Immobilization of yeast alcohol dehydrogenase on magnetic nanoparticles for improving its stability

Yeast alcohol dehydrogenase (YADH) was immobilized covalently on Fe₃O₄ magnetic nanoparticles (10.6 nm) via carbodiimide activation. The immobilization process did not affect the size and structure of magnetic nanoparticles. The YADH-immobilized magnetic nanoparticles were superparamagnetic with a saturation magnetization of 61 emu g^–1, only slightly lower than that of the naked ones (63 emu g^–1). Compared to the free enzyme, the immobilized YADH retained 62% activity and showed a 10-fold increased stability and a 2.7-fold increased activity at pH 5. For the reduction of 2-butanone by immobilized YADH, the activation energies within 25–45 °C, the maximum specific activity, and the Michaelis constants for NADH and 2-butanone were 27 J mol^–1, 0.23 mol min^–1 mg^–1, 0.62 mM, and 0.43 M, respectively. These results indicated a structural change of YADH with a decrease in affinity for NADH and 2-butanone after immobilization compared to the free enzyme.     

Glucose oxidase and catalase activities ofPenicillium variabile P16 immobilized in polyurethane sponge

Conidia ofPenicillium variabile P16 were immobilized in polyurethane sponge and used in repeated-batch processes in a fluidized-bed reactor. Optimal conditions for production of glucose oxidase and catalase were: inoculum size, 10%; glucose concentration, 80 g L^–1; Ca-carbonate concentration, 15 g L^–1; temperature, 28°C and aeration rate, 4 VV^–1 min^–1. In an extended repeated-batch process, glucose oxidase activity was highest after the fourth batch and catalase activity was highest after the fifth batch. Scanning electron microscopy showed that the fungus grew only in the interior of carrier particles.     

Response surface optimization for the continuous glucose isomerization process

Production of fructose via a continuous glucose isomerization process was optimized using response surface methodology. Glucose isomerization was performed using immobilized glucose isomerase in a flow-through tubular reactor. Process factors eg pH (7.0–7.8), temperature (50–60°C), flow rate (5–17 ml min^–1) and glucose content (30–50% w/w) of the feedstock solution were simultaneously tested according to a central composite experimental design. Measured responses such as % isomerization, and fructose yield (gh^–1) has an excellent correlation with tested factors. The highest desirability,D, (geometric mean of % isomerization and fructose yield) was obtained when the feedstock (56–60°C) had 34–36% glucose, a pH of 7.4–7.8 and was pumped at 15 ml min^–1.     

Immobilization of epoxide hydrolase purified from rat liver microsomes

Summary Purified epoxide hydrolase was immobilized by covalent binding to Sephadex G-150 activated with 1,1 carbonyl diimidazole under mild conditions Km^app values of free and immobilized epoxide hydrolase were 0.5 M and 2 M respectively towards benzo(a)pyrene-4,5-oxide, whereas Vmax^app was decreased from 300 nmol·min^–1·mg^–1 to 81 nmol·min^–1·mg^–1. Immobilization enhanced stability and allowed repeated use of the enzyme.     

Nitrate removal from the effluent of a fertilizer industry using a bioreactor packed with immobilized cells of <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis>and<Emphasis Type="Italic">Comamonas testosteroni</Emphasis>

A bioreactor for the removal of nitrate nitrogen (NO₃-N) from industrial effluent is described which is comprised of a glass column (60 cm × 6 cm) packed with alginate beads containing denitrifying organisms Pseudomonas stutzeri and Comamonas testosteroni. The effluent containing high concentrations of nitrate (600–950 mg l^–1) from the fertilizer industry and fusel oil (methanol as a major component) as organic carbon were used in the process. The reactor is operated in the continuous mode by injecting the pretreated nitrate-containing effluent at the top of the column. The Hydraulic retention time (HRT) was adjusted by changing the flow rates. When nitrate-containing wastewater was treated with immobilized cells, the nitrate removal rate reached a maximum 1.66 ± 0.07 Kg NO₃-N m^–3d^–1 at an influent NO₃-N concentration of 850 mg NO_3M-N l^–1within 12 h. The denitrification activity of the immobilized cells was compared with that of the free cells.     

