Comparison of β-galactosidase immobilization by entrapment in and adsorption on poly(2-hydroxyethylmethacrylate) membranes

β-Galactosidase was immobilized in/on poly(2-hydroxyethyl methacrylate) (pHEMA) membranes by two different methods: adsorption on Cibacron F3GA derivatized pHEMA membranes (pHEMA-CB), and entrapment in the bulk of the pHEMA membranes. The maximum β-galactosidase adsorption on pHEMA-CB membranes was obtained as 95·6μgcm^-2 in 2·0mgcm^-3 enzyme solution. The adsorption phenomena appeared to follow a typical Langmuir isotherm. In the entrapment, an increase in β-galactosidase loading resulted in a consistent increase in membrane activity from 3·3×10^-2 to 17·8×10^-2Ucm^-2 pHEMA membranes. The K_m values for both immobilized β-galactosidase (adsorbed 0·32mM and entrapped 0·81mM ) were higher than that of the free enzyme (0·26mM ). The optimum reaction temperature of the adsorbed enzyme was 5°C higher than that of both the free and the entrapped enzyme. The optimum reaction pH was 7·5 for free and both immobilized preparations. After 15 successive uses the retained activity of the adsorbed and the entrapped enzymes was 80% and 95%, respectively. The storage stability of the enzyme was found to increase upon immobilization. ©1997 SCI     

β‐Galactosidase immobilization into poly(hydroxyethyl methacrylate) membrane and performance in a continuous system

The activity of β‐galactosidase immobilized into a poly(2‐hydroxyethyl methacrylate) (pHEMA) membrane increased from 1.5 to 10.8 U/g pHEMA upon increase in enzyme loading. The K_m values for the free and the entrapped enzyme were found to be 0.26 and 0.81 mM, respectively. The optimum reaction temperatures for the free and the entrapped β‐galactosidase were both found to be 50°C. Similarly, the optimum reaction pH was 7.5 for both the free and the entrapped enzyme. The immobilized β‐galactosidase was characterized in a continuous system during lactose hydrolysis and the operational inactivation rate constant (k_iop) of the entrapped enzyme was found to be 3.1 × 10⁻⁵ min⁻¹. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 72: 1367–1373, 1999     

Fourier-transform infrared assay of bile salt-stimulated lipase activity in reversed micelles

A Fourier-transform infrared (FT-IR) spectroscopic method has been developed for assaying the bile salt-stimulated human milk lipase (BSSL, EC3.1) catalyzed hydrolysis of triolein in AOT reversed micelles in iso-octane. At 37°C in 50 mmol dm^?3 AOT the molar absorbtivities for the carbonyl stretching frequencies for triolein (at 1751 cm^?1) and oleic acid (at 1714 cm^?1) were 1646 dm³ mol^?1 cm^?1 and 743 dm^?3 mol^?1 cm^?1, respectively. The rate was linearly dependent upon the concentration of enzyme in the water pool up to 10 mg cm^?3 and maximum activity was observed at a ratio (w₀) of [H₂O]:[AOT] = 16·7. Using these conditions, and in the presence of 10 mmol dm^?3 sodium taurocholate (TC), the derived Michaelis–Menten parameters V_max and K_m were 57·5 μmol min^?1 mg^?1 and 5·53 mmol dm^?3, respectively. These results are compared with those obtained in an oil-in-water microemulsion system and are discussed in terms of the relative partitioning of the enzyme and the substrate in the aqueous and oil phases and the interfacial concentration of the substrate in the two systems.     

Immobilization of invertase onto dimer acid‐co‐alkyl polyamine

Invertase was immobilized onto the dimer acid‐co‐alkyl polyamine after activation with 1,2‐diamine ethane and 1,3‐diamine propane. The effects of pH, temperature, substrate concentration, and storage stability on free and immobilized invertase were investigated. Kinetic parameters were calculated as 18.2 mM for K_m and 6.43 × 10^?5 mol dm^?3 min^?1 for V_max of free enzyme and in the range of 23.8–35.3 mM for K_m and 7.97–11.71 × 10^?5 mol dm^?3 min^?1 for V_max of immobilized enzyme. After storage at 4°C for 1 month, the enzyme activities were 21.0 and 60.0–70.0% of the initial activity for free and immobilized enzyme, respectively. The optimum pH values for free and immobilized enzymes were determined as 4.5. The optimum temperatures for free and immobilized enzymes were 45 and 50°C, respectively. After using immobilized enzyme in 3 days for 43 times, it showed 76–80% of its original activity. As a result of immobilization, thermal and storage stabilities were increased. The aim of this study was to increase the storage stability and reuse number of the immobilized enzyme and also to compare this immobilization method with others with respect to storage stability and reuse number. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 93: 1526–1530, 2004     

