Beta-galactosidase monitoring by a biosensor based on Clark electrode: its optimization, characterization and application

beta-Galactosidase is an hydrolase enzyme that catalyzes the hydrolysis of beta-galactosides into monosaccharides. Substrates of different beta-galactosidases include ganglioside GM1, lactosylceramides, lactose, and various glycoproteins. A novel aspect of the activity determination of beta-galactosidase was presented. A glucose oxidase biosensor based on Clark electrode was utilized in order to monitor beta-galactosidase. Immobilization of glucose oxidase was made by gelatin and glutaraldehyde as cross-linker. Several parameters such as glucose oxidase activity, gelatin amount, and glutaraldehyde percentage for cross-linking were optimized. The most important parameter, lactose concentration in working buffer was studied in detail. Optimum temperature, thermal stability, optimum pH, buffer system and its concentration effect on the biosensor system, repeatability, reproducibility, and storage and operational stabilities of the biosensor were identified. A linear detection range for beta-galactosidase was observed between 9.4 x 10(-5) and 3.2 x 10(-2)U/ml. Finally, beta-galactosidase activity in artificial intestinal juice was investigated by the biosensor and the results obtained were compared with a reference spectrophotometric method.     

葡萄糖氧化酶的有机相共价固定化

将葡萄糖氧化酶(GOD)在最适pH条件下冻干后,以戊二醛活化的壳聚糖为载体,分别在传统水相和1,4-二氧六环、乙醚、乙醇三种不同的有机相中进行共价固定化。通过比较水相固定化酶和有机相固定化酶的酶比活力、酶学性质及酶动力学参数,考察酶在有机相中的刚性特质对酶在共价固定化过程中保持酶活力的影响。结果表明,戊二醛浓度为0.1%、加酶量为80 mg/1 g载体、含水1.6%的1,4-二氧六环有机相固定化GOD与水相共价固定化GOD相比,酶比活力提高2.9倍,有效酶活回收率提高3倍;在连续使用7次后,1,4-二氧六环有机相固定化GOD的酶活力仍为相应水相固定化酶的3倍。在酶动力学参数方面,不论是表观米氏常数,最大反应速度还是转换数,1,4-二氧六环有机相固定化的GOD(Kmapp=5.63 mmol/L,Vmax=1.70μmol/(min.mgGOD),Kcat=0.304 s-1)都优于水相共价固定化GOD(Kmapp=7.33 mmol/L,Vmax=1.02μmol/(min.mg GOD),Kcat=0.221 s-1)。因此,相比于传统水相,GOD在合适的有机相中进行共价固定化可以获得具有更高酶活力和更优催化性质的固定化酶。该发现可能为酶蛋白在共价固定化时因构象改变而丢失生物活性的问题提供解决途径。     

Activity studies of glucose oxidase immobilized onto poly(N-vinylimidazole) and metal ion-chelated poly(N-vinylimidazole) hydrogels

Poly(N-vinylimidazole), PVIm, gels were prepared by γ-irradiation polymerization of N-vinylimidazole in aqueous solutions. These affinity gels with a water swelling ratio of 1800% for plain polymeric gel and between 30 and 80% for Cu(II) and Co(II)-chelated gels at pH 6.0 in phosphate buffer were used in glucose oxidase (GOx) adsorption–desorption studies. Different amounts of Cu(II) and Co(II) ions (maximum 3.64 mmol/g dry gel for Cu(II) and 1.72 mmol/g dry gel for Co(II)) were loaded onto the gels by changing the initial concentration of Cu(II) and Co(II) ions, and pH. GOx adsorption on these gels from aqueous solutions containing different amount of GOx at different pH was investigated in batch reactors. Immobilized glucose oxidase activity onto the poly(N-vinylimidazole), and Cu(II) and Co(II)-chelated poly(N-vinylimidazole) were investigated with changing pH and the initial glucose oxidase concentration. Maximum activity of immobilized glucose oxidase onto the PVIm, Cu(II) and Co(II)-chelated PVIm gels was investigated and pH dependence was observed to be at pH 6.5 for free enzyme, pH 7.0 for PVIm, pH 7.5 for Cu(II) and Co(II)-chelated PVIm gels, respectively. The stability of the immobilized enzyme is very high for all gels and the residual activity was higher than 93% in the first 10 days.     