Synthesis of -Dopa by Escherichia intermedia cells immobilized in a carrageenan gel

Escherichia intermedia cells were immobilized by entrapment in a carrageenan gel and used for -DOPA synthesis from catechol, pyruvate, and ammonia. A preparation containing 75 mg of cell per gram of gel retained 60–65% of its original activity. The effect of substrate concentrations on the initial rate of -DOPA synthesis was very similar for free and immobilized cells, and substrate inhibition was observed for the three substrates. In batch reactors, up to 7.8 g l⁻¹ of -DOPA was obtained in 20 h (productivity 0.39 g l⁻¹ h⁻¹). Cells immobilized in a carrageenan gel showed higher -DOPA synthesis, in both initial rates conditions and batch reactors, than cells immobilized in a polyacrylamide gel.     

An automated flow injection analysis system for on-line monitoring of glucose and L-lactate during lactic acid fermentation in a recycle bioreactor

The study concerns on-line sequential analysis of glucose and L-lactate during lactic acid fermentation using a flow injection analysis (FIA) system. Enzyme electrodes containing immobilized glucose oxidase and L-lactate oxidase were used with an amperometric detection system. A 12-bit data acquisition card with 16 analog input channels and 8 digital output channels was used. The software for data acquisition was developed using Visual C++, and was devised for sampling every hour for sequential analyses of lactate and glucose. The detection range was found to be 2–100 g l^–1 for glucose and 1–60 g l^–1 for L-lactate using the biosensors. This FIA system was used for monitoring glucose utilization and L-lactate production by immobilized cells of Lactobacillus casei subsp. rhamnosus during a lactic acid fermentation process in a recycle batch reactor. After 13 h of fermentation, complete sugar utilization and maximal L-lactate production was observed. A good agreement was observed between analysis data obtained using the biosensors and data from standard analyses of reducing sugar and L-lactate. The biosensors exhibited excellent stability during continuous operation for at least 45 days.     

Immobilization of invertase on a new type of macroporous glycidyl methacrylate

Invertase was immobilized via its carbohydrate moiety. The immobilized enzyme has a specific activity of 5500 IU g^–1, with 45% activity yield on immobilization. In a packed bed reactor, 90% 2.5 M sucrose was converted at a flow rate of 4 bed volumes h^–1. The obtained specific productivity at 40 °C of 3 kg l^–1 h^–1 is the best one so far. Long-term stability was 290 days in 2.5 M sucrose at 40 °C and at a flow rate of 3 bed volumes h^–1.     

Methyl mercury uptake by free and immobilized cyanobacterium

Methyl mercury uptake in free cells and different immobilizates of the cyanobacteriumNostoc calcicola has been examined. The general growth of the immobilized cyanobacterial cells could be negatively correlated with methyl mercury uptake. Alginate spheres proved most efficient in terms of uptake rate (0.48 nmol mg protein^–1 min^–1, 10 min) and total bioaccumulation (10.71 nmol mg protein^–1, 1 h) with a bioconcentration factor of 3.3×10³. Alginate biofilms showed a faster methyl mercury accumulation rate (0.83 nmol mg protein^–1 min^–1, 10 min) with a saturation of 10.28 nmol mg protein^–1 reached within only 30 min (bioconcentration factor, 3.1×10³). Foam preparations with a slow initial uptake approximated biofilms but were characterized by a lower bioconcentration factor (2.8×10³). Free cells, in comparison, maintained the initial slow rate of uptake (0.62 nmol mg protein^–1 min^–1, 10 min), saturating at 30 min (8.81 nmol mg protein^–1), and the resultant lowest bioconcentration factor (2.7×10³). Cell ageing (30 days) brought a drastic reduction (3-fold) in organomercury uptake by free cells while alginate spheres maintained the same potential. Foam preparations of the same age showed a significant improvement in methyl mercury uptake followed by only a marginal decline in alginate biofilms. Data are discussed in the light of the physiological efficiency and longevity of immobilized cells.     

Inversion of sucrose with β- -fructofuranosidase (invertase) immobilized on bead DEAHP-cellulose: Batch process

The inversion of sucrose with β- -fructofuranosidase (EC 3.2.1.26) immobilized by an ionic bond on bead cellulose containing weak basic N,N-diethylamino-2-hydroxypropyl groups has been investigated. The immobilized enzyme is strongly bound at an ionic strength up to 0.1 M in the pH range 3–6. The amount adsorbed is proportional to porosity and to the exchange capacity of the ion exchange cellulose, reaching values up to 200 mg/g dry carrier, with an activity in 10% sucrose solution at 30°C, pH 5, >8000 μmol min⁻¹ g⁻¹. The inversion of sucrose with immobilized β- -fructofuranosidase was carried out in a stirred reactor. The dependence of activity on pH (3–7), temperature (0–70°C) and concentration of the substrate (2–64 wt%) were determined, and the inversion was compared with that obtained using non-immobilized enzyme under similar conditions. The rate of inversion at low substrate concentration (2–19 wt%) was described by Michaelis-Menten kinetics.     

