Immobilization and Activity of Pepsin in Silicone Elastomers

Pepsin [EC, 3.4.23.1] from Porcine stomach mucosa was immobilized in silicone elastomers utilizing condensation-cure room temperature vulcanization (RTV) of silanol-terminated poly(dimethylsiloxane) (PDMS). Two network precursor chain molar masses were used in this investigation: in pepsin–silicone (A), M _n ∼26,000 g mol⁻¹ and in pepsin–silicone (B) M _n ∼750 g mol⁻¹. Tetraethyl orthosilicate (TEOS) was used as the cross-linking agent and dibutyltin dilaurate was used as the catalyst. The activity and stability of free pepsin and pepsin immobilized in PDMS were studied with respect to pH, temperature, cross-link density, solvents and storage time using a hemoglobin assay. A notable finding is that free pepsin has zero activity in neutral buffer solution (pH 7) after incubation for 5 h, while pepsin immobilized in the silicone elastomers was found to retain more than 70% of its maximum normalized activity. There was no marked improvement in the thermal stability of the PDMS immobilized pepsin when compared to free pepsin and all the three systems showed no activity at and above 70 °C. From the Lineweaver–Burk kinetic analyses, the apparent K _m (g L⁻¹ hemoglobin) for free pepsin was 4.5, for pepsin–silicone (A) was 5.1, and for pepsin–silicone (B) was 3.9, the V _max (U/mg of pepsin) for free pepsin was 14,000, for pepsin–silicone (A) was 11,710, and for pepsin–silicone (B) was 8,510, respectively after incubation in buffer solution at pH 2 and 37 °C. The activity of the free and the PDMS immobilized pepsin in six different organic solvents was also studied. The pepsin retained high activity in non-polar solvents such as n-hexane, isooctane and toluene, but the enzyme performed poorly in methanol, ethanol and tetrahydrofuran. The degree of swelling of the pepsin immobilized silicone elastomers in these solvents had no impact on the activity of the pepsin. When stored at room temperature for time periods up to 6 months, pepsin immobilized in silicone elastomers was observed to retain its full activity. The results reported herein demonstrate that cross-linked PDMS is a promising support material for the immobilization of hydrolytic enzymes such as pepsin.     

Improved catalytic hydrolysis of carboxy methyl cellulose using cellulase immobilized on functionalized meso cellular foam

Cellulase from Penicillium funiculosum was immobilized on functionalized MCF (Meso Cellular Foam) silica by imine bond formation followed by reduction using NaBH₄. The specific activities of free and immobilized enzyme were measured for hydrolysis of soluble carboxymethyl cellulose (CMC). The highest activity of MCF immobilized and native enzyme was obtained at optimum pH 5 and 4.5 respectively. Kinetic parameters, Michaelis–Menten constant (Km) and maximum reaction velocity (Vmax), were calculated as Km = 0.025 × 10⁻² mg/mL, Vmax = 5.327 × 10⁻³ U/mg for the free enzyme and Km = 0.024 × 10⁻² mg/mL, Vmax = 9.794 × 10⁻³ U/mg for MCF immobilized enzyme respectively. The reusability of immobilized enzymes showed that 66% of its activity is retained even after 15 cycles. The availability of polar groups (–NH–, –OH) and large pore size of surface modified MCF could be electrostatically stabilizing the cellulase. Functionalized MCF was found to be a promising material for stabilizing cellulase with 16.4 wt% loading of enzyme.     

