Stability and reactivity of acid phosphatase immobilized on composite beads of chitosan and ZrO2 powders

Equal weights of chitosan and ZrO2 powders were mixed in acetic acid solution to prepare the composite beads. They were then cross-linked with glutaraldehyde and stored with and without freeze-drying before use. The physicochemical properties of acid phosphatase immobilized on four types of the supports (wet/dried pure chitosan beads, wet/dried chitosan-ZrO2 composite beads) were compared. Various parameters including glutaraldehyde concentration, cross-linking time, enzyme concentration, temperature, and pH on enzyme activity were studied. It was shown that the activity yield of enzyme immobilized on the dried chitosan-ZrO2 beads was the highest, and the relative activity remained above 83.2% within pH 2.9-5.8. Regardless of wet or dried beads, the Michaelis constant KM and maximum rate of reaction Vmax of acid phosphatase immobilized on chitosan-ZrO2 composite beads were 1.8 times larger than those on pure chitosan beads. Of the four immobilized enzymes, the use of wet chitosan-ZrO2 bead as the support showed the lowest thermal deactivation energy (78 kJ mol(-1)).     

Enhanced stabilization of mungbean thiol protease immobilized on glutaraldehyde-activated chitosan beads

A thiol protease purified from mungbean seedlings was immobilized on chitosan beads cross-linked with glutaraldehyde. The yield of the immobilized enzyme was maximum (～99%) at 1% concentration each of chitosan and glutaraldehyde. The immobilized enzyme showed reusability for 15 batch reactions. Immobilization shifted the optimum pH of the enzyme to a more acidic range and enhanced its stability both at acidic as well as alkaline pH values compared to the free enzyme. The stability of the enzyme to temperature and in aqueous non-conventional medium (ethanol and DMSO) was significantly improved by the immobilization process. The immobilized enzyme exhibited mass transfer limitation reflected by a higher apparent K_m value. This study produced an immobilized biocatalyst having improved characteristics and better operational stability than the soluble enzyme. The increase in stability in the presence of high concentrations of ethanol and DMSO may make it useful for catalyzing organic reactions such as trans-esterification and trans-amidation similar to other cysteine proteinases.     

Catalytic properties of a nitrile hydratase immobilized on activated chitosan

The catalytic properties of a nitrile hydratase, isolated from a strain of Rhodococcus ruber gt1 and immobilized by covalent cross-linking with chitosan activated with 0.1% benzoquinone solution, have been investigated. The kinetic parameters of acrylonitrile hydration catalyzed by immobilized nitrile hydratase and the enzyme in a solution have been determined. It is found that the immobilization does not lead to a decrease in the maximum reaction rate (V _max), whereas the Michaelis constant (K _M) is reduced by a factor of 2.4. The possibility of reusing an immobilized enzyme for 50 consecutive cycles of acrylonitrile transformation was shown, and the nitrile hydratase activity in the 50th cycle exceeded that in the first cycle by 3.5 times. It is shown that the effect of temperature on activity depended on the concentration of the enzyme, which confirms the dissociative nature of nitrile hydratase inactivation. It was found that immobilized nitrile hydratases remain active at pH 3.0–4.0, whereas the enzyme is inactivated in a solution under these conditions. The resulting biocatalyst can be effectively used to receive acrylamide from acrylonitrile.     

Biological ferrous sulfate oxidation by A. ferrooxidans immobilized on chitosan beads

The immobilization of Acidithiobacillus ferrooxidans cells on chitosan and cross-linked chitosan beads and the biooxidation of ferrous iron to ferric iron in a packed-bed bioreactor were studied. The biofilm formation was carried out by using a glass column reactor loaded with chitosan or cross-linked chitosan beads and 9 K medium previously inoculated with A. ferrooxidans cells. The immobilization cycles on the carrier matrix with the bioreactor operating in batch mode were compared. Then, the reactor was operated using a continuous flow of 9 K medium at different dilution rates. The results indicate that the packed-bed reactor allowed increasing the flow rate of medium approximately two fold (chitosan) and eight fold (chitosan cross-linked) without cells washout, compared to a free cell suspension reactor used as control, and to reach ferric iron productivities as high as 1100 and 1500 mg l(-1) h(-1) respectively. Scanning electron microscopy micrographs of the beads, infrared spectroscopy and the X-ray diffraction patterns of precipitates on the chitosan beads were also investigated.     