Effects of aeration and of corn steep concentration on L-asparaginase production in Escherichia coli

Summary The production of L-asparaginase was investigated in Escherichia coli, growing under different conditions of aeration in a medium containing 2% or 6% corn steep. At both concentrations, excessive aeration decreased enzyme production. In the medium with 2% corn steep, L-asparaginase activity began to decline as soon as the oxygen absorption exceeded 0.22 mmol O₂ l^–1 min^–1, and when the oxygen absorption rate was 1.26 mmol O₂ l^–1 min^–1, enzyme activity reached only about 5% of maximum. In the medium with 6% corn steep, a decline of L-asperaginase activity did not appear until the oxygen absorption rate value exceeded 0.54 mmol O₂ l^–1 min^–1, at the oxygen absorption rate of 1.26 mmol O₂ l^–1 min^–1, the enzyme activity still reached about 50% of maximum.     

Photosynthetic production of the filamentous cyanobacteriumSpirulina platensis in a cone-shaped helical tubular photobioreactor

The photosynthetic productivity of the filamentous cyanobacteriumSpirulina platensis was investigated in a cone-shaped helical tubular photobioreactor. A laboratory-scale photobioreactor was constructed with a 0.255-m² basal area and a conical shape (0.64 m high × 0.57 m top diameter). The photostage comprised transparent reinforced polyvinyl chloride (PVC) tubing with spirally wound, metal-wire reinforcing in the tubing wall (31 m in length and 1.6 cm internal diameter with 0.25 cm wall thickness; total volume = 6.23 l). The inner surface of the photostage (0.651 m²) was illuminated with compact fluorescent cool white lamps; the photosynthetically active radiation (400–700 nm) energy input into the photobioreactor was 1249 KJ day^–1 (12 h day/12 h night). The operation of an air-lift photobioreactor with CO₂-enriched air (4%) at a flow rate of 0.3 l min^–1 showed a maximum daily photosynthetic efficiency of 6.83% under batch-culture conditions. This corresponded to a production rate of 15.9 g dry biomass m^–2(basal area) day^–1 or 0.51 g dry biomass l medium^–1 day^–1.     

Aerobic nitrate and nitrite reduction in continuous cultures of Escherichia coli E4

Nitrate and nitrite was reduced by Escherichia coli E4 in a l-lactate (5 mM) limited culture in a chemostat operated at dissolved oxygen concentrations corresponding to 90–100% air saturation. Nitrate reductase and nitrite reductase activity was regulated by the growth rate, and oxygen and nitrate concentrations. At a low growth rate (0.11 h^–1) nitrate and nitrite reductase activities of 200 nmol · mg^–1 protein · min^–1 and 250 nmol · mg^–1 protein · min^–1 were measured, respectively. At a high growth rate (0.55 h^–1) both enzyme activities were considerably lower (25 and 12 nmol mg^–1 · protein · min^–1). The steady state nitrite concentration in the chemostat was controlled by the combined action of the nitrate and nitrite reductase. Both nitrate and nitrite reductase activity were inversely proportional to the growth rate. The nitrite reductase activity decreased faster with growth rate than the nitrate reductase. The chemostat biomass concentration of E. coli E4, with ammonium either solely or combined with nitrate as a source of nitrogen, remained constant throughout all growth rates and was not affected by nitrite concentrations. Contrary to batch, E. coli E4 was able to grow in continuous cultures on nitrate as the sole source of nitrogen. When cultivated with nitrate as the sole source of nitrogen the chemostat biomass concentration is related to the activity of nitrate and nitrite reductase and hence, inversely proportional to growth rate.     