Glucose biosensor based on nano-SiO2 and “unprotected” Pt nanoclusters

The “unprotected” Pt nanoclusters (average size 2 nm) mixed with the nanoscale SiO₂ particles (average size 13 nm) were used as a glucose oxidase immobilization carrier to fabricate the amperometric glucose biosensor. The bioactivity of glucose oxidase (GOx) immobilized on the composite was maintained and the as-prepared biosensor demonstrated high sensitivity (3.85 μA mM⁻¹) and good stability in glucose solution. The Pt–SiO₂ biosensor showed a detection limit of 1.5 μM with a linear range from 0.27 to 4.08 mM. In addition, the biosensor can be operated under wide pH range (pH 4.9–7.5) without great changes in its sensitivity. Cyclic voltammetry measurements showed a mixed controlled electrode reaction.     

Biosensor system for continuous flow determination of enzyme activities. I. Determination of glucose oxidase and lactic dehydrogenase activities

A biosensor system for continuous flow determination of enzyme activity was developed and applied to the determination of glucose oxidase and lactic dehydrogenase activities. The glucose oxidase activity sensor was prepared from the combination of an oxygen electrode and a flow cell. Similarly, the lactic dehydrogenase activity sensor was prepared from the combination of a pyruvate oxidase membrane, an oxygen electrode, and a flow cell. Pyruvate oxidase was covalently immobilized on a membrane prepared from cellulose triacetate, 1,8-diamino-4-aminomethyloctane, and glutaraldehyde. Glucose oxidase activity was determined from the oxygen consumed upon oxidation of glucose catalyzed by glucose oxidase. Lactic dehydrogenase activity was determined from the pyruvic acid formed upon dehydrogenation of lactic acid catalyzed by lactic dehydrogenase. The amount of pyruvic acid was determined from the oxygen consumed upon oxidation of pyruvic acid by pyruvate oxidase. Calibration curves for activity of glucose oxidase and lactic dehydrogenase were linear up to 81 and 300 units, respectively. One assay could be completed within 15 min for both sensors and these were stable for more than 25 days at 5°C. The relative errors were ±4 and ±6% for glucose oxidase and lactic dehydrogenase sensors, respectively. These results suggest that the sensor system proposed is a simple, rapid, and economical method for the determination of enzyme activities.     

Immobilization of a thermostable α-amylase, Termamyl, onto nitrocellulose membrane by Cibacron Blue F3GA dye binding

Highly porous nitrocellulose membranes were prepared by a solvent casting technique for the first time to immobilize α-amylase. An affinity dye, namely Cibacron Blue F3GA (CB), was incorporated covalently within the structure. The nitrocellulose–CB derivatized membranes were used for the immobilization of a starch degrading enzyme, α-amylase. Optimum conditions of immobilization for highest apparent activity were determined as pH 6.0, temperature 50°C and initial enzyme concentration 0.317 KNU/l. Under these optimum conditions, maximum enzyme immobilization yield was around 21% of the initial amount of the enzyme in the solution. Performance of free and immobilized enzymes at the same amount was compared for repeated runs. Up to the third use, immobilized enzyme showed higher activity than that of free enzyme mainly due to higher enzyme concentration in the membrane structure, then the apparent activity decreased gradually. However, when regenerated by switching pH to cause contraction/expansion of the structure, the membrane showed the highest activity, almost 2.5 times than that of the free enzyme. This unusual feature along with inexpensive cost may well make the nitrocellulose membrane an economical material for industrial application in glucose syrup production.     

Synthesis and characterization of cyclodextrin-based polymers as a support for immobilization of Candida rugosa lipase

The objective of this study was to prepare cross-linked β-cyclodextrin polymers for immobilization of Candida rugosa lipase. The structures of synthesized macrocyclic compounds were characterized by Fourier transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA) and scanning electron microscope (SEM) techniques. Properties of the immobilized systems were assessed and their performance on hydrolytic reaction were evaluated and compared with the free enzyme. The influence of activation agents (glutaraldehyde (GA) and hexamethylene diisocyanate (HMDI)) and thermal and pH stabilities of the biocatalyst was evaluated. After the optimization of immobilization process, the physical and chemical characterization of immobilized lipase was performed. Obtained data showed that the immobilized enzyme seemed better and offered some advantages in comparison with free enzyme. It can be observed that the free lipase loses its initial activity within around 80 min at 60 °C, while the immobilized lipases retain their initial activities of about 56% by HMDI and 82% by GA after 120 min of heat treatment at 60 °C.Results showed that the specific activity of the immobilized lipase with glutaraldehyde was 62.75 U/mg protein, which is 28.13 times higher than that of the immobilized lipase with HMDI.     