Equilibrium and kinetics studies of adsorption of copper (II) on chitosan and chitosan/PVA beads

The adsorption of Cu(II) ions from aqueous solution by chitosan and chitosan/PVA beads was studied in a batch adsorption system. Chitosan solution was blended with poly(vinyl alcohol) (PVA) in order to obtain sorbents that are insoluble in aqueous acidic and basic solution. The adsorption capacities and rates of Cu(II) ions onto chitosan and chitosan/PVA beads were evaluated. The Langmuir, Freundlich and BET adsorption models were applied to describe the isotherms and isotherm constants. Adsorption isothermal data could be well interpreted by the Langmuir model. The kinetic experimental data properly correlated with the second-order kinetic model, which indicates that the chemical sorption is the rate-limiting step. The Cu(II) ions can be removed from the chitosan and chitosan/PVA beads rapidly by treatment with an aqueous EDTA solution. Results also showed that chitosan and chitosan/PVA beads are favourable adsorbers.     

Fibrous polymer-grafted chitosan/clay composite beads as a carrier for immobilization of papain and its usability for mercury elimination

Papain, which is an industrially important enzyme, has been immobilized on fibrous polymer-modified composite beads, namely poly(methacrylic acid)-grafted chitosan/clay. Characterization studies have been done using FTIR and SEM analysis. Operating parameters such as pH and initial concentration of papain have been varied to obtain the finest papain immobilized polymer-modified composite beads. The immobilization capacity of composite beads has been determined as 34.47 ± 1.18 (n = 3) mg/g. The proteolytic activity of immobilized papain was operated using bovine serum albumin (BSA) and maximum velocity (V _max) and Michaelis–Menten constant (K_m) values of the free and immobilized enzymes were determined using Lineweaver–Burk and Eadie–Hofstee equations. Usability of papain immobilized polymer-modified composite beads as adsorbents for the elimination of mercury was investigated. The maximum removal capacity of PIPMC beads has been found to be 4.88 ± 0.21 mg Hg/g when the initial metal concentration and weight of polymer-modified composite beads were 50 mg/L and 0.04 g at pH 7, respectively. Mercury removal performance of the papain immobilized polymer-modified composite beads was investigated in conjunction with Cu (II), Zn (II) and Cd (II) ions. The mercury adsorption capacity of papain immobilized polymer-modified composite beads was a slight reduction from 1.15 to 0.89 mg/g in presence of multiple metal salts.     

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.     

Hydrolysis of whey by kluyveromyces bulgaricus cells immobilized in stabilized alginate and in chitosan beads

Kluyveromyces bulgaricus cells were immobilized in matrices resisting to complexing anions. Yeast entrapped in alginate stabilized by polyethylenimine and glutaraldehyde were unable to hydrolyse whey owing to the inactivation of β-galactosidase by the stabilizing agents. Chitosan was resistant to whey medium but decreased the yeast hydrolyzing capacity by 15% with respect to alginate. The hydrolysis rate was found to be unchanged for 37 days at 21–25°C.     

Enantioselective synthesis of (S)‐naproxen using immobilized lipase on chitosan beads

S‐naproxen by enantioselective hydrolysis of racemic naproxen methyl ester was produced using immobilized lipase. The lipase enzyme was immobilized on chitosan beads, activated chitosan beads by glutaraldehyde, and Amberlite XAD7. In order to find an appropriate support for the hydrolysis reaction of racemic naproxen methyl ester, the conversion and enantioselectivity for all carriers were compared. In addition, effects of the volumetric ratio of two phases in different organic solvents, addition of cosolvent and surfactant, optimum pH and temperature, reusability, and inhibitory effect of methanol were investigated. The optimum volumetric ratio of two phases was defined as 3:2 of aqueous phase to organic phase. Various water miscible and water immiscible solvents were examined. Finally, isooctane was chosen as an organic solvent, while 2‐ethoxyethanol was added as a cosolvent in the organic phase of the reaction mixture. The optimum reaction conditions were determined to be 35 °C, pH 7, and 24 h. Addition of Tween‐80 in the organic phase increased the accessibility of immobilized enzyme to the reactant. The optimum organic phase compositions using a volumetric ratio of 2‐ethoxyethanol, isooctane and Tween‐80 were 3:7 and 0.1% (v /v/v), respectively. The best conversion and enantioselectivity of immobilized enzyme using chitosan beads activated by glutaraldehyde were 0.45 and 185, respectively.     

Preparation and characterization of epoxy-functionalized magnetic chitosan beads: laccase immobilized for degradation of reactive dyes

Cross-linked magnetic chitosan beads were prepared by phase-inversion technique in the presence of epichlorohydrin under alkaline condition, and used for covalent immobilization of laccase. The activity of the immobilized laccase on the magnetic chitosan was about 260 U (g/dry beads) with an enzyme loading of about 16.33 ± 0.39 mg [(g/dry beads) mg/g]. Kinetic parameters, V _max and K _m values were determined as 21.7 U/mg protein and 9.4 μM for free enzyme, and 15.6 U/mg protein and 19.7 μM for the immobilized laccase, respectively. The operational and thermal stabilities of the immobilized laccase were improved compared to free counterpart. The immobilized laccase was operated in a batch reactor for the decolorization of reactive dyes from aqueous solution. The laccase immobilized on magnetic chitosan beads was very effective for removal of textile dyes from aqueous solution which creates an important environmental problem in the discharged textile dying solutions.     