Kinetic behaviour and stability of glucose oxidase entrapped in liposomes

Glucose oxidase (EC 1.1.3.4) was encapsulated in liposomes (prepared from phosphatidyl choline and cholesterol) by the dehydration–rehydration method. The enzymatic activities of native and liposomal glucose oxidase were followed by the amount of H₂O₂ obtained in the enzymatic β‐D ‐glucose oxidation. Some characteristics of the liposomal and free glucose oxidase were compared. The enzyme encapsulated in liposomes showed an apparent inhibition by glucose at concentrations higher than 0.28 mol dm^?3 with a substrate inhibition constant of 0.95 ± 0.12 mol dm^?3. The enzyme entrapped showed an apparent K_m value higher than that of the free enzyme. The apparent V_max of liposomal enzyme decreased by a factor of 0.35 with respect of that of the native enzyme. The optimum temperature of the free and entrapped enzymes remained similar but the liposomal enzyme showed maximal activity at a more acid pH (5.2). The thermal and proteolytic stabilities were enhanced by encapsulation in liposomes. The stabilization factors (relationship between half‐lives of entrapped form and free enzyme) at 45, 50 and 55 °C for liposomal glucose oxidase were 2.6, 1.6 and 1.6, respectively. Copyright © 2003 Society of Chemical Industry     

Epoxy‐derived pHEMA membrane for use bioactive macromolecules immobilization: Covalently bound urease in a continuous model system

Poly(2‐hydroxyethylmethacrylate) (pHEMA) membranes were prepared by UV‐initiated photopolymerization of HEMA in the presence of an initiator (α‐α′‐azobisisobutyronitrile, AIBN). The epoxy group, i.e., epichlorohydrin, was incorporated covalently, and the urease was immobilized onto pHEMA membranes by covalent bonding through the epoxy group. The retained activity of the immobilized enzyme was found to be 27%. The K_m values were 18 and 34 mM for the free and the immobilized enzymes, respectively, and the V_max values were found to be 59.7 and 16.2 U mg⁻¹ for the free and the immobilized enzyme. The optimum pHs was 7.2 for both forms, and the optimum temperature for the free and the immobilized enzymes were determined to be 45 and 50°C, respectively. The immobilized urease was characterized in a continuous system and during urea degradation the operational stability rate constant for the immobilized enzyme was found to be 5.83 × 10⁻⁵ min⁻¹. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 77: 2000–2008, 2000     

Poly(hydroxyethyl methacrylate‐co‐glycidyl methacrylate) reactive membrane utilised for cholesterol oxidase immobilisation

Poly(2‐hydroxyethyl methacrylate‐co‐glycidyl methacrylate) p(HEMA–GMA) membrane was prepared by UV‐initiated photopolymerisation of 2‐hydroxyethyl methacrylate (HEMA) and glycidyl methacrylate (GMA) in the presence of an initiator, azobisisobutyronitrile (AIBN). Cholesterol oxidase was immobilised directly on the membrane by forming covalent bonds between its amino groups and the epoxide groups of the membrane. An average of 53 µg of enzyme was immobilised per cm² of membrane, and the bound enzyme retained about 67% of its initial activity. Immobilisation improved the pH stability of the enzyme as well as its temperature stability. The optimum temperature was 5 °C higher than that of the free enzyme and was significantly broader. The thermal inactivation rate constants for free and immobilised preparations at 70 °C were calculated as k_{i (free)} 1.06 × 10^?1 min^?1 and k_{i (imm)} 2.68 × 10^?2 min^?1, respectively. The immobilised enzyme activity was found to be quite stable in the repeated experiments. © 2002 Society of Chemical Industry     

