Simultaneous co-immobilization of glucose oxidase and catalase in their substrates

Glucose oxidase (GOD) and catalase (CAT) were simultaneously co-immobilized onto magnesium silicate (Florisil®) by covalent coupling. Glucose was added in immobilization mixture and hydrogen peroxide, which is the substrate of CAT, was produced in coupling mixture during immobilization time. Therefore, co-immobilization of GOD and CAT was carried out in the presence of both their substrates: glucose and hydrogen peroxide, respectively. The effect of glucose concentration in immobilization mixture on activities of GOD and CAT of co-immobilized samples were investigated. Maximum GOD and CAT activities were determined for samples co-immobilized in the presence of 15 and 20 mM glucose, respectively. Co-immobilization of GOD and CAT in the presence of their substrates highly improved the activity and reusability of both enzymes.     

Simultaneous co-immobilization of glucose oxidase and catalase in their substrates

Glucose oxidase (GOD) and catalase (CAT) were simultaneously co-immobilized onto magnesium silicate (florisil) by covalent coupling. Glucose was added in immobilization mixture and hydrogen peroxide which is the substrate of CAT was produced in coupling mixture during immobilization time. Therefore, co-immobilization of GOD and CAT was carried out in presence of both their substrate: glucose and hydrogen peroxide, respectively. The effect of glucose concentration in immobilization mixture on activities of GOD and CAT of co-immobilized samples were investigated. Maximum GOD and CAT activities were determined for samples co-immobilized in presence of 15 and 20 mM glucose, respectively. Co-immobilization of GOD and CAT in presence of their substrates highly improved the activity and reusability of both enzymes.     

Kinetic properties and storage stability of catalase immobilized on to florisil

The covalent immobilization of bovine liver catalase (CAT) on to florisil via glutaraldehyde was investigated. Optimum immobilization pH and temperature were determined as pH 6.0, 10 degrees C respectively, while the amount of initial CAT per g of carrier and immobilization time was determined as 5 mg g(-1) and 120 min, respectively. The Vmax values for free and immobilized CAT were found to be 1.7 x 10(5) and 2.0 x 10(4) micromol H2O2 min(-1) mg protein(-1), respectively, whereas KM values were 33.3 mM and 1722.0 mM respectively. Operational stability was determined by using a stirred batch-type column reactor. Immobilized CAT retained about 40% of its initial activity after 50 uses. It showed higher storage stability than free CAT at 4 degrees C and 25 degrees C. Its storage stability increased with increasing relative humidity (RH) from 0 to 20% of the medium. The highest storage stability was obtained in 20% RH, however, further increase in RH from 40 to 100% significantly decreased the storage stability.     

Coimobilization of superoxide dismutase, catalase and peroxidase

For preparationing the polyenzyme antioxidant complex, containing superoxide dismutase (SOD), catalase and horseradish peroxidase (HRP), the different successivities of those enzymes co-immobilization were compared. The optimum successivity is provided by simultaneous co-immobilization of covalently bound HRP with the SOD and catalase. The catalytic enzyme activity and the catalase operational stability was kinetically characterized in various samples. For one sample, the influence of ascorbate, glutathione and ethanol on the catalase kinetic parameters was studied. A possible scheme of different processes at the H2O2 decomposition in the presence of co-immobilized SOD, catalase, HRP and the substrates-reductans was discussed.     

Determination of serum glucose using co-immobilized glucose oxidase and peroxidase onto arylamine glass beads affixed on a plastic strip

Glucose oxidase (GOD) from Aspergillus niger and horseradish peroxidase (POD) were co-immobilized onto arylamine glass beads affixed on a plastic strip with a conjugation yield of 28.2 mg/g and 43% retention of their initial specific activity. The coimmobilized enzymes showed maximum activity at pH 7.5 when incubated at 37 degrees C for 15 min. A simple, specific and sensitive method for discrete analysis of the serum glucose was developed employing this strip. The minimum detection limit of the method was 5 mg/dl. Within and between assay coefficient of variations for the serum were <5.6% and <10.6% (n = 6) respondely. A good correlation (r = 0.943) was found between the glucose values obtained by the enzyme colorimetric method employing free GOD and POD and the present method. The strip lost 50% of its initial activity after its 150 regular uses for a period of one month, when stored in reaction buffer at 4 degrees C. The method is cost-effective than the enzymic colorimetric method, as the enzyme strip is reusable.     