Immobilization and stabilization of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> SRT9 lipase on tri(4-formyl phenoxy) cyanurate

Lipase was extracted and purified from Pseudomonas aeruginosa SRT9. Culture conditions were optimized and highest lipase production amounting to 147.36 U/ml was obtained after 20 h incubation. The extracellular lipase was purified on Mono QHR5/5 column, resulting in a purification factor of 98-fold with specific activity of 12307.81 U/mg. Lipase was immobilized on tri (4-formyl phenoxy) cyanurate to form Schiff’s base. An immobilization yield of 85% was obtained. The native and immobilized lipases were used for catalyzing the hydrolysis of olive oil in aqueous medium. Comparative study revealed that immobilized lipase exhibited a shift in optimal pH from 6.9 (free lipase) to 7.5 and shift in optimal temperature from 55 °C to 70 °C. The immobilized lipase showed 20–25% increase in thermal stability and retained 75% of its initial activity after 7 cycles. It showed good stability in organic solvents especially in 30% acetone and methanol. Enzyme activity was decreased by ∼60% when incubated with 30% butanol. The kinetic studies revealed increase in K _M value from 0.043 mM (native) to 0.10 mM for immobilized lipase. It showed decrease in the V _max of immobilized enzyme (142.8 μmol min⁻¹ mg⁻¹), suggesting enzyme activity decrease in the course of covalent binding. The immobilized lipase retained its initial activity for more than 30 days when stored at 4 °C in Tris-HCl buffer pH 7.0 without any significant loss in enzyme activity.     

Electrochemical preparation and properties of nanostructured Co3O4 as supercapacitor material

Nanostructured Co₃O₄ was prepared via a simple two-step process: cathodic electrodeposition of cobalt hydroxide from additive free nitrate bath and then heat treatment at 400 °C for 3 h. The prepared oxide product was characterized by powder X-ray diffraction, infrared spectroscopy, surface area measurement, scanning electron microscopy, and transmission electron microscopy. Morphological characterization showed that the oxide product was composed of porous nanoplates, and BET measurement displayed that the oxide plates have the average pore diameter and the surface area of 4.75 nm and 208.5 m² g⁻¹, respectively. The supercapacitive performance of the nanoplates was evaluated using cyclic voltammetry and charge–discharge tests. A specific capacitance as high as 393.6 F g⁻¹ at the constant current density of 1 A g⁻¹ and an excellent capacity retention (96.5% after 500 charge–discharge cycles) was obtained. These results indicate that Co₃O₄ nanoplates can be recognized as high-performance electrode materials.     

Poly(acrylamide/maleic acid)–sepiolite composite hydrogels for immobilization of invertase

Poly(acrylamide/maleic acid) and sepiolite (PAMS) composite hydrogel was prepared and used for the immobilization of invertase. In FTIR analysis, the characteristic bands of composite such as –OH, –COOH, Si–OH show the evidence of sepiolite and maleic acid. In TGA analysis, water loss, decomposition of amide side groups and breakdown of main chain regions of the composite were found. The parameters of equilibrium swelling, initial swelling rate, diffusional exponent, and diffusion coefficient were calculated and evaluated from swelling experiments in distilled water. Invertase was immobilized onto PAMS by adsorption and poly(acrylamide/maleic acid)–sepiolite–invertase (PAMSI) was prepared. Optimum pH, optimum temperature values for free invertase and PAMSI were found. It was found that K _m values of free invertase and PAMSI were 11.3 and 34.1 mM, respectively. V _max value was found that 2.0 μmol min⁻¹ for free invertase and 13.9 μmol min⁻¹ for PAMSI, respectively. PAMSI showed excellent temperature, operational and storage stability.     

Preparation and electrochemical performance of SiCN–CNTs composite anode material for lithium ion batteries

The composite of silicon carbonitride (SiCN) and carbon nanotubes (CNTs) was synthesized by sintering the mixture of polysilylethylenediamine-derived amorphous SiCN and multi-walled CNTs at a temperature of 1,000 °C for 1 h in argon. The as-prepared SiCN–CNTs material, which was used as anode active substance in a lithium ion battery, showed excellent electrochemical performance. Charge–discharge tests showed the SiCN–CNTs anode provided a high initial specific discharge capacity of 1176.6 mA h g⁻¹ and a steady specific discharge capacity of 450–400 mA h g⁻¹ after 30 charge–discharge cycles at 0.2 mA cm⁻². Both of the abovementioned values are higher than that of pure polymer-derived SiCN, CNTs, and commercial graphite at the same charge–discharge condition. It was deduced that the CNTs in the composite not only improved the electronic conductivity and offered channels and sites for the immigrating and intercalating of Li⁺ but also stabilized the structure of the composite.     