Some properties of β-D-Glucosidase from Trichoderma harzianum C1R1

β-D-Glucosidase from Trichoderma harzianum C₁R₁ consists of several isocomponents having isoelectric points in the pH range of 4.85-7.50. All the components exhibit both cellobiase and 4-nitrophenyl β-D-glucosidase (4NPGase) activity. The enzyme affinity for cellobiose (K_m = 3.92 mmol dm^?3) is 14.5 times weaker than for 4NPG (K_m = 0.27 mmol dm^?3). The hydrolysis of both substrates is competitively inhibited by glucose, the inhibition of 4NPG hydrolysis (K₁ = 2.00 mmol dm^?3) being about 4.2 times stronger compared to the hydrolysis of cellobiose (K₁ = 8.43 mmol dm^?3). The 4NPG hydrolysis is also competitively inhibited by the presence of cellobiose and D-glucono-1,5-lactone (K_i(cellobiose) = 5.00 mmol dm^?3; K_{i(D-glucono-1,5-lactone}) = 22 μmol dm^?3). The optimal hydrolysis conditions are the same for both substrates (pH 4.5,55° C). The half-lives of thermal inactivation at 61° C are 27 and 10min for cellobiase and 4NPGase, respectively.     

Photoelectrocatalytic humic acid degradation kinetics and effect of pH,applied potential and inorganic ions

This study addresses the removal of humic acid (HA) dissolved in an aqueous medium by a photoelectrocatalytic process. UV₂₅₄ removal and the degradation of color (Vis₄₀₀) followed pseudo‐first order kinetics. Rate constants were 1.1 × 10^?1 min^?1, 8.3 × 10^?2 min^?1 and 2.49 × 10^?2 min^?1 (R² > 0.97) for UV₂₅₄ degradation and 1.7 × 10^?1 min^?1, 6.5 × 10^?2 min^?1 and 2.0 × 10^?2 min^?1 for color removal from 5 mg dm^?3, 10 mg dm^?3 and 25 mg dm^?3 HA respectively. Following a 2 h irradiation time, 96% of the color, 98% of the humic acid and 85% of the total organic carbon (TOC) was removed from an initial 25 mg dm^?3 HA solution in the photoanode cell. Photocatalytic removal on the same photoanode was also studied in order to compare the two methods of degradation. Results showed that the photoelectrocatalytic method was much more effective than the photocatalytic method especially at high pH values and with respect to UV₂₅₄ removal. The effect of other important reaction variables, eg pH, external potential and electrolyte concentration, on the photoelectrocatalytic HA degradation was also studied. Copyright © 2003 Society of Chemical Industry     

Application of chitosan‐entrapped β‐galactosidase in a packed‐bed reactor system

The model enzyme β‐galactosidase was entrapped in chitosan gel beads and tested for hydrolytic activity and its potential for application in a packed‐bed reactor. The chitosan beads had an enzyme entrapment efficiency of 59% and retained 56% of the enzyme activity of the free enzyme. The Michaelis constant (K_m) was 0.0086 and 0.011 μmol/mL for the free and immobilized enzymes, respectively. The maximum velocity of the reaction (V_max) was 285.7 and 55.25 μmol mL^?1 min^?1 for the free and immobilized enzymes, respectively. In pH stability tests, the immobilized enzyme exhibited a greater range of pH stability and shifted to include a more acidic pH optimum, compared to that of the free enzyme. A 2.54 × 16.51‐cm tubular reactor was constructed to hold 300 mL of chitosan‐immobilized enzyme. A full‐factorial test design was implemented to test the effect of substrate flow (20 and 100 mL/min), concentration (0.0015 and 0.003M), and repeated use of the test bed on efficiency of the system. Parameters were analyzed using repeated‐measures analysis of variance. Flow (p < 0.05) and concentration (p < 0.05) significantly affected substrate conversion, as did the interaction progressing from Run 1 to Run 2 on a bed (p < 0.05). Reactor stability tests indicated that the packed‐bed reactor continued to convert substrate for more than 12 h with a minimal reduction in conversion efficiency. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 91: 1294–1299, 2004     