A highly sensitive flow-through amperometric immunosensor based on the Peroxidase chip and enzyme-channeling principle

A concept based on the Peroxidase-chip (P-chip), antibody co-immobilization, competitive and enzyme-channeling principle was exploited to develop an integrated flow-through amperometric biosensor for detection of environmental pollutants such as s-triazine herbicides. In this concept, recombinant peroxidase is immobilized on the gold electrode (P-chip) in such a way that direct electron transfer is achieved. The recognition and quantitation the target analyte is realized through the competition between the simazine-glucose oxidase (GOD) conjugate and free simazine for the binding sites of the monoclonal antibody co-immobilized with peroxidase on the gold electrode. The arrangement allows to generate a specific signal in the presence of glucose through the channeling of H2O2 produced by GOD conjugate bound to the antibody. The immunosensor exhibited 50% signal decrease (IC50 value) at approximately 0.02 microg l(-1). A concentration of 0.1 ng l(-1) gave a signal clearly distinguishable from the blank whereas the ELISA using the same antibody had a typical detection limit of about 1 microg l(-1), which is four orders of magnitude higher compared to the presented biosensor system. The results demonstrated that gene engineering biomolecules, in this case recombinant peroxidase, might be attractive reagents for the development of electrochemical immunosensors.     

Co-immobilization of lipase, glycerol kinase, glycerol-3-phosphate oxidase and peroxidase onto alkylamine glass beads through glutaraldehyde coupling

A method for co-immobilizing lipase from porcine pancreas, glycerol kinase (GK) from Cellulomonas spp., glycerol-3-phosphate oxidase (GPO) from Aerococcus viridans and peroxidase from horseradish onto zirconia-coated alkylamine glass beads through glutaraldehyde coupling has been described. The co-immobilized enzymes retained 71.4% of initial specific activity with a conjugation yield of 43.6 mg/g support. The optimum pH and Km for triolein increased, while Vmax was decreased slightly, but incubation temperature for maximum activity remained unaltered after co-immobilization. The co-immobilized enzymes showed increased thermal and storage stabilities in cold, compared to their native form. Among the various metal salts tested, only CuSO4 caused inhibition of both free and co-immobilized enzymes. The co-immobilized enzymes showed better suitability over mixture of individually immobilized enzymes in determination of serum triglyceride.     

Kinetic characteristics of extracellular catalase from Penicillium piceum F-648 and variants of fungi,adapted to hydrogen peroxide

A comparative kinetic study of extracellular catalases produced by Penicillium piceum F-648 and their variants adapted to H2O2 was performed in culture liquid filtrates. The specific activity of catalase, the maximum rate of catalase-induced H2O2 degradation (Vmax),Vmax/KM ratio, and the catalase inactivation rate constant in the enzymatic reaction (kin, s-1) were estimated in phosphate buffer (pH 7.4) at 30 degrees C. The effective constant representing the rate of catalase thermal inactivation (kin*, s-1) was determined at 45 degrees C. In all samples, the specific activity and KM for catalase were maximum at a protein concentration in culture liquid filtrates of 2.5-3.5 x 10(-4) mg/ml. The effective constants describing the rate of H2O2 degradation (k, s-1) were similar to that observed in the initial culture. These values reflected a twofold decrease in catalase activity in culture liquid filtrates. We hypothesized that culture liquid filtrates contain two isoforms of extracellular catalase characterized by different activities and affinities for H2O2. Catalases from variants 5 and 3 with high and low affinities for H2O2, respectively, had a greater operational stability than the enzyme from the initial culture. The method of adaptive selection for H2O2 can be used to obtain fungal variants producing extracellular catalases with improved properties.     