Comparison of the potencies of (+)- and (−)-2-ethylhexanoic acid in causing peroxisome proliferation and related biological effects in mouse liver

Male C57BL/6 mice were exposed to 1% (w/w) (+)- or (?)-2-ethylhexanoic acid or an equimolar mixture of these enantiomers in their diet for 4 or 10 days. A significant increase in liver weight and a 2- to 3-fold increase in the protein content of the mitochondrial fraction were seen in all cases. Peroxisomal palmitoyl-CoA oxidation was increased 2- to 3.5-fold after 4 days of treatment and 4- to 5-fold after 10 days, while the corresponding increases in peroxisomal lauroyl-CoA oxidase activity were 2- to 3-fold and 9- to 12-fold, respectively. Peroxisomal catalase activity was unchanged, whereas the microsomal and cytosolic activities were increased 2- to 3-fold and 6- to 16-fold, respectively. These treatments also induced microsomal ω-hydroxylation of lauric acid 7-fold and soluble epoxide hydrolase activity in the mitochondrial and cytosolic fractions, as well as microsomal epoxide hydrolase activity about 50–100%. The only significant differences observed between the effects of (+)-2-ethylhexanoic acid and its (?)-enantiomer were on peroxisomal palmitoyl-CoA oxidation and lauroyl-CoA oxidase activity after 4 days of treatment. In both these cases the (+)-enantiomer resulted in increases which were 50–75% greater than those seen with the (?)-form. © 1994 Wiley-Liss, Inc.     

Glucose concentration determination by means of fluorescence emission spectra of soluble and insoluble glucose oxidase: some useful indications for optical fibre-based sensors

By using soluble and insoluble glucose oxidase, the changes in intrinsic emission fluorescence in the visible spectral region were studied as a function of glucose concentration. Insoluble glucose oxidase (GOD) was obtained by entrapment in a gelatine membrane or by covalent attachment on an agarose membrane grafted with hexamethylendiamine. The intensity of the fluorescence emission peak at 520 nm or the value of the integral fluorescence area from 480 to 580 nm were taken as physical parameters representative of the glucose concentration during the enzyme reaction. By using these parameters, linear calibration curves for glucose concentration were obtained. The extension of the calibration curve and the sensitivity of the adopted systems were found to be dependent on the enzyme state (free or immobilized) and on the immobilization method. In particular, it was found that the extent of the linear range of the calibration curves is increased of one order of magnitude when the glucose oxidase is immobilized, while the sensitivity of the measure is decreased of one order of magnitude by the immobilization process. Measures carried out by using the integral fluorescence area resulted more sensitive than those obtained with the peak size. Useful indications for the construction of optical fibre-based sensors were drawn from the reported results.     

Novel polymeric microspheres: Synthesis,enzyme immobilization,antimutagenic activity,and antimicrobial evaluation against pathogenic microorganisms

New polymeric microspheres containing azomethine ( 1a ‐ 1c and 2a ‐ 2c ) were synthesized by condensation to compare the enzymatic properties of the enzyme glucose oxidase (GOx) and to investigate antimutagenic and antimicrobial activities. The polymeric microspheres were characterized by elemental analysis, infrared spectra (FT‐IR), proton nuclear magnetic resonance spectra, thermal gravimetric analysis, and scanning electron microscopy analysis. The catalytic activity of the glucose oxidase enzyme follows Michaelis‐Menten kinetics. Influence of temperature, reusability, and storage capacity of the free and immobilized glucose oxidase enzyme were investigated. It is determined that immobilized enzymes exhibit good storage stability and reusability. After immobilization of GOx in polymeric supports, the thermal stability of the enzyme increased and the maximum reaction rate (V_max) decreased. The activity of the immobilized enzymes was preserved even after 5 months. The antibacterial and antifungal activity of the polymeric microspheres were evaluated by well‐diffusion method against some selected pathogenic microorganisms. The antimutagenic properties of all compounds were also examined against sodium azide in human lymphocyte cells by micronuclei and sister chromatid exchange tests.     