Lack of effect of thrombin on fibrin(ogen)-endothelial cell interaction

In the present study we investigate the fibrin(ogen)-endothelial cell binding and the effect of thrombin on the endothelial cells in relation to fibrin(ogen) binding capacity. Endothelial cell fibrinogen binding was concentration and time-dependent, reaching saturation at 1.4 M of added ligand. At equilibrium, the number of fibrinogen molecules bound per endothelial cell in the monolayer was 5.8±0.7×10⁶. When endothelial cells were activated by different concentrations of thrombin (0–0.1 NIH units ml^–1), no increase in fibrinogen binding capacity was observed at all the thrombin concentration tested. Whereas disruption of endothelial cell monolayers was observed at thrombin concentrations higher than 0.05 NIH units ml^–1, no increase in the amount of fibrinogen bound was observed. Therefore, resting and thrombin-activated endothelial cells show the same fibrinogen binding capacity.The adhesion of endothelial cells in suspension on immobilized fibrinogen or fibrin was studied to ascertain whether the behavior of fibrin is similar to that of fibrinogen. The extent of endothelial cell attachment to immobilized fibrinogen and fibrin was similar (4275±130 cells cm^–2 for fibrinogen and 4350±235 cells cm^–2 for fibrin) and represent approximately 40% of the added endothelial cells. However, endothelial cell adhesion to immobilized fibrin was significantly faster than endothelial cell adhesion to immobilized fibrinogen. The maximum binding rate was 66±9 and 46±8 cells cm^–2 min^–1 for fibrin and fibrinogen, respectively. Therefore, the fibrinopeptides released by thrombin from fibrinogen induce qualitative changes which enhance the fibrin interaction with the endothelial cells.     

Cell immobilization in composite agar layer microporous membrane structures: growth kinetics of gel-entrapped cultures and cell leakage limitation by a microporous membrane

Agar discs containing different amounts of viable Escherichia coli cells (from 10 to 10⁶ organisms·g^–1 agar) were incubated in a nutrient medium and the growth of agar-entrapped bacteria and free (released) cells was monitored. The study was repeated with composite immobilized-cell structures obtained by placing a microporous membrane filter between the gel matrix and the incubation medium. In both cases, immobilized cells grew exponentially and reached a peak concentration an order of magnitude higher than that of free (suspended) cell cultures. The maximum specific growth rates of entrapped bacteria, ranging between 0.0115 min^–1 and 0.0145 min^–1, i.e., slightly higher than that of control free cultures (0.011 min^–1), showed no clear dependence on the initial cell loading (ICL). The microporous filter proved efficient in limiting cell leakage since it noticeably lengthened the leakage time at a given ICL. This efficiency, however, decreased at high ICL and high growth rate of immobilized organisms. Correspondence to: G.-A. Junter     

Degradation of 2-methylnaphthalene by free and immobilized cells of Pseudomonas sp. strain NGK1

A Pseudomonas sp. strain NGK1 (NCIM 5120) capable of utilizing 2-methylnaphthalene (2-MN) was immobilized in various matrices namely, polyurethane foam (PUF), alginate, agar and polyvinyl alcohol (PVA) (1.5 × 10¹² c.f.u. g^–1 beads). The degradation rates of 25 and 50 mM 2-MN by freely suspended cells (2 × 10¹¹ c.f.u. ml^–1) and immobilized cells in batches, semi-continuous with shaken culture and continuous degradation in a packed-bed reactor were compared. The PUF-immobilized cells achieved higher degradation of 25 and 50 mM of 2-MN than freely suspended cells and the cells immobilized in alginate, agar or PVA. The PVA- and PUF-immobilized cells could be reused for more than 30 and 20 cycles respectively, without losing any degradation capacity. The effect of dilution rates on the rate of degradation of 25 and 50 mM 2-MN with freely suspended and immobilized cells were compared in the continuous system. Increase in dilution rate increased the degradation rate only up to 1 h^–1 in free cells with 25 mM 2-MN and no significant increase was observed with 50 mM 2-MN. With immobilized cells, the degradation rate increased with increase in dilution rate up to 1.5 h^–1 for 25 mM and 1 h^–1 for 50 mM 2-MN. These results revealed that the immobilized cell systems are more efficient than freely suspended cells for biodegradation of 2-MN.     

Degradation of caffeine by immobilized cells of Pseudomonas putida strain C 3024

Summary A caffeine-resistant strain of Pseudomonas putida was isolated from soil and was grown with caffeine as the sole source of carbon, energy and nitrogen. Cells were immobilized in agar gel particles which were continuously supplied with a caffeine solution (0.52 g · l^–1, D=1.0 h^–1) in a homogeneously mixed aerated reaction vessel. In the presence of the ATPase inhibitor arsenate the caffeine was removed by the immobilized cells at an average rate of 0.25 mg caffeine · h^–1 · (mg cell carbon)^–1 during 6 days. Thereafter a rapid decline of activity was observed. From a similar system without arsenate supplied with a growth medium containing a limiting amount of caffeine (0.13 g · l^–1) the caffeine was almost completely oxidized by the immobilized cells. The concentration of the remaining caffeine was 1.4 mg · l^–1, which is much lower than the substrate constant for caffeine (9.7 mg · l^–1) observed with freshly harvested suspended resting cells.     

