Immobilization of L-glutamate oxidase and peroxidase for glutamate determination in flow injection analysis system

Streptomyces SP.N 14, isolated from soil samples, produced extracellular L-glutamate oxidase (GOD) in liquid culture. After a two-step ammonium sulfate purification and dextran G-150 chromatography, the specific activity was reached at 28.2 U/mg. The partial purified enzyme and horseradish peroxidase (HRP) were covalently coupled to alkylamine controlled pore glass (CPG) by means of glutaraldehyde. About 200–300 U/g of immobilized GOD and 300–400 U/g of immobilized HRP were obtained. The immobilized enzymes were packed into a teflon tube and used in flow injection analysis (FIA) for glutamate in broth. A good linear range was observed for this immobilized enzyme system at 0.1–2.0 mM, and the precision was 2.8% (n = 25). More than 80 samples were measured within an hour. One enzyme column with about 4 U of immobilized GOD and 5 U of immobilized HRP, applied for 50 assays/d, has been used for more than 50 d. The concentration of L-glutamate remaining lower than 2.0 mM, the determination of glutamate in this system was not affected by pH and temperature within the range of 6.0–7.0 and 25–35‡C, respectively. The system was applied to determine L-glutamate in broth samples during L-glutamate fermentation, and good correlation was achieved between results obtained with the system and with the Warburg’s method.     

Enhancing enzymatic properties by the immobilization method

Effects of some immobilized carriers on enzymatic properties have been studied. The following results were obtained: (1) When cholinesterase was immobilized on the hydrophobic carrier with either α-naphthylamine, benzylamine, orp-methylbenzylamine groups, the affinities of immobilized cholinesterase for toxic organophosphors, GB (Isopnopy 1-methylphophonofluoridate) and Vx [o-ethyl-S-(2-diisopnoylomino-thyl) methyl phosphonothiolate], were enhanced 60–90 times and 700–1200 times, respectively, whereas the thermal stability of the immobilized cholinesterase increased to 110%. Approximately 82–88% activity of the immobilized cholinesterase remained after continuously operating for 8 h; and (2) Lipase was immobilized on the carrier that was made up of 6% polyethylenimine, 1% alginate gel, and 1% glutaraldehyde. The initial reaction rate of the esterification of lauric acid with lauric alcohol catalyzed by this kind of immobilized lipase was increased 21 times, as compared to lipase powder. About 72% esterification activity of lipase remained after continuous operating for 10 d.     

The immobilization of Cytochrome c on MWNT–PAMAM–Chit nanocomposite incorporated with DNA biocomposite film modified glassy carbon electrode for the determination of nitrite

A novel and sensitive biosensor was developed for the determination of nitrite. Firstly, multi-walled carbon nanotubes–poly(amidoamine)–chitosan (MWNT–PAMAM–Chit) nanocomposite along with the incorporation of DNA was used to modify the glassy carbon electrode. Then the immobilization of Cyt c was accomplished using electrochemical deposition method by consecutive cyclic voltammetry (CV) scanning in a neutral Cyt c solution. CV behaviors of the modified electrodes showed that the MWNT–PAMAM–Chit nanocomposite is a good platform for the immobilization of DNA and Cyt c in order, at the same time, an excellent promoter for the electron transfer between Cyt c and the electrode. At high potential, the immobilized Cyt c could be further oxidized into highly reactive Cyt c π-cation by two-step electrochemical oxidation, which could oxidize NO₂ ⁻ into NO₃ ⁻ in the solution. Therefore, a nitrite biosensor based on the biocatalytic oxidation of the immobilized Cyt c was fabricated, which showed a fast response to nitrite (less than 5 s). The linear range of 0.2–80 μM and a detection limit of 0.03 μM was obtained. Finally, the application in food analysis using sausage as testing samples was also investigated.     