Amperometric determination of glucose,based on the direct electron transfer between glucose oxidase and tin oxide

An amperometric glucose biosensor was designed for the detection of glucose in blood, urine, beverages, and fermentation systems. In typical glucose biosensors that employ enzymes, mediators are used for efficient electron transfer between the enzymes and the electrode. However, some of these mediators are known to be toxic to the enzymes and also must be immobilized on the surface of the electrode. We propose a mediator-free glucose biosensor that uses a glucose oxidase immobilized on a tin oxide electrode. Direct electron transfer is possible in this system because the tin oxide has redox properties similar to those of mediators. The method for immobilization of the glucose oxidase onto the tin oxide is also very simple. Tin oxide was prepared by the anodization and annealing of pure tin, and this provides a large surface area for the immobilization step because of its porosity. Glucose oxidase was immobilized onto the tin oxide using the membrane entrapment method. The proposed method provides a simple process for fabricating the enzyme electrode. Glucose oxidase immobilized onto the tin oxide, prepared in accordance with this method, has a relatively large current response when comparedto those of other glucose biosensors. The sensitivity of the biosensor was 19.55 μA/mM, and a linear response was observed between 0∼3 mM glucose. This biosensor demonstrated good reproducibility and stability.     

Analysis of micromolar concentrations of glucose by an interference free flow injection based biosensor

A fast, sensitive, interference-free, single enzyme single reagent glucose biosensor, operated in flow injection analysis (FIA) mode, was developed. The method used involved formation of colored complex of titanium sulfate reagent with the peroxide generated by glucose oxidase immobilized in a packed bed reactor. The color developed was detected spectrophotometrically in a flow cuvette. The system could measure down to 0.5 mg glucose l^–1 and the response was reproducible and linear in the range 1 mg l^–1 to 100 mg l^–1. The analysis time for a 500 l sample was 35 s and was free of interference from a number of substances tested. Analysis results using an off-line batch kit were observed to be in agreement with the developed system for determination of glucose in blood plasma samples.     

Glucose oxidase overproducing mutants of Penicillium variabile (P16)

Abstract Conidia of Penicillium variabile P16 were subjected to mutagenesis and selection for glucose oxidase production on media containing o -dianisidine. Studies of the relationship between dose of UV irradiation and conidial survival and frequency of mutation showed that the best frequency of positive mutation (17%) was obtained in correspondence to a conidial survival of 52%. Out of 54 overproducing mutants tested in shaken flasks, M-80.10 showed the highest level of glucose oxidase activity (127% higher than the wild-type). M-80.10 mutant, transferred every 15 days to fresh medium and tested monthly for 8 months, appeared stable. The time course of growth and enzyme production by the mutant M-80.10 showed an increase of the glucose oxidase activity in the culture medium up to 19 U ml⁻¹ after 96 h of fermentation.     

Improvement of covalent immobilization procedure of β‐galactosidase from Kluyveromyces lactis for galactooligosaccharides production: Modeling and kinetic study

Galactooligosaccharides (GOS) are prebiotics produced from lactose through an enzymatic reaction. Employing an immobilized enzyme may result in cost reductions; however, the changes in its kinetics due to immobilization has not been studied. This study experimentally determined the optimal reaction conditions for the production of GOS from lactose by β‐galactosidase (EC 3.2.1.23) from Kluyveromyces lactis covalently immobilized to a polysiloxane‐polyvinyl alcohol (POS‐PVA) polymer activated with glutaraldehyde (GA), and to study the transgalactosylation kinetics. Yield immobilization was 99 ± 1.1% with 78.5 ± 2.4% enzyme activity recovery. An experimental design 24 with 1 center point and 2 replicates was used. Factors were lactose [L], enzyme concentration [E], pH and temperature (T). Response variables were glucose and galactose as monosaccharides [G1], residual lactose [Lac]r and GOS as disaccharides [G2] and trisaccharides [G3]. Best conditions were pH 7.1, 40 °C, 270 gL^?1 initial lactose concentration and 6 U mL^?1 enzyme concentration, obtaining 25.46 ± 0.01 gL^?1 yield of trisaccharides. Although below the HPLC‐IR detection limit, tetrasaccharides were also identified after 115 min of reaction. The immobilization protocol was then optimized by diminishing total reactant volumes : support ratio, resulting in improved enzyme activity synthesizing 43.53 ± 0.02 gL^?1 of trisaccharides and 13.79 ± 0.21 gL^?1 of tetrasaccharides, and after four cycles remaining relative activity was 94%. A reaction mechanism was proposed through which a mathematical model was developed and rate constants were estimated, considering a pseudo steady‐state hypothesis for two concomitant reactions, and from this simplified analysis, the reaction yield could eventually be improved. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1568–1578, 2017     

Heterooligosaccharide synthesis catalyzed by α-glucosidase from Bacillus stearothermophilus

