Influence of the microenvironment on the activity of enzymes immobilized on Teflon membranes grafted by γ-radiation

The effect of the microenvironment and immobilization method on the activity of immobilized β-galactosidase was investigated. Immobilization was done on Teflon membranes grafted with different acrylic monomers by γ-radiation and activated by two different coupling agents through the functional groups of the grafted monomers. 2-Hydroxyethyl methacrylate (HEMA) and methacrylic acid (MAA) were grafted on the membrane, and 1,6-hexamethylenediamine (HMDA) was used as a spacer. Glutaraldehyde or cyanuric chloride were used as coupling agents to bind the enzyme to the membrane. Four different catalytic membranes were obtained using the same solid support. Direct comparison between the isothermal behaviour of the biocatalyst in its free and immobilized form was carried out. In particular the dependence of the isothermal activity on the temperature and pH was studied and the kinetic parameters determined. The influence of the microenvironment on the observed activity of the four membranes was evidenced and discussed. The way of improving the yield of these catalytic membranes is discussed also.     

Enzyme immobilization by fouling in ultrafiltration membranes: Impact of membrane configuration and type on flux behavior and biocatalytic conversion efficacy

Enzyme-immobilization in membranes accomplished by fostering membrane fouling was evaluated. Four different membrane configurations and five membranes were compared for immobilization of alcohol dehydrogenase (ADH) in terms of enzyme loading, permeate flux and final biocatalytic conversion. The membrane configuration impacted the efficiency of the enzyme-immobilization as well as the biocatalytic-membrane reaction, and the “sandwich mode”, with an extra polypropylene support above the membrane skin layer, worked best due to its high flux and stable conversion. Among the membranes, a GR51PP polysulphone membrane allowed for the highest flux during the reaction with the enzyme-immobilized membrane. At the same time, the lowest enzyme loading and low reaction stability were achieved for this membrane. Satisfactory enzyme loadings, stable conversions, but low flux rates were obtained for the PLTK and PLGC regenerated cellulose membranes. With these two highly hydrophilic membranes, the ADH enzyme activity was fully retained even after 24 h of storage of the membrane. Filtration blocking and resistance models were used to analyze the fouling/immobilization mechanisms and give explanations for the different results. The work confirms that fouling-induced enzyme immobilization is a promising option for enhancing biocatalytic productivity, and highlights the significance of the membrane type and configuration for optimal performance.     

Nonisothermal bioreactors in the treatment of vegetation waters from olive oil: laccase versus syringic acid as bioremediation model

Laccase from Trametes versicolor was immobilized by diazotization on a nylon membrane grafted with glycidil methacrylate, using phenylenediamine as spacer and coupling agent. The behavior of these enzyme derivatives was studied under isothermal and nonisothermal conditions by using syringic acid as substrate, in view of the employment of these membranes in processes of detoxification of vegetation waters from olive oil mills. The pH and temperature dependence of catalytic activity under isothermal conditions has shown that these membranes can be usefully employed under extreme pH and temperatures. When employed under nonisothermal conditions, the membranes exhibited an increase of catalytic activity linearly proportional to the applied transmembrane temperature difference. Percentage activity increases ranging from 62% to 18% were found in the range of syringic acid concentration from 0.02 to 0.8 mM, when a difference of 1 degrees C was applied across the catalytic membrane. Because the percentage activity increase is strictly related to the reduction of the production times, the technology of nonisothermal bioreactors has been demonstrated to be an useful tool also in the treatment of vegetation waters from olive oil mills.     