Cooperative kinetics of human prostatic acid phosphatase

The steady-state kinetics of hydrolysis reaction catalysed by human prostatic acid phosphatase (PAP) by using 1-naphthyl phosphate, phenyl phosphate and phosphotyrosine as substrates has been studied at pH 5.5. The substrate binding curves were sigmoidal and Hill cooperation coefficient h was higher than 1 for each of the examined compounds. Thus, human prostatic acid phosphatase kinetics exhibits positive cooperativity towards the studied substrates. The extent of cooperativity was found to depend on the substrate used and on enzyme concentration. The highest cooperativity of PAP was observed for 1-naphthyl phosphate and the lowest for phosphotyrosine. When prostatic phosphatase concentration increased, Hill cooperation coefficient (h) and half saturation constant (K(0.5)) both grew, but the catalytic constant (k(cat)) remained constant, for each of the substrates studied. Ligand-induced association-dissociation equilibrium of the active oligomeric species (monomer-dimer-tetramer-oligomers) is suggested.     

The effect of porosity on the catalytic performance of constant-mass, spherical beads containing immobilized enzyme

The mass balances to a spherical bead with increasing porosity, ? (obtained by plain expansion of an otherwise compact bead), containing an immobilized enzyme and surrounded by a stagnant film are developed in dimensionless form for the case of Michaelis-Menten kinetics by considering three alternative situations in terms of pore structure (either setting the pore number, the pore radius or the pore length as a constant). The pore pattern of the porous bead does not play a major role in the variation of the lowest concentration of substrate ever reached in the bulk of the bead, which increases as ? increases and eventually levels off when ? approaches unity. The ratio between the rate of reaction brought about by the immobilized enzyme within the porous bead and that obtained for a compact bead is greater when ? is higher, and a vertical asymptote is apparently reached when the porosity approaches unity, a trend that is similarly observed for all pore patterns considered.     

Induction of antitumor activities in spleen cells from mice and rats activated with lipopolysaccharide immobilized on beads

Summary Lipopolysaccharides (LPS) were coupled to polystyrene beads in order to apply the LPS without toxicity. The antitumor activity of the LPS-immobilizing beads was studied in experiments in vitro and in vivo. In vitro studies showed that spleen cells from C3H/HeN mice stimulated by beads immobilizing LPS fromEscherichia coli produced cytolytic activity as strong as that of lymphokine-activated killer (LAK) cells. Spleen cells from Sprague-Dawley rats stimulated by beads immobilizing LPS fromSalmonella minnesota produced cytolytic activity stronger than that of LAK cells. However, spleen cells stimulated by beads immobilizing each component of the LPS separately could not induce cytolysis. Contact stimulation, even for a brief period, sufficed for cytolytic activity, and was enhanced by culture for 48–72 h. Through in vivo studies, the suppression of tumor growth and a prolongation of the survival time were observed in tumorbearing mice injected with spleen cells activated by beads immobilizing LPS fromE.coli, and in mice injected with LAK cells. The effect of the activated spleen cells was stronger than that of the LAK cells. In rats bearing metastatic tumors, spleen cells activated by beads immobilizing LPS fromS.minnesota suppressed lung metastases more strongly than did LAK cells. These findings indicate that LPS immobilized by beads induced killer cells more strongly than interleukin-2. Ex vivo immunomodulation with LPS-immobilizing beads can be applied usefully as an anticancer treatment.     

Application of chitosan beads immobilized Rhodococcus sp. NCIM 2891 cholesterol oxidase for cholestenone production

The cell-bound cholesterol oxidase from the Rhodococcus sp. NCIM 2891 was purified three fold by diethylaminoethyl–sepharose chromatography. The estimated molecular mass (SDS-PAGE) and K_m of the enzyme were ∼55.0 kDa and 151 μM, respectively. The purified cholesterol oxidase was immobilized on chitosan beads by glutaraldehyde cross-linking reaction and immobilization was confirmed by Fourier transform infrared spectroscopy, scanning electron microscopy and energy dispersive X-ray analysis. The optimum temperature (45 °C, 5 min) for activity of the enzyme was increased by 5 °C after immobilization. Both the free and immobilized cholesterol oxidases were found to be stable in many organic solvents except for acetone. Fe²⁺ and Pb²⁺ at 0.1 mM of each acted as inhibitors, while Ag⁺, Ca²⁺, Ni²⁺ and Zn²⁺ activated the enzyme at similar concentration. The biotransformation of cholesterol (3.75 mM) with the cholesterol oxidase immobilized beads (3.50 U) leads to ∼88% millimolar yield of cholestenone in a reaction time of 9 h at 25 °C. The immobilized enzyme retains ∼67% activity even after 12 successive batches of operation. The biotransformation method thus, shows a great promise for the production of pharmaceutically important cholestenone.     