Lignin peroxidase H2 from Phanerochaete chrysosporium: purification, characterization and stability to temperature and pH

The wood-destroying fungus Phanerochaete chrysosporium secretes extracellular enzymes known as lignin peroxidases that are involved in the biodegradation of lignin and a number of environmental pollutants. Several lignin peroxidases are produced in liquid cultures of this fungus. However, only lignin peroxidase isozyme H8 has been extensively characterized. In agitated nutrient nitrogen-limited culture, P. chrysosporium produces two lignin peroxidases in about equal proportions. The molecular weights of these two major proteins (H2 and H8) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 38,500 (H2) and 42,000 (H8). The isoelectric points of these enzymes were 4.3 for H2 and 3.65 for H8. All subsequent experiments in this study were performed with H2 as it contributed the most (42%) to total activity and had the highest specific activity (57.3 U/mg). The Km values of lignin peroxidase H2 for H2O2 and veratryl alcohol were calculated to be 47 microM and 167 microM at pH 3.5, respectively. The pH optima for veratryl alcohol oxidase activity were pH 2.5 at 25 degrees C, pH 3.0 at 35 degrees C, and pH 3.5 at 45 degrees C. In the same manner the temperature optimum shifted from 25 degrees C at pH 2.5 to 45 degrees C at pH 3.5 and approximately 45-60 degrees C at pH 4.5. During storage the resting enzyme was relatively stable for 48 h up to 50 degrees C. Above this temperature the enzyme lost all activity within 6 h at 60 degrees C. At 70 degrees C all activity was lost within 10 min. The resting enzyme retained approximately 80% of its initial activity when stored at 40 degrees C for 21 h at a pH range of 4.0-6.5. Above pH 7.5 and below 4.0, the enzyme lost all activity in less than 5 h. During turnover the enzyme remained active at pH 5.5 for over 2 h whereas the enzyme activity was lost after 45 min at pH 2.5. The oxidation of veratryl alcohol was inhibited by EDTA, azide, cyanide, and by the catalase inhibitor 3-amino-1,2,4-triazole, but not by chloride. In the absence of another reducing substrate incubation of lignin peroxidase H2 with excess H2O2 resulted in partial and irreversible inactivation of the enzyme. The spectral characteristics of lignin peroxidase H2 are similar to those of other peroxidases. The suitability of lignin peroxidases for industrial applications is discussed.     

Co-immobilization of dextransucrase and dextranase in alginate

Dextransucrase from Leuconostoc mesenteroides and dextranase from Penicillium lilacinum were co-immobilized and used to produce isomaltooligosaccharides from sucrose. The enzymes were co-immobilized by encapsulating soluble dextransucrase and dextranase covalently attached to Eupergit C in alginate (beads, fibers, and capsules). The alginate capsule co-immobilization was done in the presence of soluble starch and resulted in a high immobilization yield (71%), and the enzymes retained their activities during 20 repeated batch reactions and for a month in storage at 4 °C. The presence of starch was essential for the stability of dextransucrase in alginate capsules. Furthermore, it is important that the dextranase be pre-immobilized prior to alginate capsule co-immobilization to prevent dextranase leakage and inactivation of dextransucrase. The co-immobilized enzymes formed oligosaccharides from sucrose, which can be used as prebiotics. In addition, the oligosaccharides that were produced after the addition of sucrose reacted with the alginate fiber-encapsulted dextransucrase, thus increasing the amount of prebiotics. Co-immobilization in alginate fiber and beads also resulted in high yields (70 and 64%), but enzymatic activities decreased by 74 and 99%, respectively, after a month in storage at 4 °C. The newly developed alginate capsule method for co-immobilization of dextransucrase and dextranase is simple yet effective and has the potential for industrial-scale production of isomaltooligosaccharides.     