Enzymatic activity and stability of D-fructose dehydrogenase and sarcosine dehydrogenase immobilized onto giant vesicles

Stable vesicles with diameters between about 1 and 10 mum were prepared by a particular emulsification technology that involved the use of the surfactants Span 80 and Tween 80 and the phospholipid lecithin (phosphatidylcholine from soybeans). Two membrane enzymes, d-fructose dehydrogenase from Gluconobacter sp. (FDH) and sarcosine dehydrogenase from Pseudomonas putida (SDH), were for the first time immobilized onto the bilayer membranes of these type of vesicles; and the catalytic activity and enzymatic stability were measured and compared with the enzymes in a vesicle-free solution. The enzyme activity as well as stability considerably increased upon immobilization. In particular, immobilized FDH at 25 degrees C was stable for at least 20 days, while the activity of the free enzyme dropped to about 20% of its initial value during the same period of time.In contrast to FDH and SDH, immobilization of sorbitol dehydrogenase from Gluconobacter suboxydans (SODH) was not successful, as no improved activity or stability could be obtained.     

Immobilization of DNA on microporous membranes using UV-irradiation]

Various techniques of DNA immobilization onto nitrocellulose and nylon microporous membranes have been compared. Despite a strong primary adsorption of DNA onto these membranes during blotting procedures, poor retention of the target DNA and low hybridization signals are obtained after hybridization and washings. Covalent cross-linking of DNA upon UV irradiation leads to a quantitative immobilization of target DNA. Quantum yield of DNA photoimmobilization estimated for a single base in DNA is about 10(-4). UV irradiation dose sufficient for immobilization of DNA fragment of a known length can be calculated by the formula Ilc = (22.3 +/- 4.8) c/l, where l is the DNA fragment length (in base pairs), c is the DNA part (%) to be immobilized. The UV irradiation dose about 0.6-0.8 kJ/m2 is optimal for most hybridization experiments. Doses higher than 0.8-1 kJ/m2 may cause a loss in the hybridization efficiency. Under optimal immobilization conditions, hybridization signals increasing five-fold for nitrocellulose membranes and fifty-fold for uncharged nylon membranes as compared with baking these membranes in vacuum.     

Immobilization of Candida cylindracea lipase on poly lactic acid,polyvinyl alcohol and chitosan based ternary blend film: Characterization,activity, stability and its application for N-acylation reactions

The ecofriendly ternary blend polymer film was prepared from the chitosan (CH), polylactic acid (PLA) and polyvinyl alcohol (PVA). Immobilization of Candida cylindracea lipase (CCL) was carried out on ternary blend polymer via entrapment methodology. The ternary blend polymer and immobilized biocatalyst were characterized by using N₂ adsorption–desorption isotherm, SEM, FTIR, DSC, and (%) water content analysis through Karl Fischer technique. Biocatalyst was then subjected for the determination of practical immobilization yield, protein loading and specific activity. Immobilized biocatalyst was further applied for the determination of biocatalytic activity for N-acylation reactions. Various reaction parameters were studied such as effect of immobilization support (ratio of PLA:PVA:CH), molar ratio (dibutylamine:vinyl acetate), solvent, biocatalyst loading, time, temperature, and orbital speed rotation. The developed protocol was then applied for the N-acylation reactions to synthesize several industrially important acetamides with excellent yields. Interestingly, immobilized lipase showed fivefold higher catalytic activity and better thermal stability than the crude extract lipase CCL. Furthermore various kinetic and thermodynamic parameters were studied and the biocatalyst was efficiently recycled for four successive reuses. It is noteworthy to mention that immobilized biocatalyst was stable for period of 300 days.     

Monolithic bioreactors: Effect of chymotrypsin immobilization on its biocatalytic properties

The effect of different modes of α-chymotrypsin attachment to the surface of methacrylate-based ultrashort monolithic minicolumns on enzyme activity has been studied. The immobilization of protease was carried out via direct covalent binding of chymotrypsin, as well as via its attachment through small and polymer spacers. It was established that the lowest enzyme activity against N-benzoyl-l-tyrosine ethyl ester was found for bioreactor obtained via direct attachment of chymotrypsin to the surface of GMA–EDMA minidisks, whereas the highest parameter close to that determined for dissolved enzyme was found in the case of bioreactor prepared by the introduction of copolymer of 2-deoxy-N-methacryloylamido-d-glucose with N-vinylpyrrolidone and acrolein as a long and flexible polymer spacer. Additionally, the effect of flow rate of substrate recirculation on bioconversion efficiency was examined. Independently on immobilization method, the increase of flow rate led to the raise of biocatalytic efficiency.     