Immobilization of proteolytic enzyme on highly porous activated carbon derived from rice bran

Highly porous activated carbon (HPAC) was used as carrier matrix for immobilization of acid protease (AP). Immobilization of acid protease on mesoporous activated carbon (AP-HPAC) performs as best enzyme carrier. At pH 6.0, 250 mg acid protease g⁻¹ HPAC was immobilized. The optimum temperature for both free and immobilized AP activities were 50 °C. After incubation at 50 °C, the immobilized AP maintained about 50% of its initial activity, while the free enzyme was completely inactivated. When testing the reusability of AP-HPAC combination immobilized system, a significant catalytic efficiency was maintained along more than five consecutive reaction cycles. The highly porous nature of the carbon permits significant higher loadings of enzyme, which results in a higher enzyme-support strength and increased stability. The changes in the AP, HPAC and AP-HPAC were confirmed by Fourier Transform Infrared spectroscopy (FT-IR). Furthermore, scanning electron microscopy (SEM) allowed us to observe that the morphology of the surface of HPAC and the AP-HPAC.     

Pseudo-capacitance properties of porous metal oxide nanoplatelets derived from hydrotalcite-like compounds

Nanoplatelets of metal oxides with interesting porous structure were obtained by thermal treatment of Ni/Al hydrotalcite. Structural and surface properties of the porous oxides were characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM and HRTEM), and N₂ adsorption–desorption. The electrochemical performance of the electrodes was investigated by cyclic voltammetry, electrochemical impedance spectroscopy and constant current charge–discharge measurements. Ni/Al hydrotalcite calcined at 450 °C (NA-450) displayed a maximum specific capacitance (419.0 F g⁻¹) due to the porous structure with the highest specific surface area (142.3 m² g⁻¹) and small pore size (4.4 nm). The present study shows the potential of NiO nanoplatelets composite material for electrochemical pseudo-capacitors.     

Electrochemical determination of ascorbic acid at the surface of a graphite electrode modified with multi-walled carbon nanotubes/tetradecyltrimethylammonium bromide

A multi-walled carbon nanotubes (MWCNTs)–tetradecyltrimethylammonium bromide (TTAB) film-coated graphite electrode (GE) was fabricated, and the electrochemical oxidation of ascorbic acid (AA) was studied in Britton–Robinson (B–R) buffer (pH 7.0) using cyclic, square wave, and differential pulse voltammetry (CV, SWV, and DPV). An electroanalytical study of AA and acetaminophen (ACOP) and of several mixtures of these compounds in different ratios was made. A sensitive linear voltammetric response for AA was obtained for the concentration range of 5 × 10⁻⁷ to 1.7 × 10⁻⁴ mol L⁻¹, with a correlation coefficient of 0.992, and the detection limit for AA was found to be 1.1 × 10⁻⁷ mol L⁻¹ using DPV. The relative standard deviation (RSD) was 2.7%, suggesting that the film electrode has excellent day-to-day reproducibility. The proposed voltammetric approach was also applied to the determination of the AA concentration in commercial tablets.     

Immobilization and enzymatic activity of β-glucosidase on mesoporous SBA-15 silica

The mesoporous silicate SBA-15 has shown to be a good support for the immobilization of β-glucosidase from almonds, an enzyme with high molecular weight (ca. 130 kDa for the dimer). An enzyme loading of 430 mg per gram of support (3.2 ± 0.2 μmol g⁻¹ of SBA-15) was achieved at 7 h. The optimum pH for the immobilization was 3.5. The electrostatic interactions between the surface of SBA-15 and the enzyme molecules were the driving force of the adsorption process. The immobilized β-glucosidase presented enzymatic activity on the hydrolysis of the 4-nitrophenyl-β-d-glucopyranoside at 3.5 pH. The catalytic activity was similar to the free enzyme for reaction time of 30 min. When the reaction pH was higher (5.5 pH) the enzyme was desorbed.     