Co-immobilization of lipase, glycerol kinase, glycerol-3-phosphate oxidase and peroxidase on to aryl amine glass beads affixed on plastic strip for determination of triglycerides in serum

Commercial lipase, glycerol kinase (GK), glycerol-3-phosphate oxidase (GPO) and peroxidase (POD) have been co-immobilized covalently on to arylamine glass beads affixed on a plastic strip through diazotization with a conjugation yield of 89.1 mg/g support and 64.1% retention of specific activity. The co-immobilized enzymes showed maximum activity at pH 7.5, when incubated at 40 degrees C for 20 min. The strip was employed for determination of serum triglycerides (Tgs). The minimum detection limit of the method was 0.20 mM/L. The recovery of added Tgs was 88.0%. Within day and between day coefficient of variations were <7.0 % and <11.0%, respectively. A good correlation (r = 0.982) was observed between total serum Tgs values obtained by present method and the most commonly used enzymic colorimetric method, employing free enzymes. Among the various serum substances tested at their physiological concentrations, only cholesterol, ascorbic acid and bilirubin caused 30%, 15%, and 20% inhibition of strip-bound enzymes, respectively. The strip lost 50% of its activity after 150 regular uses over a period of 33 days, when stored in reaction buffer at 4 degrees C. The method reported here has the advantage over other existing methods, as it provides higher sensitivity, better stability and reusability of co-immobilized enzymes and is also economical.     

褐飞虱高温条件下应激反应及体内保护酶系活性的研究

利用34℃、36℃、38℃3个温度处理褐飞虱的若虫和不同型成虫,研究其在热休克过程中体内保护酶系活性及脂质过氧化物含量的变化。结果表明,热休克对4龄若虫过氧化氢酶（CAT）活性影响大于3龄、5龄,而其成虫期CAT活性则随日龄增加而增加,36℃为CAT清除H2O2最适温度。高龄若虫全内谷胱甘肽过氧化物本(GSH-px）活性高于低龄若虫,老熟成虫体内GSH－px清除H2O2能力强于初羽化成虫,超氧化物歧化酶（SOD）活性与处理温度呈正相关,而同一处理温度下SOD活性随成虫龄期和成虫日龄增加而降低,长型成虫体内CAT、GSH－px、SOD活性均高于短翅型,而雌、雄成虫间CAT、GSH－px、SOD活性无明显差异（α＞0.05）。褐飞虱体内脂质过氧化物（LPO）含量随处理温度升高而上升,同一处理温度下随若虫龄期和成虫日龄的增加而增加,短型成虫体内LPO含量高于长翅型。     

Comparative kinetic characteristics of catalase of Penicillium species molds

Extracellular catalases produced by fungi of the genus Penicillium: P. piceum, P. varians and P. kapuscinskii were purified by consecutive filtration of culture liquids. The maximum reaction rate of H2O2 decomposition, the Michaelis constants and specific catalytic activities of isolated catalases were determined. The operational stability was characterized by effective rate of catalase inactivation during enzymatic reaction (kin at 30 degrees C). The thermal stability was determined by the rate of enzyme thermal inactivation at 45 degrees C (k*[symbol: see text]H, s-1). Catalase from P. piceum displayed the maximum activity, which was higher than the activity of catalase from bovine liver. The operational stability of catalase from P. piceum was twofold to threefold higher than the stability of catalase from bovine liver. The physicochemical characteristics of catalases of fungi are better than the characteristics of catalase from bovine liver and intracellular catalase of yeast C. boidinii.     

Purification and thermodynamic characterization of glucose oxidase from a newly isolated strain of Aspergillus niger

An intracellular glucose oxidase (GOD) was isolated from the mycelium extract of a locally isolated strain of Aspergillus niger NFCCP. The enzyme was partially purified to a yield of 28.43% and specific activity of 135 U mg(-1) through ammonium sulfate precipitation, anion-exchange chromatography, and gel filtration. The enzyme showed high specificity for D-glucose, with a K(m) value of 25 mmol L(-1). The enzyme exhibited optimum catalytic activity at pH 5.5. Optimum temperature for GOD-catalyzed D-glucose oxidation was 40 degrees C. The enzyme displayed a high thermostability having a half-life (t(1/2)) of 30 min, enthalpy of denaturation (H*) of 99.66 kJ mol(-1), and free energy of denaturation (G*) of 103.63 kJ mol(-1). These characteristics suggest that GOD from A. niger NFCCP can be used as an analytical reagent and in the design of biosensors for clinical, biochemical, and diagnostic assays.     

