Application of the enzymes

The value of proteases in cleansing tissue wounds has been appreciated for several hundred years. Wounds were sometimes cleansed in the past by application of protease-containing maggot saliva. Nowadays, this is usually more acceptably achieved by topical application of the enzyme to the wound surface. In some cases, the enzyme is formulated in an aqueous-based cream, and in others it is impregnated into special bandages. Trypsin, papain, collagenase and various microbial enzymes have been used in this regard. 

The applicability of the enzyme-labile anchor group was demonstrated by the synthesis of tetrahydro-yS-carbohns (58) employing the Pictet-Spengler reaction (Scheme 10.10). 

Formulations. Any formulation is a compromise between the previously mentioned requirements. For example, the fermentation broth may contain enzyme-stabilizing substances, but the application of the enzyme or precipitation problems in the formulation may demand a high degree of purification that eliminates the stabilizers. Alternatively, the pH necessary for good microbial or physical stability may differ from the pH that gives optimum enzyme stability, or a preservative that is effective at the optimum pH for enzyme stability may have a denaturing effect on the enzyme. 

Stability, duration, sensitivity, interference, and availability of substrates to contact enzymes are the criteria for the success of an enzyme sensor. These criteria depend on sources of enzymes, immobilization techniques, and transducers used. Food matrices are much more complicated than the clinical samples, hence, these criteria become extremely important for the application of the enzyme sensor in food analysis. An extensive list of the response time, detection limits, and stability of biosensors was summarized by Wagner (59). 

Application of the enzyme and definition of limitations (e.g. substrate specificity, activity, operational stability)... 

When, in 1832, Wohler and Liebig first discovered the cyanide-catalyzed coupling of benzaldehyde that became known as the benzoin condensation , they laid the foundations for a wide field of growing organic chemistry [1]. In 1903, Lapworth proposed a mechanistical model with an intermediate carbanion formed in a hydrogen cyanide addition to the benzaldehyde substrate and subsequent deprotonation [2]. In the intermediate active aldehyde , the former carbonyl carbon atom exhibits an inverted, nucleophilic reactivity, which exemplifies the Umpo-lung concept of Seebach [3]. In 1943, Ukai et al. reported that thiazolium salts also surprisingly catalyze the benzoin condensation [4], an observation which attracted even more attention when Mizuhara et al. found, in 1954, that the thiazolium unit of the coenzyme thiamine (vitamin Bi) (1, Fig. 9.1) is essential for its activity in enzyme biocatalysis [5]. Subsequently, the biochemistry of thiamine-dependent enzymes has been extensively studied, and this has resulted in widespread applications of the enzymes as synthetic tools [6]. 

In this paper we report the electrochemical polymerization of the PPy-GOD film on the glassy carbon (GC) electrode in enzyme solution without other supporting electrolytes and the electrochemical behavior of the synthesized PPy-GOD film electrode. Because the GOD enzyme molecules were doped into the polymer, the film electrode showed a different cyclic voltammetric behavior from that of a polypyrrole film doped with small anions. The film electrode has a good catalytic behavior to glucose, which is dependent on the film thickness and pH. The interesting result observed is that the thin PPy-GOD film electrode shows selectivity to some hydrophilic pharmaceutical drugs which may result in a new analytical application of the enzyme electrode. 

The solution was allowed to react with the enzyme for at least 4 hours. Just prior to the application of the enzyme-degraded starch to the yarn, the paddle wheel was turned on for stirring and the gear pump turned on for recirculation. Both were run for 1 hr to achieve a uniform fluid mixture. 

This, maybe somewhat peculiar, example demonstrates that the use of digestible catalysts, like the enzyme phytase, further broadens the field of catalysis, i.e. not in a visible production rmit but in the species or product where the catalytic conversion is really needed. Other examples in the food industry are the application of the enzymes chymosin, invertase and amylase for cheese, bonbon and bread manufacturing, respectively. 

Stefuca V, Gemeiner P, Kurillova L, Danielsson B, Bales V (1990) Application of the enzyme thermistor to the direct estimation of intrinsic kinetics using the saccharose-immobilized invertase system. Enzyme Microb Technol 12 830 - 835 Stefuca V, Welwardova A, Gemeiner P, Jakubova A (1994) Application of enzyme flow microcalorimetry to the study of microkinetic properties of immobilized biocatalyst. Biotech-nolTech 8 497-502... 

Satoh I (1988) Biomedical Applications of the Enzyme Thermistor in Lipid Determinations. In Mosbach K (ed) Methods in Enzymology, vol 137. Immobilized Enzymes and Cells, Part D. Academic Press, Orlando, p 217... 

The recovered broth has to be formulated into a final product to comply with requirements appropriate to its final application. In formulation, whether for a solid or liquid product, different issues are addressed. Maintaining the activity of enzyme from the time of manufacture to the time of application through storage is one of the main factors tested during formulation. Other stability issues such as microbial stability and physical stability are important as well. Depending on the final application of the enzyme, the physical appearance can be customized (e.g., colored). A specific example of formulation is immobilization (see also Chapter 2). 

The various enzyme products are marketed on the basis of activity. Sometimes the activity is determined by a standard enzyme assay, but more frequently the activity is determined by methods based upon the industrial application of the enzyme. Large consumers with well-equipped laboratories find it expedient to purchase concentrates, but smaller plants prefer to purchase enzymes at standardized activities. Materials compatible with the intended application are added to the enzyme products by the enzyme manufacturer to adjust the activity to the required value. Stabilizers, activators and other materials to improve the response of the enzyme are also added. In the case of enzymes for food applications, the usual bacteriological tests (e.g. absence of Salmonella, gas formers, etc.) are applied to the products. 

When the practical application of the enzyme for the transformation of a substrate is considered, an important goal is to attain the best efficiency, defined as the maximum amount of substrate degraded per minimum units of enzyme inactivated during the reaction. 

A flow-through viscometer developed for application as a sensor in automated analyses consisted of a glass capillary connected to the sample flow circuit with thin-walled tubes at both ends. These tubes separate the fluid to be tested from a hydraulic fluid, and this construction ensures the absence of dead space and a minimal test volume. Development of a mathematical model describing the enzymatic degradation of macromolecules resulted in a reciprocal equation allowing rectilinear presentation of the calibration data. The application of the enzyme to the assay of amylase was described. 

The product quality demands are highly dependent on the application of the enzyme. Detergent enzymes typically have requirements for their physical appearance (color, odor) and also for compatibility with the detergent. Impurities related to the process (e.g., the presence of unfermented sugar from the media) and microorganism (e.g., metabolites produced during the fermentation) can influence the quality of the product. Dealing with these issues typically requires addition of one or more purification steps in the process. 

The purpose of this review is to provide an overview of enzymes from extreme thermophiles with particular emphasis on thermostability characteristics, and with some reference to the commercial applicability of the enzymes. An extreme thermophile is defined here as an organism with a growth optimum of 65 °C or higher. This limits the scope of the review to bacteria, since no extremely thermophilic fungi or algae have been isolated, and excludes much of the work carried out on Bacillus stearothermophilus. 

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