Entrapped enzyme

Films or membranes of silkworm silk have been produced by air-drying aqueous solutions prepared from the concentrated salts, followed by dialysis (11,28). The films, which are water soluble, generally contain silk in the silk I conformation with a significant content of random coil. Many different treatments have been used to modify these films to decrease their water solubiUty by converting silk I to silk II in a process found usehil for enzyme entrapment (28). Silk membranes have also been cast from fibroin solutions and characterized for permeation properties. Oxygen and water vapor transmission rates were dependent on the exposure conditions to methanol to faciUtate the conversion to silk II (29). Thin monolayer films have been formed from solubilized silkworm silk using Langmuir techniques to faciUtate stmctural characterization of the protein (30). ResolubiLized silkworm cocoon silk has been spun into fibers (31), as have recombinant silkworm silks (32). 

The exact procedure for entrapment will depend on the nature of the catalyst and the required sol-gel, but for enzyme entrapment it generally involves the hydrolysis or partial hydrolysis of an alkoxysilane precursor, giving a precursor sol-gel solution, followed by addition of more solvent and of the catalyst. Figure 5.7 provides a simple... 

Groger, H., Copan, E., Bathuber, A. and Vorlop, K. (2001) Asymmetric synthesis of an (R)-cyanohydrin using enzymes entrapped in lens-shaped gels. Organic Letters, 13, 1969-1971. 

Fig. 3.10 Relative activities of endo-l,3-P-D-glucanase L V in aqueous solution (1) and in the immobilized state (2) as well as immobilized endo-l,3-P-D-glucanase L0 (3) vs. the time of testing. The enzyme entrapment was performed as described in Refs. [55,56], The silica matrix...

A second process of enzyme entrapment in LDH was also performed using colloidal solutions of LD H. Such colloidal solutions can be prepared by delamination... 

In this communication a study of the catalytic behavior of the immobilized Rhizomucor miehei lipase in the transesterification reaction to biodiesel production has been reported. The main drawbacks associated to the current biodiesel production by basic homogeneous catalysis could be overcome by using immobilized lipases. Immobilization by adsorption and entrapment have been used as methods to prepare the heterogeneous biocatalyst. Zeolites and related materials have been used as inorganic lipase supports. To promote the enzyme adsorption, the surface of the supports have been functionalized by synthesis procedures or by post-treatments. While, the enzyme entrapping procedure has been carried out by sol-gel method in order to obtain the biocatalyst protected by a mesoporous matrix and to reduce its leaching after several catalytic uses. 

The way of enzyme entrapment has been described79 proposing the application of sol-gel matrices. The optodes of urea sensor were prepared by the sol-gel method and were stored in a refrigerator. As the pH sensitive dye the bromothymol blue was used. Since it is best acting in pH range 6 to 7.6, the pH of sol-gel bulks obtained in the experiment was chosen as pH 6. Before measurements, the optodes were incubated in the temperature 36.6°C. 

J. Wang, D. Leech, M. Ozsoz, S. Martinez, and M.R. Smyth, One step fabrication of glucose sensors with enzyme entrapment within poly(ester-sulfonic acid), coatings. Anal. Chim. Acta 245, 139-143 (1991). 

A platinum disk electrode was electrolytically platinized in a platinum chloride solution to increase the surface area and enhance the adsorption power. The platinized platinum electrode was then immersed in a solution containing 10 mg ml l ADH. 0.75 mM and 6.2 mM NAD. After sufficient adsorption of these molecules on the electrode surface, the electrode was transferred into a solution containing 0.1 M pyrrole and 1 M KC1. Electrochemical polymerization of pyrrole was conducted at +0.7 V vs. Ag/AgCl. The electrolysis was stopped at a total charge of 1 C cm 2. An enzyme-entrapped polypyrrole membrane was deposited on the electrode surface. 

Usually, activities of enzymes (hydrogenases included) are investigated in solutions with water as the solvent. However, enhancement of enzyme activity is sometimes described for non-aqueous or water-limiting surroundings, particular for hydrophobic (or oily) substrates. Ternary phase systems such as water-in-oil microemulsions are useful tools for investigations in this field. Microemulsions are prepared by dispersion of small amounts of water and surfactant in organic solvents. In these systems, small droplets of water (l-50nm in diameter) are surrounded by a monolayer of surfactant molecules (Fig. 9.15). The water pool inside the so-called reverse micelle represents a combination of properties of aqueous and non-aqueous environments. Enzymes entrapped inside reverse micelles depend in their catalytic activity on the size of the micelle, i.e. the water content of the system (at constant surfactant concentrations). 