Immobilization of pepsin on an acrylamide/2‐hydroxyethyl methacrylate copolymer and its use in casein hydrolysis

Pepsin was immobilized through covalent bonding on a copolymer of acrylamide and 2‐hydroxyethyl methacrylate via the individual and simultaneous activation of both groups. The extent of enzyme coupling upon the activation of both the amino and hydroxyl groups of the copolymer resulted in a synergistic effect. However, the order of activation of the support was critical. The covalently bound enzyme retained more than 50% of its activity even after six cycles. The storage stability of the covalently bound enzyme was 60% after storage for 1 month, whereas the free enzyme lost all of its activity within 10 days of storage at 35°C. The Michaelis constant (K_m) and maximum reaction velocity (V_max) were 1.1 × 10^?6 and 0.87 for the free enzyme and 1.2 × 10^?6 and 0.98 for the covalently bound enzyme when the enzyme concentration was kept constant and the substrate concentration was varied. Similarly, K_m and V_max were 6.73 × 10^?11 and 0.47 for the free enzyme and 7.59 × 10^?11 and 0.545 for the covalently bound enzyme when the substrate concentration was kept constant and the enzyme concentration was varied; this indicated no conformational change during coupling, but the reaction was concentration‐dependent. The hydrolysis of casein was carried out with a fixed‐bed reactor (17 cm × 1 cm). Maximum hydrolysis (90%) was obtained at a 2 cm³/min flow rate at 35°C with a 1 mM casein solution. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 96: 1544–1549, 2005     

The effect of external stimuli on the invertase adsorption capacity of poly(N-vinyl-2-pyrrolidone-co-itaconic acid) hydrogels

Polyelectrolyte Poly(N-vinyl-2-pyrrolidone-co-itaconic acid) hydrogels (P(VP/IA)) with varying compositions were prepared from ternary VP/IA/water mixtures. The effect of external stimuli such as pH of the solution, temperature, substrate concentration of solution, and storage stability on the invertase adsorption capacity of P(VP/IA) hydrogels was investigated. The adsorption capacity of the hydrogels was found to increase from 4.4 to 18.4 mg invertase/g dry gel with increasing amount of IA in the gel system, while P(VP) gel adsorbed only 3.1 mg invertase/g dry gel. Kinetic parameters were calculated as Michaelis-Menten constant K_m = 20.6 mmol L^–1 and maximum velocity V_max = 6.44×10^–5 mol dm^–3 min^–1 for free enzyme and in the range of K_m = 26.4–41.1 mmol L^–1 and V_max = 6.35·10^–5–6.66·10^–5 mol dm^–3 min^–1, depending on the amount of IA in the hydrogel. Enzyme activities were found to increase from 59.0% to 72.0% with increasing amount of IA in the gel system and retained their activities for one month storage. The enzyme activities, after storage for one month at 4°C, were found to be 21.0% and 59.0–74.0% of the initial activity values for free and adsorbed enzyme, respectively. The optimal pH values for free and adsorbed enzymes were determined as 4.56 and 5.00, respectively. The optimum temperature for free and adsorbed enzymes was 55°C. Adsorption studies have shown that not only the gel composition but also the stimuli, temperature and pH of the solution play an important role on the invertase adsorption capacity of poly(VP/IA) hydrogels.     

Removal of aqueous phenolic compounds from a model system by oxidative polymerization with turnip (Brassica napus L var purple top white globe) peroxidase