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.     

Biocomposite of subtilisin Carlsberg with chitosan as an effective biocatalyst for hydrolysis and synthesis of peptides

New biocatalysts, preparations of subtilisin Carlsberg immobilized on chitosan (a deacetylated derivative of chitin), were obtained. The enzyme content, hydrolytic activity, and ability to catalyze peptide bond formation in organic solvents were characterized for these preparations. The influence of the form and composition of the biocomplosite (content of the enzyme and glutaraldehyde, the cross-linking agent) and buffer pH on the biocatalytic properties of the immobilized enzyme was studied in the reactions of peptide bond hydrolysis. The synthase activity of the preparations was investigated in the reaction of synthesis of Z-Ala-Ala-Leu-Phe-pNA in a 6:4 DMF-acetonitrile mixture in dependence on the reaction time. The yield of this product was 100% after only 40 min.     

Advantages in using immobilized thermophilic beta-glycosidase in nonisothermal bioreactors

Catalytic membranes, obtained by immobilizing thermophilic beta-glycosidase onto nylon supports, were used in a nonisothermal bioreactor to study the effect of temperature gradients on the rate of enzyme reaction. Two experimental approaches were carried out to explain the molecular mechanisms by which the temperature gradients affect enzyme activity. The results showed that the thermophilic enzyme behaved as the mesophilic beta-galactosidase, exhibiting an activity increase which was linearly proportional to the transmembrane temperature difference. The efficiency of the system proposed was determined by calculating two constants, alpha and beta, which represent respectively the percentage increase of enzyme activity when a temperature difference of 1 degrees C or a temperature gradient of 1 degrees C cm-1 were applied across the catalytic membrane. The increase of enzyme activity in nonisothermal bioreactors entailed a proportional reduction of production times. The advantages in using thermophilic enzymes immobilized in nonisothermal bioreactors are also discussed.     

Membrane microreactor in biocatalytic transesterification of triolein for biodiesel production

Transesterification is a principal chemical reaction that occurs in biodiesel production. We developed a novel biocatalytic membrane microreactor (BMM) for continuous transesterification by utilizing an asymmetric membrane as an enzyme-carrier for immobilization. The BMM was developed by pressure driven filtration of lipase from Pseudomonas fluorescens, which is suitable for highly efficient biocatalytic transesterification. Lipase solution was allowed to permeate through an asymmetric membrane with NMWL 300 kDa composed of polyethersulfone. The performances of BMM were studied in biodiesel synthesis via transesterification of triolein with methanol. Transesterification was carried out by passing a solution of triolein and methanol through the asymmetric membrane. The degree of triolein conversion using this microreactor was ca. 80% with a reaction time of 19 min. The BMM system displayed good stability, with no activity decay over a period of 12 day with continuous operation. Results from triolein transesterification clearly demonstrate the potential of an asymmetric membrane as an enzyme carrier material. Enzyme activity (mmol/h·g_lipase) was approximately 3 fold higher than that of native free lipase.     

Development of Silica Gel-Supported Modified Macroporous Chitosan Membranes for Enzyme Immobilization and Their Characterization Analyses

The present work was aimed at developing stability enhanced silica gel-supported macroporous chitosan membrane for immobilization of enzymes. The membrane was surface modified using various cross-linking agents for covalent immobilization of enzyme Bovine serum albumin. The results of FT-IR, UV–vis, and SEM analyses revealed the effect of cross-linking agents and confirmed the formation of modified membranes. The presence of silica gel as a support could provide a large surface area, and therefore, the enzyme could be immobilized only on the surface, and thus minimized the diffusion limitation problem. The resultant enzyme immobilized membranes were also characterized based on their activity retention, immobilization efficiency, and stability aspects. The immobilization process increased the activity of immobilized enzyme even higher than that of total (actual) activity of native enzyme. Thus, the obtained macroporous chitosan membranes in this study could act as a versatile host for various guest molecules.     

