Enzyme using entrapment technique

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). 

Enzymes, when immobilized in spherical particles or in films made from various polymers and porous materials, are referred to as immobUized enzymes. Enzymes can be immobilized by covalent bonding, electrostatic interaction, crosslinking of the enzymes, and entrapment in a polymer network, among other techniques. In the case of batch reactors, the particles or films of immobilized enzymes can be reused after having been separated from the solution after reaction by physical means, such as sedimentation, centrifugation, and filtration. Immobilized enzymes can also be used in continuous fixed-bed reactors, fluidized reactors, and membrane reactors. 

Most of the membrane segregated enzyme systems previously examined suffer some constitutive drawbacks which limit their yield and area of application. When enzymes are entrapped within the sponge of asymmetric membranes, product and substrate mass transfer occur mainly by a diffusive mechanism reactor performance is then controlled only by means of the amount and kind of charged enzyme, and the fluid dynamics of the solution in the core of the fibers. UF or RO fluxes, moreover, result in enzyme losses. Enzyme crosslinking in the membrane pores can reduce these losses, but it can lead to an initial activity loss, as compared to that of the native enzyme. Of course, once the enzyme is deactivated, it makes the reactor useless for further operation. Such immobilization techniques are seldom useful for microbial cells due to their large size. 

For this technique to be of more general use it must also be applicable to other enzymes. We have began by extending the procedure to urease, an enzyme whose entrapment in polypyrrole, as well as use in a conductometric biosensor for urea has already been reported (35, 36). We have found that urease was readily electrodeposited and that the activity of the deposited enzyme was easily seen by admittance measurements (Figure. 4). The apparent Km for the immobilis enzyme was calculated fi om the double-reciprical plot (y = 0.17 + O.SOx, regression coefficient = 0.9994, n = 9) to be 2.9 mM, which can be compared to the range 3-5.1 mM for the soluble enzyme (36). A control electrode produced at the same time displayed negligible response to urea. 

Figure 10.12 (a) Immobilization of enzyme using the entrapment technique in a matrix and (b) in a fiber. 

With these assumptions in hand, interpretation of real assay data involves plotting a model-derived value for concentration of NAD at the enzyme surface (NAD ). The value for the Mnad+ can be fitted to allow the Lineweaver-Burk plot to intercept the x-axis at a value that yields the value of Km as determined in solution. The value for Vmax is then read as the intercept at the y-axis (Figure 12.2). This approach permits derivation of a Vmax for the electrode that is independent of the effects of mass transfer. If one further assumes that the immobilization process does not affect the turnover rate of the immobilized enzyme (relative to its activity in solution), then this value of Vmax (which represents the total activity of all bound enzyme) can also be used to estimate the amount of immobilized enzyme. This model can be particularly useful when fabricating electrodes using immobilization techniques that entrap a fraction of enzyme from bulk solutions, such as direct physical absorption or co-immobilization within gels. 

The above technique leads to a bulk polymer gel swollen with water. The gel may be chopped into fine pieces while swollen or, alternately, dried, crushed and sieved to give moderately uniform particles. The polymerization may also be done in film form, thus yielding a membrane containing the entrapped enzyme. Another popular technique involves suspension polymerization using a non-aqueous medium such as toluene or chloroform. Suspension pol3nnerization is particularly attractive because it provides an easy way to remove heat rapidly from the polymerization and yields a product of enzjnne entrapped in spherical beads whose size is controlled by the agitation and other conditions of the suspension polymerization. 

When the reaction product is soluble in water, enzyme regeneration is difficult to achieve, since the enzyme is often lost during isolation of the product. One way to overcome this problem is application of immobilised enzyme systems. The enzyme is either covalently or ionically attached to an insoluble carrier material or is entrapped in a gel. Depending on the size of the particles used, a simple filtration and washing procedure can be used to separate the immobilised enzyme from the dissolved product A well-known example of this technique is the industrial production of 6-APA. 

It is well known that pine enzymes change then behaviour and stability when they are immobilised. In the past two decades the immobilisation of microorganisms, cells and parts of cells has gradually been introduced into microbiology and biotechnology. The cell immobilisation techniques are modifications of the techniques developed for enzymes. However, the larger size of microbes has influenced the techniques. As for immobilised enzymes, two broad types of method have been used to immobilise microorganisms attachment to a support and entrapment. 

Going back to the entrapment into the water pool, work on enzymes in liposomes has been and is an active research field. Most of the work presented in the literature concerns the entrapment of one enzyme at a time. In particular, the excellent review by Walde and Ischikawa, 2001 provides a rich account and discussion of the various techniques used to incorporate enzymes inside liposomes, and their possible applications in chemistry, medicine, and industry. Table 10.1 is a modification and simplification of one of their tables (Walde and Ischikawa, 2001, Table 6). This... 

Many different types of techniques for protein immobilization have been developed using, in most cases, enzyme sensors. Early studies of enzyme biosensors often employed thick polymer membranes (thickness 0.01-1 mm) in which enzymes are physically entrapped or chemically anchored. The electrode surface was covered with the enzyme-immobilized polymer membranes to prepare electrochemical enzyme sensors. Although these biosensors functioned appropriately to... 

It is desirable under certain circumstances to use an enzyme in what is called an immobilized form. The enzyme is attached covalently or by entrapment in a polymer matrix. A contaminated fluid that comes into contact with the polymer is thus acted upon by the enzyme to produce a desired effect. We will discuss the advantages in subsequent chapters. While this technique lowers the efficiency of the enzyme, it extends its useful life by orders of magnitude, and the enzyme is not thrown away with the bathwater. ... 

One of the key factors in biosensor design is the immobilisation technique used to attach the biorecognition molecule to the transducer surface so as to render it in a stable and functional form. The challenge is to have a stable layer (or layers) of biorecognition molecules that do not desorb from the surface and that retain their activity. Entrapment or encapsulation techniques avoid the chemical changes that usually change the structure of the enzymes and modify their recognition capacity. 

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