Intracellular glucose oxidase from Penicillium funiculosum 46.1

A method for isolating extracellular glucose oxidase from the fungus Penicillium funiculosum 46.1, using ultrafiltration membranes, was developed. Two samples of the enzyme with a specific activity of 914-956 IU were obtained. The enzyme exhibited a high catalytic activity at pH above 6.0. The effective rate constant of glucose oxidase inactivation at pH 2.6 and 16 degrees C was 2.74 x 10(-6) s-1. This constant decreased significantly as pH of the medium increased (4.0-10.0). The temperature optimum for glucose oxidase-catalyzed beta-D-glucose oxidation was in the range 30-65 degrees C. At temperatures below 30 degrees C, the activation energy for beta-D-glucose oxidation was 6.42 kcal/mol; at higher temperatures, this parameter was equal to 0.61 kcal/mol. Kinetic parameters of glucose oxidase-catalyzed delta-D-glucose oxidation depended on the initial concentration of the enzyme in the solution. Glucose oxidase also catalyzed the oxidation of 2-deoxy-D-glucose, maltose, and galactose.     

Kinetics of catalase inactivation induced by ultrasonic cavitation

Kinetic patterns of sonication-induced inactivation of bovine liver catalase (CAT) were studied in buffer solutions (pH 4-11) within the temperature range from 36 to 55 degrees C. Solutions of CAT were exposed to low-frequency (20.8 kHz) ultrasound (specific power, 48-62 W/cm2). The kinetics of CAT inactivation was characterized by effective first-order rate constants (s-1) of total inactivation (kin), thermal inactivation (*kin), and ultrasonic inactivation (kin(us)). In all cases, the following inequality was valid: kin > *kin. The value of kin(us) increased with the ultrasound power (range, 48-62 W/cm2) and exhibited a strong dependence on pH of the medium. On increasing the initial concentration of CAT (0.4-4.0 nM), kin(us) decreased. The three rate constants were minimum within the range of pH 6.5-8; their values increased considerably at pH < 6 and pH > 9. At 36-55 degrees C, temperature dependence of kin(us) was characterized by an activation energy (Eact) of 19.7 kcal/mol, whereas the value of Eact for CAT thermoinactivation was equal to 44.2 kcal/mol. Bovine serum and human serum albumins (BSA and HSA, respectively) inhibited sonication-induced CAT inactivation; complete prevention was observed at concentrations above 2.5 micrograms/ml. Dimethyl formamide (DMFA), a scavenger of hydroxyl radicals (HO.), prevented sonication-induced CAT inactivation at 10% (kin and *kin increased with the content of DMFA at concentrations in excess of 3%). The results obtained indicate that free radicals generated in the field of ultrasonic cavitation play a decisive role in the inactivation of CAT, which takes place when its solutions are exposed to low-frequency ultrasound. However, the efficiency of CAT inactivation by the radicals is determined by (1) the degree of association between the enzyme molecules in the reaction medium and (2) the composition thereof.     

Enhancement of thiamine release during synthetic mutualism between <Emphasis Type="Italic">Chlorella sorokiniana</Emphasis> and <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> growing under stress conditions

Thiamine release during synthetic mutualism between Chlorella sorokiniana co-immobilized in alginate beads with the microalgae growth-promoting bacterium Azospirillum brasilense was measured under stress conditions of pH, light intensity, and nitrogen starvation in short-term experiments. Thiamine release in the co-immobilized treatment was significantly higher at acidic pH compared to thiamine released by either microorganism alone. Under slightly alkaline pH, C. sorokiniana released the highest amount of thiamine. At stressful pH 6, the co-immobilized treatment released a higher quantity of thiamine than the sum of thiamine released by either microorganisms when immobilized separately. Release of thiamine by C. sorokiniana alone or co-immobilized was light intensity dependent; with higher the light intensity, more thiamine was released. Extreme light intensity negatively affected growth of the microalgae and release of thiamine. Nitrogen starvation during the first 24 h of culturing negatively affected release of thiamine by both microorganisms, where C. sorokiniana was more severely affected. Partial or continuous nitrogen starvation had similar negative effects on C. sorokiniana, but co-immobilization improved thiamine release. These results indicate that thiamine is released during synthetic mutualism between C. sorokiniana and A. brasilense, and this happens specifically during the alleviation of pH stress in the microalgae.     