Immobilization of Naringinase in PVA–Alginate Matrix Using an Innovative Technique

A synthetic polymer, polyvinyl alcohol (PVA), a cheap and nontoxic synthetic polymer to organism, has been ascribed for biocatalyst immobilization. In this work PVA–alginate beads were developed with thermal, mechanical, and chemical stability to high temperatures (<80 °C). The combination of alginate and bead treatment with sodium sulfate not only prevented agglomeration but produced beads of high gel strength and conferred enzyme protection from inactivation by boric acid. Naringinase from Penicillium decumbens was immobilized in PVA (10%)–alginate beads with three different sizes (1–3 mm), at three different alginate concentrations (0.2–1.0%), and these features were investigated in terms of swelling ratio within the beads, enzyme activity, and immobilization yield during hydrolysis of naringin. The pH and temperature optimum were 4.0 and 70 °C for the PVA–alginate-immobilized naringinase. The highest naringinase activity yield in PVA (10%)–alginate (1%) beads of 2 mm was 80%, at pH 4.0 and 70 °C. The Michaelis constant (K _Mapp) and the maximum reaction velocity (V _maxapp) were evaluated for both free (K _Mapp = 0.233 mM; V _maxapp = 0.13 mM min⁻¹) and immobilized naringinase (K _Mapp = 0.349 mM; V _maxapp = 0.08 mM min⁻¹). The residual activity of the immobilized enzyme was followed in eight consecutive batch runs with a retention activity of 70%. After 6 weeks, upon storage in acetate buffer pH 4 at 4 °C, the immobilized biocatalyst retained 90% of the initial activity. These promising results are illustrative of the potential of this immobilization strategy for the system evaluated and suggest that its application may be effectively performed for the entrapment of other biocatalysts.     

Lactose Hydrolysis by β-Galactosidase Covalently Immobilized to Thermally Stable Biopolymers

Lactose has been hydrolyzed using covalently immobilized β-galactosidase on thermally stable carrageenan coated with chitosan (hydrogel). The hydrogel’s mode of interaction was proven by Fourier transform infrared spectroscopy, differential scanning calorimetry (DSC), and Schiff’s base formation. The DSC thermogram proved the formation of a strong polyelectrolyte complex between carrageenan and chitosan followed by glutaraldehyde as they formed one single peak. The modification of carrageenan improved the gel’s thermal stability in solutions from 35 °C to 95 °C. The hydrogel has been proven to be efficient for β-galactosidase immobilization where 11 U/g wet gel was immobilized with 50% enzyme loading capacity. Activity and stability of free and immobilized β-galactosidase towards pH and temperature showed marked shifts in their optimum pH from 4.5–5 to 5–5.5 and temperature from 50 °C to 45–55 °C after immobilization, which reveals higher catalytic activity and reasonable stability at wider pHs and temperatures. The apparent K _m of the immobilized enzyme increased from 13.2 to 125 mM, whereas the V _max increased from 3.2 to 6.6 μmol/min compared to the free enzyme, respectively. The free and immobilized enzymes showed lactose conversion of 87% and 70% at 7 h, respectively. The operational stability showed 97% retention of the enzyme activity after 15 uses, which demonstrates that the covalently immobilized enzyme is unlikely to leach. The new carrier could be suitable for immobilization of other industrial enzymes.     

Synthesis of Poly(<Emphasis Type="Italic">o</Emphasis>-anisidine)/H<Subscript>2</Subscript>SO<Subscript>4</Subscript> Film for the Development of Glucose Biosensor

The poly(o-anisidine)–sulfuric acid–glucose oxidase (POA–H₂SO₄–GOx) electrode has been investigated in the present work. Platinum electrode was used for the synthesis of poly (o-anisidine)–sulfuric acid (POA–H₂SO₄) film using galvanostatic method with 0.2 M o-anisidine, 1.0 M H₂SO₄ solution, 1.0 pH and 2 mA/cm² applied current density. The synthesized film was characterized using electrochemical technique, conductivity measurement, UV–visible spectroscopy, Fourier transform infrared spectroscopy, and scanning electron microscopy. GO_X was immobilized on synthesized POA–H₂SO₄ film by cross-linking via glutaraldehyde in phosphate and acetate buffer. The Michaelis–Menten constant ( K_\textm￠K_{\text{m}}^\prime ) was determined for the immobilized enzyme. The glucose oxidase electrode shows the maximum current response at pH 5.5 and potential 0.6 V. The sensitivity of POA–H₂SO₄–GO_X electrode in phosphate and acetate buffer has been recorded. The results of this study reveal that the phosphate buffer gives fast response as compared to acetate buffer in amperometric measurements.     