Selective determination of uric acid in the presence of ascorbic acid at poly(<Emphasis Type="Italic">p</Emphasis>-aminobenzene sulfonic acid)-modified glassy carbon electrode

The electrocatalytic behavior of uric acid has been investigated with a glassy carbon electrode modified with p-aminobenzene sulfonic acid through electrochemical polymerization. This resulting electrode shows an excellent electrocatalytic response to uric acid and ascorbic acid, with a peak-to-peak separation of 0.267 V in a 0.1 mol L⁻¹ phosphate buffer solution (PBS) at pH 7.0. These results indicate that the proposed electrode can eliminate the serious interference of ascorbic acid, which coexists with uric acid in body fluids. Differential pulse voltammetry (DPV) was used for detecting uric acid with selectivity and sensitivity. The anodic peak current of uric acid was proportional to its concentration in the range of 1.2 × 10⁻⁷–8.0 × 10⁻⁴ mol L⁻¹, with a detection limit of 4.0 × 10⁻⁸ mol L⁻¹. The proposed method has been applied with satisfactory results to the determination of uric acid in human urine without any pretreatment.     

A porous ruthenium silica catalyst modified with amino benzoic acid for the oxidation of butanol with molecular oxygen

Silica was extracted from rice husk (RH) and modified with ruthenium and amino benzoic acid. The catalysts RH–Ru and RH–Ru–A (incorporated with 5% of 4-(methylamino)benzoic acid) were synthesized from rice husk via solvent extraction and sol–gel technique. XRD diffractogram showed both catalysts were amorphous. BET results showed that the surface area of RH–Ru–A (73.9 m² g⁻¹) was smaller compared with RH–Ru (138 m² g⁻¹). After calcinations at 700 °C, RH–Ru-700 and RH–Ru–A-700 showed a lower specific surface area, i.e. 21.8 and 19.0 m² g⁻¹, respectively. The SEM micrograph of RH–Ru-700 showed the presence of elongated nano fibers, while RH–Ru–A-700 showed the presence of large regular pore structures. RH–Ru and RH–Ru–A were used as catalyst for the oxidation of 1-butanol with molecular oxygen as the oxidant. The oxidation yielded only one product, i.e. 1-butanal. Although the yield of 1-butanal was less than 10% both catalyst showed great potential for the oxidation of primary alcohols into aldehydes at moderate reaction conditions. A plausible mechanism was suggested for the catalyzed oxidation.     

Lipase-Catalyzed Synthesis of Saccharide–Fatty Acid Esters Using Suspensions of Saccharide Crystals in Solvent-Free Media

Saccharide–fatty acid esters, important biobased and biodegradable emulsifiers in foods, cosmetics, and pharmaceuticals, were produced with high yields and productivity via immobilized Rhizomucor miehei lipase-catalyzed esterification in solvent-free systems at 65 °C. Preliminary experiments demonstrated high rates of reaction occurred in the presence of acetone near or above its boiling point, due to the formation of 10–200 μm suspensions of saccharide particles. Subsequently, a two-step process was developed to produce a solvent-free supersaturated solution of 1.5–2.0 wt% saccharide that remained stable for ≥10–12 h. The solvent-free suspensions were used in a bioreactor system at 65 °C, consisting of a reservoir open to the atmosphere that contained molecular sieves, a peristaltic pump, and a packed bed bioreactor, operated under continuous recirculation. At 10 h intervals, suspensions were re-formed by treating the substrate/product mixture with additional acyl acceptor and applying strong agitation. Using this system and approach, a product mixture containing 88% fructose oleate was formed, of which 92% was monoester, within 6 days. This equates to a productivity of 0.2 mmol h⁻¹ g⁻¹, which is similar to values reported for synthesis in the presence of solvent.     