Thermal inactivation of glucose oxidase. Mechanism and stabilization using additives

Thermal inactivation of glucose oxidase (GOD; beta-d-glucose: oxygen oxidoreductase), from Aspergillus niger, followed first order kinetics both in the absence and presence of additives. Additives such as lysozyme, NaCl, and K2SO4 increased the half-life of the enzyme by 3.5-, 33.4-, and 23.7-fold respectively, from its initial value at 60 degrees C. The activation energy increased from 60.3 kcal mol-1 to 72.9, 76.1, and 88.3 kcal mol-1, whereas the entropy of activation increased from 104 to 141, 147, and 184 cal x mol-1 x deg-1 in the presence of 7.1 x 10-5 m lysozyme, 1 m NaCl, and 0.2 m K2SO4, respectively. The thermal unfolding of GOD in the temperature range of 25-90 degrees C was studied using circular dichroism measurements at 222, 274, and 375 nm. Size exclusion chromatography was employed to follow the state of association of enzyme and dissociation of FAD from GOD. The midpoint for thermal inactivation of residual activity and the dissociation of FAD was 59 degrees C, whereas the corresponding midpoint for loss of secondary and tertiary structure was 62 degrees C. Dissociation of FAD from the holoenzyme was responsible for the thermal inactivation of GOD. The irreversible nature of inactivation was caused by a change in the state of association of apoenzyme. The dissociation of FAD resulted in the loss of secondary and tertiary structure, leading to the unfolding and nonspecific aggregation of the enzyme molecule because of hydrophobic interactions of side chains. This confirmed the critical role of FAD in structure and activity. Cysteine oxidation did not contribute to the nonspecific aggregation. The stabilization of enzyme by NaCl and lysozyme was primarily the result of charge neutralization. K2SO4 enhanced the thermal stability by primarily strengthening the hydrophobic interactions and made the holoenzyme a more compact dimeric structure.     

Glucose oxidase immobilization on a novel cellulose acetate-polymethylmethacrylate membrane

Glucose oxidase (GOD) was immobilized on cellulose acetate-polymethylmethacrylate (CA-PMMA) membrane. The immobilized GOD showed better performance as compared to the free enzyme in terms of thermal stability retaining 46% of the original activity at 70 degrees C where the original activity corresponded to that obtained at 20 degrees C. FT-IR and SEM were employed to study the membrane morphology and structure after treatment at 70 degrees C. The pH profile of the immobilized and the free enzyme was found to be similar. A 2.4-fold increase in Km value was observed after immobilization whereas Vmax value was lower for the immobilized GOD. Immobilized glucose oxidase showed improved operational stability by maintaining 33% of the initial activity after 35 cycles of repeated use and was found to retain 94% of activity after 1 month storage period. Improved resistance against urea denaturation was achieved and the immobilized glucose oxidase retained 50% of the activity without urea in the presence of 5M urea whereas free enzyme retained only 8% activity.     

Enhanced bio-decolorization of azo dyes by co-immobilized quinone-reducing consortium and anthraquinone

In the present study, the accelerating effect of co-immobilized anthraquinone and quinone-reducing consortium was investigated in the bio-decolorization process. The anthraquinone and quinone-reducing consortium were co-immobilized by entrapment in calcium alginate. The co-immobilized beads exhibited good catalytic activity and increased the decolorization rate for many kinds of azo dyes. The reusability of the co-immobilized beads was evaluated with repeated-batch decolorization experiments. After ten repeated experiments, the decolorization rate of co-immobilized beads retained over 92.8% of their original value. Furthermore, acclimatized quinone-reducing consortium was analyzed by denaturing gradient gel electrophoresis (DGGE) and 16S rDNA gene sequencing to get the complete picture of its diversity. This study explored a great improvement of conventional treatment system and the new bio-treatment concept.