Enantioselective Resolution of γ-Lactam by a Whole Cell of <Emphasis Type="Italic">Microbacterium hydrocarbonoxydans</Emphasis> (L29-9) Immobilized in Polymer of PVA–Alginate–Boric Acid

Using immobilized cells of a novel strain of Microbacterium hydrocarbonoxydans L29-9 in polymers of polyvinyl alcohol (PVA)–alginate–boric acid, enantioselective resolution of racemic γ-lactam to produce (−)γ-lactam was successfully carried out. A 6:1 ratio of PVA:sodium alginate not only prevented agglomeration of the matrix but also produced beads with high gel strength. The optimum biotransformation conditions were 1 g/L substrate, pH 7.0, reaction temperature of 30 °C, and reaction time of 3 h. After every two cycles, the immobilized cell beads were separated and immersed in 0.5 mM KCl solution at 4 °C for preservation. At optimum conditions, the enantiomeric excess and the yield of (−)γ-lactam were >99% and 34%, respectively. The beads showed a slight decrease in the enantiomeric excess when re-used up to 14 cycles (the enantioselectivity of the immobilized cells decreased slightly after 14 cycles of usage).     

Immobilization of prostaglandin synthetase by hydrophobic adsorption

In this article, the immobilization of prostaglandin synthetase onn-alkyl or aryl amino-agar beads by hydrophobic adsorption is reported. The effects of different hydrophobic groups in the agar beads, pH of buffer, concentration of salts on the adsorption of prostaglandin synthetase, and the properties of immobilized prostaglandin synthetase were also studied. The results showed that 20–35 mg of microsome containing PG synthetase (protein content 8–15 mg) could be adsorbed on each gram ofn-dodecylamino-agar beads after suction drying the gel in the buffer of pH 5.5 (containing 0.5 mol/L KC1), 0.1 mol/L citric-phosphate at 4‡C. The remaining immobilized enzyme activity was over 80%. The optimum pH of immobilized PG synthetase is 8.0, similar to that of the native enzymes. The thermostability of immobilized PG synthetase in the buffer containing 0.5 mol/L KC1 was increased. Immobilized PG synthetase was used as a catalyst of synthesis of prostaglandin E₁. The preservation of activity after 10 working cycles was 86.2%.     

Characterization of Sol-gel bioencapsulates for ester hydrolysis and synthesis

Candida rugosa lipase was entrapped in silica sol-gel particles prepared by hydrolysis of methyltrimethoxysilane and assayed by p-nitrophenyl palmitate hydrolysis, as a function of pH and temperature, giving pH optima of 7.8 (free enzyme) and 5.0–8.0 (immobilized enzyme). The optimum temperature for the immobilized enzyme (50–55°C) was 19°C higher than for the free enzyme. Thermal, operational, and storage stability were determined with n-butanol and bytyric acid, giving at 45°C a half-life 2.7 times greater for the immobilized enzyme; storage time was 21 d at room temperature. For ester synthesis, the optimum temperature was 47°C, and high esterification conversions were obtained under repeated batch cycles (half-life of 138 h).     