Gregoriadis G, Leathwood PD, Ryman BE. Enzyme entrapment in liposomes. FEBS Lett 1971 14 95. 

Vofi H,Miethe P (1992) Enzymes entrapped in liquid crystals a novel approach for bio-catalysis in nonaqueous media. In Tramper J, Vermae MH, Beet HH, Stockar UV (eds) Biocatalysis in non-conventional media, progress in biotechnology. Elsevier, London 8 739... 

O Driscoll, K.F., Techniques of enzyme entrapment in gels, Methods EnzymoL, 44, 169-183, 1976. 

Activity and stability measurements of the enzyme entrapped inside POPC vesicles against externally added Bz-Tyr-pNA. No activity against the larger substrate Suc-Ala-Ala-Pro-Phe-pNA or casein. Inhibition of externally present enzyme by an inhibitor protein. 

Martinek, K., Levashov, A. V, Klyachko, N. L., Pantin, V. L, and Berezin, I. V. (1981). The principles of enzyme stabilization. 6. Catalysis by water-soluble enzymes entrapped into reversed micelles of surfactants in organic solvents. Biochem. 

Sata, S., Murakata, T., Ochrfuji, M. and Snznki, T. (1994) Development of immobilized enzyme entrapped within inorganic matrix and its catalytic activity in organic meditrm. 

I. V. Berezin, and K. Martinek, Catalysis by enzymes entrapped into hydrated surfactant aggregates having lamellar or cylindrical (hexagonal) or ball-shaped (cubic) structure in organic solvents,... 

The most common method for enzyme entrapment is by polymerizing acrylamide in the presence of the enzyme. The result is a flexible porous polymeric gel, which traps the enzyme but allows the diffusion of substrates and products (Figure 5.11). Whole cells are also similarly entrapped in alginate, a natural polymer which occurs in... 

Previously, Nafion was used in electroanalysis as electrode covering membrane to immobilize cationic species (7,8) and as a barrier to anionic species (9,10). In biosensor field, the main limitation in the use of Nafion for enzyme entrapment is related to the solubility of the Nafion in lower aliphatic alcohols which are not compatible with the enzyme activity (11). Until now, only glucose oxidase was successfully immobilized in Nafion. 

To aleviate the dissolution of the enzymatic film during the glucose assay procedure, a covering membrane made by addition of 10 pi of Nafion 0.5% was used. The amperometric current increases until a constant value of 20 pA is reached on the fourth glucose assay (Fig. IB). The addition of Nafion film atop of the AQ-enzyme layer doesn t seem to denaturate the enzyme entrapped in the AQ film because the current reached a similar value as the current observed in absence of Nafion. On the other hand, the use of Nafion as covering membrane gives a thicker film that leads to mass transport limitation of the substrates in the AQ film (20). It results in a longer time to obtain the steady-state current. 

Electrical Wiring of Enzymes Entrapped in Polymeric Matrices on Electrode Snpports... 

Enzyme entrapment in polymers functionalized with redox relay units... 

Enzymes entrapped by sol-gel processing have received increased attention in recent years [116]. Sol-gel matrices offer several advantages for enzyme entrapment and the development of new biosensors including inherent low-temperature processing... 

The large size of the pores of MCM-41 has also allowed the entrapment of enzymes, such as cytochrome c, papain and trypsin [193]. Enzyme entrapment has been extensively performed with sol-gel materials. The types of applications of redox catalysis using enzyme-mesoporous materials is expected to parallel the sol-gel materials, which is discussed in the last section of this chapter. 

Polysalts have been prepared as microcapsules for enzyme entrapment (biocatalysts), in the separation of proteins as well as in surfactant binding. Protein separation is particularly selective, with biomacromolecules separated according to their isoelectric point. Coaeervation is the preferred method for water soluble drugs where no modification of the absorption kinetics are warranted. 

Veronese, F.M. Mammucari, C. Schiavon, F. Schiavon, O. Lora, S. Secundo, F. Chilin, A. Guiotto, A. Pegylated enzyme entrapped in poly(vinyl alcohol) hydrogel for biocatalytic application. II Farmaco 2001, 56, 541-547. 

Thionine-polypyrrole layer topped with enzyme entrapped polypyrrole layer Platinum disk... 

Gregoriadis G, Leathwood PD, Ryman BE (1971) Enzyme entrapment in liposomes. FEBS Lett 14(2) 95-99 Gregoriadis G (1976) The carrier potential of liposomes in biology and medicine (second of two parts). N Engl J Med 295(14) 765-70 Gregoriadis G (1976) The carrier potential of liposomes in biology and medicine (first of two parts). N Engl J Med 295(13) 704-10... 

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