Production of isomaltooligosaccharides by a-glucosidase immobilized in chitosan beads and by polyethyleneimine-glutaraldehyde treated mycelia of Aspergillus carbonarious

Partially purified a-glucosidase from Aspergillus carbonarious, immobilized on glutaraldehyde-activated chitosan beads in a packed bed reactor, produced isomaltooligosaccharides at a yield of 60% (w/w) using 30% (w/v) maltose solution. Using intact mycelia attached with polyethyleneimine-glutaraldehyde, produced isomaltooligosaccharides at a yield of 46% (w/w) using 30% (w/v) maltose solution. Batchwise reaction stabilities were improved for chitosan beads immobilized enzyme and polyethyleneimine-glutaraldehyde treated mycelia as compared to mycelia without any treatment.     

Preparation and characterization of immobilized phospholipase A2 on chitosan beads for lowering serum cholesterol concentration

Phospholipase A₂ (PLA₂) from cobra venom, which can hydrolyze the S_N2 ester bond of 1,2-diacylphosphatides, was immobilized by covalent binding to porous chitosan beads. Immobilization has to be carried out by using the carboxylic groups instead of the amine groups of the enzyme to get reasonable activity retention (higher than 50%). The effects of amount of activating reagent EDC and enzyme loading during the immobilization step were investigated. Since EDC could modify important Asp groups in the enzyme, the EDC/enzyme weight ratio should be less than 10. Although the activity retention of immobilized enzyme increased with enzyme/bead weight ratio, this ratio should be kept to a minimum at 1×10⁻³ to optimize coupling yield of enzyme activity and reduce internal diffusion resistance. The kinetic properties and stability of the immobilized enzyme were determined. The immobilized PLA₂ was packed into a column to hydrolyze phospholipid in a circulating packed-bed reactor. The flow rate of the substrate solution should be set at 37.5 cm/min (superficial velocity) to eliminate external diffusion resistance, under which condition the column reactor could be reused up to 10 times with less than 20% loss of activity. Since enzymatic hydrolysis of phospholipid on low density lipoprotein (LDL) particle surface with PLA₂ could result in faster plasma clearance of the modified LDL particles, an in vitro bioreactor containing immobilized PLA₂ should be able to lower serum cholesterol concentration. A significant decrease in total serum cholesterol concentration in hypercholesterolemic rabbits was observed after 90-min treatment.     

乙酸对土壤胶体矿物吸附酸性磷酸酶的影响

研究了不同pH值、不同浓度乙酸对酸性磷酸酶在土壤胶体和矿物表面吸附的影响,结果表明,在pH2～8的乙酸体系中,酶在胶体矿物表面的最大吸附pH一般出现在蛋白的等电点和矿物的零电荷点(PZC)之间,各土壤胶体和粘粒矿物对酶的吸附量大小顺序为针铁矿》黄棕壤>砖红壤>高岭石>二氧化锰,乙酸浓度对酶在胶体矿物表面的吸附量和吸附结合能具有较显著影响,在0～200mmol·L^-1范围内,随着乙酸浓度的增加,酶吸附量呈现先升高、后降低、再稳定的趋势,而吸附结合能的变化与此相反,并就乙酸对酶在胶体矿物表面吸附影响的可能机理进行了初步探讨。     

Stability and biological activity of catecholamines and 5-hydroxytryptamine immobilized to sepharose and glass beads.

[14C]Noradrenaline, [14C]adrenaline, and 5-[14C]hydroxytryptamine were complexed to several types of Sepharose derivatives through an amide or a diazo linkage. Complexes were also made with arylamine glass beads through a diazo linkage. After the reaction the complexes were extensively washed with distilled water or dilute HCl or both, plus other solvents. Radioactivity appeared in the storage solutions and the amount increased with time. When initial low levels of release were achieved by extensive washing, the addition of a different solvent produced a marked increase in release. The biological activity of these complexes was tested on isolated rabbit aortic strips and rat fundus strips. All complexes caused the tissues to contract. The biological activity, however, closely paralleled the release of the ligands from the complexes and the release of ligand was demonstrated through the use of oil immersion and dialysis sac. These data do not support the recent claims that catecholamines bound to glass beads exert their pharmacological effects as covalently bound complexes on isolated tissue. The lack of stability of these complexes indicates the need for caution in drawing conclusions as to the site or the mechanism of action of immobilized catecholamines and other drugs.