Immobilization of Phenylalanine Dehydrogenase and Its Application in Flow-Injection Analysis System for Determination of Plasma Phenylalanine

Phenylalanine dehydrogenase (l-PheDH) from Sporosarcina ureae was immobilized on DEAE-cellulose, modified initially with 2-amino-4,6-dichloro-s-triazine followed by hexamethylenediamine and glutaraldehyde. The highest activity of immobilized PheDH was determined as 95.75 U/g support with 56% retained activity. The optimum pH value of immobilized l-PheDH was shifted from pH 10.4 to 11.0. The immobilized l-PheDH showed activity variations close to the maximum value in a wider temperature range of 45–55 °C, whereas it was 40 °C for the native enzyme. The pH and the thermal stability of the immobilized l-PheDH were also better than the native enzyme. At pH 10.4 and 25 °C, K _m values of the native and the immobilized l-PheDH were determined as K _{m Phe} = 0.118, 0.063 mM and K _{m NAD}⁺ = 0.234, 0.128 mM, respectively. Formed NADH at the exit of packed bed reactor column was detected by the flow-injection analysis system. The conversion efficiency of the reactor was found to be 100% in the range of 5–600 μM Phe at 9 mM NAD⁺ with a total flow rate of 0.1 mL/min. The reactor was used for the analyses of 30 samples each for 3 h per day. The half-life period of the reactor was 15 days.     

Statistical Optimization of Alpha-Amylase Production with Immobilized Cells of <Emphasis Type="Italic">Streptomyces erumpens</Emphasis> MTCC 7317 in <Emphasis Type="Italic">Luffa cylindrica</Emphasis> L. Sponge Discs

The purpose of this investigation was to study the effect of Streptomyces erumpens cells immobilized in various matrices, i.e., agar–agar, polyacrylamide, and luffa (Luffa cylindrica L.) sponge for production of α-amylase. Luffa sponge was found to be 21% and 51% more effective in enzyme yield than agar–agar and polyacrylamide, respectively. Response surface methodology was used to evaluate the effect of three main variables, i.e., incubation period, pH, and temperature on enzyme production with immobilized luffa cells. The experimental results showed that the optimum incubation period, pH, and temperature were 36h, 6.0, and 50 °C, respectively. The repeated batch fermentation of immobilized cells in shake flasks showed that S. erumpens cells were more or less equally physiologically active on the support even after three cycles of fermentation (3,830–3,575 units). The application of S. erumpens crude enzyme in liquefying cassava starch was studied. The maximum hydrolysis of cassava starch (85%) was obtained with the application of 4ml (15,200 units) of crude enzyme after 5 h of incubation.     

Recombinant firefly luciferase in Escherichia coli

The authentic recombinant luciferase, the luciferase with the structure similar to that of the native protein, was obtained using random mutagenesis, and its properties were studied in comparison with several fusion proteins. Thermoinactivation curves of the recombinant luciferases within the 10–50°C temperature interval showed that thermoinactivation involves reversible and irreversible steps. Immobilization of the recombinant Luciola mingrelica and Photinus pyralis firefly luciferases on BrCN-activated sepharose was carried out. Immobilization resulted in the preparation of enzymes with high catalytic activity. Physicochemical properties and analytical characteristics of the immobilized recombinant and native luciferases were studied. The catalytic properties of the immobilized recombinant L. mingrelica luciferase were close to those of the native luciferase but the former enzyme appeared to be significantly more stable. The immobilized recombinant luciferases can be used for ATP assay within 0.01–10000 nM range.     

Effect of Concentration and Substrate Flow Rate on Isomaltulose Production from Sucrose by <Emphasis Type="Italic">Erwinia</Emphasis> sp. Cells Immobilized in Calcium-Alginate Using Packed Bed Reactor

Isomaltulose was obtained from sucrose solution by immobilized cells of Erwinia sp. D12 using a batch and a continuous process. Parameters for sucrose conversion into isomaltulose were evaluated using both experimental design and response surface methodology. Erwinia sp. D12 cells were immobilized in different alginates, and the influence of substrate flow rate and concentration parameters to produce isomaltulose from sucrose were observed. Response surface methodology demonstrated that packed bed columns containing cells immobilized in low-viscosity sodium alginate (250 cP) presented a mean isomaltulose conversion rate of 47%. In a continuous process, both sucrose substrate concentration and substrate flow rate parameters had a significant effect (p < 0.05) and influenced the conversion of sucrose into isomaltulose. Higher conversion rates of sucrose into isomaltulose, from 53–75% were obtained using 75 g of immobilized cells at a substrate flow rate of 0.6 mL/min.     