Immobilization of soybean (Glycine max) urease on alginate and chitosan beads showing improved stability: Analytical applications

The soybean (Glycine max) urease was immobilized on alginate and chitosan beads and various parameters were optimized and compared. The best immobilization obtained were 77% and 54% for chitosan and alginate, respectively. A 2% chitosan solution (w/v) was used to form beads in 1N KOH. The beads were activated with 1% glutaraldehyde and 0.5 mg protein was immobilized per ml of chitosan gel for optimum results. The activation and coupling time were 6 h and 12 h, respectively. Further, alginate and soluble urease were mixed to form beads and final concentrations of alginate and protein in beads were 3.5% (w/v) and 0.5 mg/5 ml gel. From steady-state kinetics, the optimum temperature for urease was 65 °C (soluble), 75 °C (chitosan) and 80 °C (alginate). The activation energies were found to be 3.68 kcal mol⁻¹, 5.02 kcal mol⁻¹, 6.45 kcal mol⁻¹ for the soluble, chitosan- and alginate-immobilized ureases, respectively. With time-dependent thermal inactivation studies, the immobilized urease showed improved stability at 75 °C and the t_1/2 of decay in urease activity was 12 min, 43 min and 58 min for soluble, alginate and chitosan, respectively. The optimum pH of urease was 7, 6.2 and 7.9 for soluble, alginate and chitosan, respectively. A significant change in K_m value was noticed for alginate-immobilized urease (5.88 mM), almost twice that of soluble urease (2.70 mM), while chitosan showed little change (3.92 mM). The values of V_max for alginate-, chitosan-immobilized ureases and soluble urease were 2.82 × 10² μmol NH₃ min⁻¹ mg⁻¹ protein, 2.65 × 10² μmol NH₃ min⁻¹ mg⁻¹ protein and 2.85 × 10² μmol NH₃ min⁻¹ mg⁻¹ protein, respectively. By contrast, reusability studies showed that chitosan–urease beads can be used almost 14 times with only 20% loss in original activity while alginate–urease beads lost 45% of activity after same number of uses. Immobilized urease showed improved stability when stored at 4 °C and t_1/2 of urease was found to be 19 days, 80 days and 121 days, respectively for soluble, alginate and chitosan ureases. The immobilized urease was used to estimate the blood urea in clinical samples. The results obtained with the immobilized urease were quite similar to those obtained with the autoanalyzer^®. The immobilization studies have a potential role in haemodialysis machines.     

Phytate dephosphorylation by free and immobilized cells ofSaccharomyces cerevisiae

Summary Saccharomyces cerevisiae in the form of baker's yeast, cells cultivated on a yeast extract-peptone-glucose medium, as well as cells immobilized in 18% (w/v) polyacrylamide gel showed the ability to hydrolyze 1.727 mM sodium phytate solution at 45°C, pH 4.6, in a stirred tank reactor. Seventy percent yield of dephosphorylation was observed after 2 h using a baker's yeast concentration of 5.8 g dry matter per 100 ml. Hydrolytic activity at 1.8–2.0 M Pi min^–1 was observed between 1st and 3rd h of the reaction in cells cultured 24 or 48 h. No inhibition by the substrate was found at sodium phytate concentrations of 0.587–1.727 mM. After 1.5 h of hydrolysis a single, well distinguished peak ofmyo-inositol-triphosphate was the main product found. By means of immobilization the stability of the biocatalyst was enhanced 3.3-fold and reached its half-life at 64 ninety-minute runs.     

Characterization of <Emphasis Type="Italic">Mucor miehei</Emphasis> lipase immobilized on polysiloxane-polyvinyl alcohol magnetic particles

Mucor miehei lipase was immobilized on magnetic polysiloxane-polyvinyl alcohol particles by covalent binding. The resulting immobilized biocatalyst was recycled by seven assays, with a retained activity around 10% of its initial activity. K_m and V_max were respectively 228.3 M and 36.1 U mg of protein^–1 for immobilized enzyme. Whereas the optimum temperature remained the same for both soluble and immobilized lipase (45 °C), there was a shift in pH profiles after immobilization. Optimum pH for the immobilized lipase was 8.0. Immobilized enzyme showed to be more resistant than soluble lipase when assays were performed out of the optimum temperature or pH.