Turnip roots, which are readily available in Mexico, are a good source of peroxidase, and because of their kinetic and biochemical properties have a high potential as an economic alternative to horseradish peroxidase (HRP). The efficiency of using turnip peroxidase (TP) to remove several different phenolic compounds as water‐insoluble polymers from synthetic wastewater was investigated. The phenol derivatives studied included phenol, 2‐chlorophenol, 3‐chlorophenol, o‐cresol, m‐cresol, 2,4‐dichlorophenol and bisphenol‐A. The effect of pH, substrate concentration, amount of enzyme activity, reaction time and added polyethylene glycol (PEG) was investigated in order to optimize reaction conditions. A removal efficiency ≥85% was achieved for 0.5 mmol dm^?3 phenol derivatives at pH values between 4 and 8, after a contact time of 3 h at 25 °C with 1.28 U dm^?3 of TP and 0.8 mmol dm^?3 H₂O₂. Addition of PEG (100–200 mg dm^?3) significantly reduced the reaction time required (to 10 min) to obtain >95% removal efficiency and up to 230% increase in remaining TP activity. A relatively low enzyme activity (0.228 U dm^?3) was required to remove >95% of three phenolic solutions in the presence of 100–200 mg dm^?3 PEG. TP showed efficient and fast removal of aromatic compounds from synthetic wastewaters in the presence of hydrogen peroxide and PEG. These results demonstrate that TP has good potential for the treatment of phenolic‐contaminated solutions. © 2002 Society of Chemical Industry     

Production of the fungal exopolysaccharide scleroglucan by cultivation of Sclerotium glucanicum in an airlift reactor with an external loop

Sclerotium glucanicum NRRL 3006 was cultivated in a 120 dm³ working volume airlift reactor with external recirculation loop for the production of the exopolysaccharide (EPS), scleroglucan. When culture pH was not controlled EPS production, at a range of air flow rates, was poor (<2 kg m^?3). Biomass formation generally increased with increasing aeration rate. When culture pH was controlled at 4·5 ± 0·2 EPS production was maximal at an air flow rate of 100 dm³ min^?1 and fell as air flow rate decreased. Operation of the reactor at a high (100 dm³ min^?1) air flow rate for the early part of the process (up to 96 h), followed by a low air flow rate (20 dm³ min^?1), led to reduced biomass and oxalate formation, and a slight increase in EPS concentration relative to operation at a constant air flow rate of 100 dm³ min^?1. EPS production in this pneumatically-agitated reactor was equal to the highest levels reported in small-scale stirred tank reactors.     

Hydrolysis of lecithin by phospholipase A2 in mixed reversed micelles of lecithin and sodium dioctyl sulphosuccinate

The hydrolysis of egg yolk lecithin was studied as catalysed by porcine pancreatic phospholipase A₂ (phosphatide 2-acyl-hydrolase, EC 3.1.1.4) entrapped in reversed micelles of lecithin and sodium dioctyl sulphosuccinate (AOT) in isooctane. The influence of relevant parameters such as temperature, pH, water content, and buffer, AOT, lecithin, calcium and enzyme concentrations was investigated. The order in which the reactants were mixed showed a strong influence on catalytic activity. Higher activities were obtained if the enzyme, water and calcium were first solubilized and pre-equilibrated in plain AOT reversed micelles before the addition of the substrate. Maximum activity was obtained at 750 mmol dm^?3 calcium in the water pool of the micelles, which is an extremely high concentration when compared with the values reported in the literature. This was related to the formation of divalent chelates of calcium with AOT that compete with the binding of the co-factor to the enzyme. Accordingly, the optimum concentration of calcium in the inner core of the micelles decreased from 750 to 500 mmol dm^?3 when the AOT concentration was lowered from 15 to 10 mmol dm^?3.     

Production of optically pure L-alanine by immobilized Pseudomonas sp. BA2 cells

The conditions for immobilizing the new L -aminoacylase-producing bacterial strain, Pseudomonas sp. BA2, by entrapment in κ-carrageenan gel, were investigated. The optimal gel concentration and cell load were determined. The addition of CoCl₂ and N-acetyl-L -alanine to the immobilizing matrix enhanced L -aminoacylase activity. The enzymatic properties of immobilized Pseudomonas sp. BA2 were investigated. Enzyme activity in immobilized cells was optimal at a pH of 6·5 using 0·15 mol dm⁻³ Tris–maleate buffer at 45°C. The presence of 0·7 mmol dm⁻³ CoCl₂ in the enzymatic reaction mixture improved L -aminoacylase activity. The immobilized cell preparation was used for the production of L -alanine from N-acetyl-DL -alanine in a batch reactor. Conversions of 100% were obtained using substrate concentrations ranging from 20 to 200 mmol dm⁻³. The reactor production was 0·74 mol h⁻¹ g cell⁻¹ dm⁻³ which is noticeably higher than that previously reported in the literature. © 1998 Society of Chemical Industry     