Glucoamylase covalently coupled to porous glass

Glucoamylase (EC 3.2.1.3) was immobilized to alkylamine porous glass with glutaraldehyde. The choice and pretreatment of carrier and conditions for immobilization have been investigated. The immobilized enzyme contained about 4.0–8.0% protein and its activity was about 1000–1700 U/g. Some characteristics of the immobilized enzyme and the native enzyme have been comparatively investigated. The optimum temperature and the pH stability of the preparation were almost identical to the native one. However, the optimum pH of bound glucoamylase shifted 1.3 pH units toward the alkaline side compared to the native one. The Michaelis constant(K _m ) of bound glucoamylase for soluble starch was about four times higher than that of the native enzyme, whileK _m values for maltose approached those of the native material. At 45‡C the half-life of IMG was 104 days under operational conditions. Alkaline protease, α-amylase, asparaginase, and penicillin acylase were also chemically coupled to porous glass by the same method and high relative activities were obtained.     

Immobilized enzymes and cells in poly(N-vinyl caprolactam)-based hydrogels

A one-step mild method for entrapping animal cells and enzymes in macroporous composite poly (N-vinyl caprolactam)-calcium alginate (PVCL-CaAlg) hydrogels is described. Some properties of immobilized enzymes, such as thermal and storage stabilities and stability in water/organic media were investigated. Composite PVCL-CaAlg gels were successfully applied to immobilize a number of proteases, namely, trypsin, α-chymotrypsin, carboxypeptidase B, and thrombin. Thermal stability of the immobilized preparations obtained by entrapment in hydrogel beads allowed us to use them at 65–80†C, while the native enzymes were completely inactivated at 50–55°C. Various applications of enzymes and cells immobilized in beads weredemonstrated. Immobilized trypsin and carboxypeptidase B were applied to prepare human insulin from recombinant proinsulin. The hydrogel beads with entrapped α-chymotrypsin were used in enantioselective hydrolysis of Shiff's base of D,L-phenylalanine ethyl ester (SBPH) in acetonitrile/water medium. Thrombin immobilized in PVCL-based hydrogel films was shown to be a promising compound for wound treatment. To prepare pure preparations of monoclonal antibodies (MAb) several hybridoma cell lines, including hybridoma cell lines producing MAb to interleukin-2, were successfully cultivated in the hydrogel beads.     

Dry Red Wine Making Using Yeast Immobilized on Cork Pieces

The commercial Saccharomyces cerevisiae Uvaferme 299 wine yeast was immobilized on cork pieces to produce a biocatalyst for use in dry red wine making. The immobilized biocatalyst was suitable for clarified and non-clarified grape must fermentation at ambient and low temperatures (0–25 °C). Fermentation times were low and very low amounts of residual sugar were detected, showing suitability for dry wine production. The presence of suspended solids in the non-clarified must did not affect the activity of the immobilized cells. Complete fermentation of sugars at 0 °C was possible with immobilized Uvaferme 299, although not a cryotolerant strain, indicating a cryoprotective effect of cork. The presence of cork did not affect the composition of major volatiles with methanol and acetaldehyde kept at low levels. Reduction of amyl alcohols on total volatiles was also observed in wines fermented at low temperatures. Differences in the headspace aroma profile in wines produced by immobilized and free cells were found by GC–MS analysis, but no cork taint compounds were detected.     