Effects of various factors on capacitive properties of VO<Subscript><Emphasis Type="Italic">x</Emphasis></Subscript>·<Emphasis Type="Italic">n</Emphasis>H<Subscript>2</Subscript>O powders in aqueous electrolyte

In this work, effects of drying temperature, pH of aqueous electrolyte and current density on capacitive performance of VO _x ·nH₂O material were firstly investigated. VO _x ·nH₂O powders were prepared by a melt quenching method. The samples were characterized by X-ray diffraction analysis (XRD) and Fourier transform infrared (FTIR). The capacitive properties of VO _x ·nH₂O samples were examined by cyclic voltammetry and galvanostatic charge/discharge test. VO _x ·nH₂O sample which was obtained at the drying temperature of 80 °C, delivers a maximum specific capacitance of 227.3 F g⁻¹ and exhibits excellent capacity retention in the potential range of −0.3 to 0.7 V at a current density of 200 mA g⁻¹ in NaNO₃ solution with pH 2.     

Oxidation of Carbon Monoxide Over SBA-15-Confined Copper,Palladium and Iridium Nanocatalysts

Abstract  

Copper, palladium and iridium nanoparticles were synthesised within the pore channels of selectively grafted mesoporous silica SBA-15. The support and catalysts were characterised by different techniques. The synthesized catalyst were able to catalyse oxidation of carbon monoxide with activity values as high as 7.0 × 10⁻³ mmol g⁻¹ cat s⁻¹ at 353 K. Carbon monoxide conversion was found to increase with decreasing nano particle size.     

Electrochemical investigation of Li–Al anodes in oligo(ethylene glycol) dimethyl ether/LiPF<Subscript>6</Subscript>

1 M LiPF₆ dissolved in oligo(ethylene glycol) dimethyl ether with a molecular weight 500 g mol⁻¹ was investigated as a new electrolyte (OEGDME500, 1 M LiPF₆) for metal deposition and battery applications. At 25 °C a conductivity of 0.48 × 10⁻³ S cm⁻¹ was obtained and at 85 °C, 3.78 × 10⁻³ S cm⁻¹. The apparent activation barrier for ionic transport was evaluated to be 30.7 kJ mol⁻¹. OEGDME500, 1 M LiPF₆ allows operating temperature above 100 °C with very attractive conductivity. The electrolyte shows excellent performance at negative and positive potentials. With this investigation, we report experimental results obtained with aluminum electrodes using this electrolyte. At low current densities lithium ion reduction and re-oxidation can be achieved on aluminum electrodes at potentials about 280 mV more positive than on lithium electrodes. In situ X-ray diffraction measurements collected during electrochemical lithium deposition on aluminum electrodes show that the shift to positive potentials is due to the negative Gibbs free energy change of the Li–Al alloy formation reaction.     

Facile Solvothermal Synthesis Ni(OH)<Subscript>2</Subscript> Nanostructure for Electrochemical Capacitors

Nickel hydroxide nanosheets were successfully synthesized by facile solvothermal method without any template. The structure and morphology of the as-prepared sample were characterized by X-ray diffraction, Fourier transform infrared spectroscopy and transmission electron microscopy. The observations revealed the formation of hexagonal phase β-Ni(OH)₂ nanosheets with an average diameter of about 100–120 nm. Electrochemical studies were carried out using cyclic voltammetry and galvanostatic charge–discharge tests, respectively. A maximum specific capacitance of 2,342 F g⁻¹, which is the highest reported for a β-Ni(OH)₂ electrode, could be achieved in 6 mol L⁻¹ KOH electrolyte within the potential range of 0–0.50 V (vs. SCE) for the obtained β-Ni(OH)₂ electrode at 0.4 A g⁻¹, suggesting its potential application in the electrode material for electrochemical capacitors.     