Production of salicylate hydroxylase from Pseudomonas putida UUC-1 and its application in the construction of a biosensor

The enzyme salicylate hydroxylase was produced from Pseudomonas putida UUC-1 in an 80 dm³ 16 h batch fermentation process yielding, ～ 9 K units per 60 dm³. This enzyme preparation has been studied for its application in the construction of biosensor systems, e.g. carbon past electrodes, screen-printed carbon electrodes, and disposable carbon enzyme electrodes, which will be used for the rapid estimation of salicylate in blood sample. The linear range of the disposable carbon enzyme electrode in response to salicylate was achieved to 1·8 mmol dm^?3.     

Removal of phosphate by adsorption onto oyster shell powder—kinetic studies

Kinetic studies on the removal of phosphate by adsorption onto oyster shell powder have been investigated at 24 °C. The results showed that the equilibrium occurred in 10 min and the equilibrium data followed the Freundlich isotherm. Freundlich constants were found to be k_f, 1.4 × 10^?2, and n, 0.71. The phosphate removal was not influenced by pH over the range 5.0–10.5. Continuous agitation studies at 24 °C and 530 rpm reached equilibrium after 7.7 days, when 24 g dm^?3 of oyster shell powder reduced the phosphate concentration from 50 to 7.0 mg dm^?3. The Lagergren rate constant for the slow adsorption process was observed to be 3.81 × 10^?4 dm³ min^?1. Comparison with calcium carbonate, GR grade, showed that oyster shell powder and CaCO₃ behave more or less in the same way. Copyright © 2004 Society of Chemical Industry     

Production of L‐methionine by immobilized pellets of Aspergillus oryzae in a packed bed reactor

Production of L ‐methionine by immobilized pellets of Aspergillus oryzae in a packed bed reactor was investigated. Based on the determination of relative enzymatic activity in the immobilized pellets, the optimum pH and temperature for the resolution reaction were 8.0 and 60 °C, respectively. The effects of substrate concentration on the resolution reaction were also investigated and the kinetic constants (K_m and V_m) of immobilized pellets were found to be 7.99 mmol dm^?3 and 1.38 mmol dm^?3 h^?1, respectively. The maximum substrate concentration for the resolution reaction without inhibition was 0.2 mol dm^?3. The L ‐methionine conversion rate reached 94% and 78% when substrate concentrations were 0.2 and 0.4 mol dm^?3, respectively, at a flow rate of 7.5 cm³ h^?1 using the small‐scale packed bed reactor developed. The half‐life of the L ‐aminoacylase in immobilized pellets was 70 days in continuous operation. All the results obtained in this paper exhibit a practical potential of using immobilized pellets of Aspergillus oryzae in the production of L ‐methionine. © 2002 Society of Chemical Industry     

Utilization of response surface methodology to optimize the culture media for the production of rhamnolipids by Pseudomonas aeruginosa AT10

Pseudomonas aeruginosa AT10 produced a mixture of surface‐active rhamnolipids when cultivated on mineral medium with waste free fatty acids as carbon source. The development of the production process to an industrial scale included the design of the culture medium. A 2⁴ full factorial, central composite rotational design and response surface modelling method (RSM) was used to enhance rhamnolipid production by Pseudomonas aeruginosa AT10. The components that are critical for the process medium were the carbon source, the nitrogen source (NaNO₃), the phosphate content (K₂ HPO₄/KH₂PO₄ 2:1) and the iron content (FeSO₄·7H₂O). Two responses were measured, biomass and rhamnolipid production. The maximum biomass obtained was 12.06 g dm^?3 DCW, when the medium contained 50 g dm^?3 carbon source, 9 g dm^?3 NaNO₃, 7 g dm^?3 phosphate and 13.7 mg dm^?3 FeSO₄·7H₂O. The maximum concentration of rhamnolipid, 18.7 g dm^?3, was attained in medium that contained 50 g dm^?3 carbon source, 4.6 g dm^?3 NaNO₃, 1 g dm^?3 phosphate and 7.4 mg dm^?3 FeSO₄·7H₂O. © 2002 Society of Chemical Industry