α-Glucosidase from Bacillus stearothermophilus was used as a catalyst for oligosaccharide synthesis by reversed hydrolysis. The yield of disaccharides and trisaccharides depended strongly on the units of enzyme activity added, and on the stability of the enzyme under reaction conditions. When glucose was the only saccharide present in the reaction mixture with α-glucosidase, isomaltose (51%), nigerose (25%), maltose (14%) and kojibiose (10%) were formed. In 50% glucose solution, disaccharide concentrations reached up to 400 mmol/l and trisaccharides were also produced. When other saccharides (mannose or xylose), in addition to glucose, were present in the reaction mixture, both homodisaccharides and heterodisaccharides were formed, their quantity being dependent on the glucose/saccharide acceptor ratios. The highest yields of oligosaccharides were observed with glucose alone, consistent with the observation that the enzyme stability was highest with glucose as the sole saccharide.     

Catalytic properties of the immobilized Talaromyces thermophilus β-xylosidase and its use for xylose and xylooligosaccharides production

The present study explores the efficiency of Talaromyces thermophilus β-xylosidase, in the production of xylose and xylooligosaccharides. The β-xylosidase was immobilized by different methods namely ionic binding, entrapment and covalent coupling and using various carriers. Chitosan, pre-treated with glutaraldehyde, was selected as the best support material for β-xylosidase immobilization; it gave the highest immobilization and activity yields (94%, 87%, respectively) of initial activity, and also provided the highest stability, retaining 94% of its initial activity even after being recycled 25 times. Shifts in the optimal temperature and pH were observed for the immobilized β-xylosidase when compared to the free enzyme. The maximal activity obtained for the immobilized enzyme was achieved at pH 8.0 and 53 °C, whereas that for the free enzyme was obtained at pH 7.0 and 50 °C. The immobilized enzyme was more thermostable than the free β-xylosidase. We observed an increase of the K_m values of the free enzyme from 2.37 to 3.42 mM at the immobilized state. Native and immobilized β-xylosidase were found to be stimulated by Ca²⁺, Mn²⁺ and Co²⁺ and to be inhibited by Zn²⁺, Cu²⁺, Hg²⁺, Fe²⁺, EDTA and SDS. Immobilized enzyme was found to catalyze the reverse hydrolysis reaction, forming xylooligosaccharides in the presence of a high concentration of xylose. In order to examine the synergistic action of xylanase and β-xylosidase of T. thermophilus, these two enzymes were co-immobilized on chitosan. A continuous hydrolysis of 3% Oat spelt xylan at 50 °C was performed and better hydrolysis yields and higher amount of xylose was obtained.     

Effects of immobilization on the kinetics of enzyme-catalyzed reactions. I. Glucose oxidase in a recirculation reactor system.

Glucose oxidase from Aspergillus niger was immobilized on nonporous glass beads by covalent bonding and its kinetics were studied in a packed-column recycle reactor. The optimum pH of the immobilized enzyme was the same as that of soluble enzyme; however, immobilized glucose oxidase showed a sharper pH-activity profile than that of the soluble enzyme. The kinetic behavior of immobilized glucose oxidase at optimum pH and 25 degrees C was similar to that of the soluble enzyme, but the immobilized material showed increased temperature sensitivity. Immobilized glucose oxidase showed no loss in activity on storage at 4 degrees C for nearly ten weeks. On continuous use for 60 hr, the immobilized enzyme showed about a 40% loss in activity but no change in the kinetic constant.     

Simultaneous enzymatic/electrochemical determination of glucose and L-glutamine in hybridoma media by flow-injection analysis

A split-stream flow-injection analysis system is described for simultaneous determination of glucose and L-glutamine in serum-free hybridoma bioprocesses media. Amperometric measurement of glucose is based on anodic oxidation of hydrogen peroxide produced by immobilized glucose oxidase within a triple layer membrane of an integrated flow-through glucose-selective biosensor. Determination of L-glutamine is based on quantitating ammonium ions produced in a flow-through enzymes reactor containing immobilized glutaminase enzyme, and subsequent downstream potentiometric detection of these ions by a nonacting-based ion-selective polymer membrane electrode. Endogenous potassium and ammonium ion interference in the L-glutamine determination are eliminated by using a novel in-line tubular cation-exchange membrane unit to exchange these interferent species for cations undetectable by the membrane electrode. The first generation split-steam flow-injections system can assay 12 samples/h using direct injections of 50 muL of media samples, with linear responses to glucose in the range of 0.03 to 30mM, and log-linear response to L-glutamine from 0.1 to 10 mM. (c) 1993 Wiley & Sons, Inc.