Some Biochemical Properties of Lipase from Bay Laurel (<Emphasis Type="Italic">Laurus nobilis</Emphasis> L.) Seeds

Lipase was isolated from bay laurel (Laurus nobilis L.) seeds, some biochemical properties were determined. The bay laurel oil was used as the substrate in all experiments. The pH optimum was found to be 8.0 in the presence of this substrate. The temperature optimum was 50 °C. The specific activity of the lipase was found to be 296 U mg protein⁻¹ in optimal conditions. The enzyme activity is quite stable in the range of pH 7.0–10. The enzyme was stable for 1 h at its optimum temperature, and retained about 68% of activity at 60 °C during this time. K _m and V _max values were determined as 0.975 g and 1.298 U mg protein⁻¹, respectively. Also, storage stability and metal effect on lipolytic activity were investigated. Enzyme activity was maintained for 9, 12, and 42 days at room temperature, 4 and −20 °C, respectively. Ca²⁺, Co²⁺, Cu²⁺, Fe²⁺, and Mg²⁺ lightly enhanced bay laurel lipase activity.     

A Calcium(II)-Based <Emphasis Type="SmallCaps">l</Emphasis>-Arginine for ATP Binding and Hydrolysis

The supramolecular recognition of Ca(II) and N _α-4-tosyl-l-arginine methyl ester hydrochloride (TAME) with ATP were investigated using ¹H and ³¹P NMR spectra. In the Ca(II)–ATP–TAME ternary system, Ca²⁺ and TAME bind with ATP via the phosphate chain and adenine ring of ATP. The binding forces are mainly electrostatic and cation (Ca²⁺)–π and π–π stacking interaction. Furthermore, the hydrolysis of ATP catalyzed by Ca(II) and TAME was studied at pH 7.0 and 60 °C using ³¹P NMR spectra. Kinetics studies show that the ATP hydrolysis rate constant is 0.1035 h⁻¹ in the Ca(II)–TAME–ATP ternary system, whereas the value is 8.5 × 10⁻³ h⁻¹ under the same conditions without TAME and Ca²⁺. The Ca(II) ions and TAME accelerate the ATP hydrolysis process about 12-fold. The proposed mechanism of ATP hydrolysis catalyzed by Ca²⁺–TAME occurs through an addition–elimination reaction sequence. These results can help us get more useful information at the molecular level about the key amino acid residue(s) and metal ions that serve as cofactors in the ATPase effect on ATP hydrolysis/synthesis.     

A Novel Dinucleating Ligand-Containing Hexabenzimidazoles and Its Dinuclear Zinc Complex Bridged by a Carboxylate Group for the Hydrolysis of 2-Hydroxypropyl-p-nitrophenyl phosphate

A dinuclear zinc(II) complex with the ligand bis{tris[2-(1-methylbenzimidazole-2-yl)ethyl]-methylamine}nitrilotriacetic acid sodium salt, L, was synthesized and characterized. Complex formation of L with Zn²⁺ in aqueous acetone was studied by Zn²⁺ titration using ¹H NMR and UV–vis spectroscopies. Analysis of the titration data indicates the formation of a dizinc complex. The ν_as(COO⁻) and ν_s(COO⁻) stretches were observed at 1572 and 1450 cm⁻¹, respectively. The low separation of the stretches, Δ_exp = 115 cm⁻¹, is an indication of chelating coordination of the carboxylate group between the two zinc(II) ions. The catalytic activity of [LZn₂]³⁺ 1, as a model for phosphatase that catalyze chemical transformation of phosphate ester, in the hydrolysis of the RNA model, 2-hydroxypropyl p-nitrophenyl phosphate, was examined in aqueous acetone buffer solution, pH 7.0–9.5. The mechanism of the catalytic hydrolysis suggests that the rate of acceleration is due to what is called double Lewis acid activation.