Distribution of immobilized enzymes on porous membranes

In this article, the results from a theoretical and experimental investigation of enzyme immobilization in porous membranes are reported. A theoretical model of the immobilization process, which accounts for restricted diffusion of enzyme in the pores of the membrane, has been developed. The model predicts the effect of immobilization kinetics and time of immobilization on the enzyme distribution in the pores of the membrane. The immobilization of glucose oxidase and glucose oxidase-biotin conjugate on porous alumina membranes was experimentally investigated. Enzyme uptake data was correlated to the theory to determine the rate constant of imobilization and the distribution of the enzyme in the pore. Immobilization studies were carried out for enzyme adsorption and for enzyme attachment by covalent coupling. The distribution of enzyme was experimentally studied by assembling five membranes in the diffusion cell. Following immobilization, the membranes were separated and each was assayed for activity. The amount of active enzyme present in each membrane yielded a discrete distribution that compared well with that predicted by theory. (c) 1992 John Wiley & Sons, Inc.     

Bacterial cellulose as carrier for immobilization of laccase: Optimization and characterization

Bacterial cellulose (BC) has attracted attention as a new functional material due to its excellent mechanical strength, tridimensional nanostructure, high purity, and increased water absorption, compared to plant cellulose. In this work, commercial laccase was immobilized on BC and the influence of enzyme concentration, contact time, and pH was optimized toward the recovery activity of immobilized laccase. This optimization was carried out using a 3³ experimental design and response surface methodology. Enzyme concentration played a critical role in laccase immobilization. Under optimized conditions (0.15 μL L^?1 of enzyme concentration, 4.8 h of contact time, pH 5.4), the predicted and experimental response were equal to 47.88 and 49.30%, respectively. The thermal stability of the immobilized laccase was found to increase notably at 60 and 70°C presenting stabilization factor equal to 1.79 and 2.11, respectively. The immobilized laccase showed high operational stability, since it retained 86% of its initial activity after seven consecutive biocatalytic cycles of reaction with 2,2′‐azinobis‐(3‐ethylbenzothiazoline‐6‐sulfonic acid). Kinetic studies showed that the values of Michaelis–Menten constant and maximum reaction rate decreased upon immobilization (9.9‐ and 1.6‐fold, respectively). Globally, the use of immobilized laccase on BC offers an interesting tool for industrial biocatalytic applications.     

Optimization of catechol production by membrane-immobilized polyphenol oxidase: a modeling approach

Although previous research has focused on phenol removal efficiencies using polyphenol oxidase in nonimmobilized and immobilized forms, there has been little consideration of the use of polyphenol oxidase in a biotransformation system for the production of catechols. In this study, polyphenol oxidase was successfully immobilized on various synthetic membranes and used to convert phenolic substrates to catechol products. A neural network model was developed and used to model the rates of substrate utilization and catechol production for both nonimmobilized and immobilized polyphenol oxidase. The results indicate that the biotransformation of the phenols to their corresponding catechols was strongly influenced by the immobilization support, resulting in differing yields of catechols. Hydrophilic membranes were found to be the most suitable immobilization supports for catechol production. The successful biocatalytic production of 3-methylcatechol, 4-methylcatechol, catechol, and 4-chlorocatechol is demonstrated.     

