second cooling step was ?30 °C, 38% of the cells excluded trypan blue, 29% were morphologically intact, and 30% showed spontaneous contractions after thawing, expressed as percentages of the control, A much lower survival was found after cooling to ?50 °C.Histological and electron microscopical study of the heart immediately after thawing revealed no differences between hearts cooled to ?20, ?30, or ?196 °C. Also no marked differences were observed between the morphological integrity after freezing and thawing of the atrium, the left and right ventricle walls, and the ventricular septum. The survival data suggest the presence of nonmorphologically detectable alterations in cells frozen to ?196 °C, compared to cells frozen to ?30 °C. The morphological investigations indicate no essential differences in resistance of atrial and ventricular cells to the freezing process.Experiments involving neonatal rat hearts cooled to ?196 °C, according to the method which gave optimal preservation of the isolated cells, revealed that after thawing cells are present from which growing and contracting cultures can be derived. It appears that cells in the neonatal rat heart are more resistant to freezing to ?196 °C than cells in the adult rat heart.