Laccase from Coriolus hirsutus as alternate label for enzyme immunoassay

A new label—laccase from the fungus Coriolus hirsutus—was applied for solid-phaseenzyme-linked immunosorbentassays of the pesticide 2,4-dichlorophenoxyacetic acid (2,4-D). Two proposed assays are based on (1) competitive binding of antibody-laccase conjugate with immobilized 2,4-D-protein conjugate and 2,4-D in tested sample, and (2) competition of 2,4-D and 2,4-D-laccase conjugate for binding with immobilized antibodies. Kinetic and concentration dependencies for these reactions were studied, and the ELISAs were optimized in accordance with the data obtained. The elaborated systems perm it the detection of 2,4-D in concentrations down to 10–20ng/mL; time of the assays is 1.5–2 h. The main advantage of the laccase label, in comparison with the widely used peroxidase one, lies in the lack of hydrogen peroxide from substrate mixture, because dissolved oxygen plays the role of oxidizer.     

Immobilization ofLactobacillus bulgaricus in a hollowfiber bioreactor for production of lactic acid from acid whey permeate

Lactobacillus bulgaricus was immobilized in the shell side of an industrial hollow-fiber ultrafiltration module. Acid whey permeate, containing 46 g/L lactose supplemented with 10 g/L yeast extract, was pumped through the tube side at dilution rates of 0.2–2.5/h. At a cell concentration of 100 g/L, productivity was 1.5–5 g lactic acid/L/h.     

Immobilization of Yarrowia lipolytica Lipase—a Comparison of Stability of Physical Adsorption and Covalent Attachment Techniques

Lipase immobilization offers unique advantages in terms of better process control, enhanced stability, predictable decay rates and improved economics. This work evaluated the immobilization of a highly active Yarrowia lipolytica lipase (YLL) by physical adsorption and covalent attachment. The enzyme was adsorbed on octyl–agarose and octadecyl–sepabeads supports by hydrophobic adsorption at low ionic strength and on MANAE–agarose support by ionic adsorption. CNBr–agarose was used as support for the covalent attachment immobilization. Immobilization yields of 71, 90 and 97% were obtained when Y. lipolytica lipase was immobilized into octyl–agarose, octadecyl–sepabeads and MANAE–agarose, respectively. However, the activity retention was lower (34% for octyl–agarose, 50% for octadecyl–sepabeads and 61% for MANAE–agarose), indicating that the immobilized lipase lost activity during immobilization procedures. Furthermore, immobilization by covalent attachment led to complete enzyme inactivation. Thermal deactivation was studied at a temperature range from 25 to 45°C and pH varying from 5.0 to 9.0 and revealed that the hydrophobic adsorption on octadecyl–sepabeads produced an appreciable stabilization of the biocatalyst. The octadecyl–sepabeads biocatalyst was almost tenfold more stable than free lipase, and its thermal deactivation profile was also modified. On the other hand, the Y. lipolytica lipase immobilized on octyl–agarose and MANAE–agarose supports presented low stability, even less than the free enzyme.     

Electrochemical detection of phenolic compounds using composite film of multiwall carbon nanotube/surfactant/tyrosinase on a carbon paste electrode

A new tyrosinase-based biosensor was developed for detection of phenolic compounds using composite film of multiwall carbon nanotube (MWCNT)/dimethylditetradecylammonium bromide (DTDAB)/tyrosinase (Tyr) on a Nafion-incorporated carbon paste electrode. The biosensor showed a sensitive electrochemical response to the reduction of the oxidation products of different phenolic compounds (phenol, catechol, p-cresol, and p-chlorophenol) by dissolved O₂ in the presence of the immobilized enzyme. The effects of pH, operating potential, MWCNT concentration, and the DTDAB/Tyr ratio on electrochemical response were explored for optimum analytical performance. The biosensor exhibited a linear response range of 1.5–25.0, 2.0–15.0, 2.0–15.0, and 2.5–25.0 μM and sensitivity of 2,900, 3,100, 3,100, and 1,500 μA/mM for phenol, catechol, p-cresol, p-chlorophenol, respectively. In addition, the response of the enzyme electrode showed Michaelis–Menten behavior at concentrations of the phenolic compounds higher than 5.0 μM. The stability and the application of the biosensor were also evaluated.