Nitrilase immobilization and transposition from a micro-scale batch to a continuous process increase the nicotinic acid productivity

In recent years, many biocatalytic processes have been developed for the production of chemicals and pharmaceuticals. In this context, enzyme immobilization methods have attracted attention for their advantages, such as continuous production and increased stability. Here, enzyme immobilization methods and a collection of nitrilases from biodiversity for the conversion of 3-cyanopyridine to nicotinic acid were screened. Substrate conversion over 10 conversion cycles was monitored to optimize the process. The best immobilization conditions were found with cross-linking using glutaraldehyde to modify the PMMA beads. This method showed good activity over 10 cycles in a batch reactor at 30 and 40°C. Finally, production with a new thermostable nitrilase was examined in a continuous packed bed reactor, showing very high stability of the biocatalytic process at a flow rate of 0.12 ml min^–1 and a temperature of 50°C. The complete conversion of 3-cyanopyridine was obtained over 30 days of operation. Future steps will concern reactor scale-up to increase the production rate with reasonable pressure drops.     

Hollow-fiber enzyme reactor operating under nonisothermal conditions

A hollow-fiber enzyme reactor, operating under isothermal and nonisothermal conditions, was built employing a polypropylene hollow fiber onto which beta-galactosidase was immobilized. Hexamethylenediamine and glutaraldehyde were used as spacer and coupling agent, respectively. Glucose production was studied as a function of temperature, substrate concentration, and size of the transmembrane temperature gradient. The actual average temperature differences across the polypropylene fiber, to which reference was done to evaluate the effect of the nonisothermal conditions, were calculated by means of a mathematical approach, which made it possible to know, using computer simulation, the radial and axial temperature profiles inside the bioreactor and across the membrane. Percent activity increases, proportional to the size of the temperature gradients, were found when the enzyme activities under nonisothermal conditions were compared to those measured under comparable isothermal conditions. Percent reductions of the production times, proportional to the applied temperature gradients, were also calculated. The advantage of employing nonisothermal bioreactors in biotechnological industrial process was discussed.     

Enzyme immobilization by radiation-induced polymerization of hydrophobic glass-forming monomers at low temperatures.

Enzyme immobilization was studied by means of radiation-induced polymerization of hydrophobic glass-forming monomers at low temperatures. The polymerized hydrophobic composite was generally obtained in microspheric form. Enzymatic activity showed little decrease with repeated use in these systems. The particle size of the microsphere increased with increasing monomer concentration, and activity yield had a maximum at an optimum monomer concentration. Immobilization by copolymerization of hydrophilic and hydrophobic comonomers was also investigated and a maximum activity yield was found at a certain monomer concentration. A model scheme for immobilization at low temperatures was proposed and discussed.     

Epoxidation of propene by microbial cells immobilized on inorhanic supports

Immobilization of gas-utilizing microorganism strains (Mycobacteria, Rhodococcus, methane-utilizers) on inorganic supports based on alumina, silicates, and carbon was carried out to develop heterogeneous biocatalysts for the biotechnologic processes, including the process of propene epoxidation. Adsorption ability of these microorganisms, biocatalytic properties of resting and immobilized bacterial cells, and effect of immobilization tehniques on biocatalysis were studied. An approach of double immobilization using inorganic materials (supports and gel) was proposed as simple, universal, and available methopd to immobilize bacterial cells, resulting in a higher retention (up to 100%) of cells' enzymatic activity and enhanced stability.     

Immobilization of Bacillus licheniformis cells, producers of thermostable α-amylase, on polymer membranes

Cells of Bacillus licheniformis 44MB82-G immobilized on different polymer membranes were used for production of thermostable α-amylase. The α-amylase yields of the membrane-immobilized cells were affected by the reactive chemical groups of the carriers and the spacer size. Formaldehyde-activated polysulphone membranes (PS-FA) were the most suitable for effective immobilization. The highest amylase yield (62% increase of the control) and operational stability (97% residual activity after 480 h repeated batch cultivation) were obtained with this system. This was confirmed by scanning electron micrographs. An additional increase of α-amylase production by PS-FA-membrane immobilized cells was achieved in a fluidized-bed reactor. Received 20 March 1997/ Accepted in revised form 08 January 1998