||A problem of paramount importance in analytical chemistry is selectivity, particularly at low analyte concentrations and in the presence of interfering substances. High selectivity, even in trace analysis, has been gained by progress in analytical instrumentation such as high-performance liquid chromatography, gas chromatography, mass spectrometry and atomic absorption spectroscopy. However, these powerful instruments are not applicable for in-situ operation and can only be used in specialized laboratories. The development of highly selective sensors that are easy to handle is therefore an important problem. Biosensors may provide solution to this problem. Biosensors are devices incorporating a biologically active element in intimate contact with a physico-chemical signal transducer and an electronic signal processor. The goal of this combination is to utilize the high selectivity of biological sensing for analytical purposes in various fields of research and technology. Of all types of biosensors, catalytic sensors based on the molecular analyte recognition and conversion by enzymes have been most intensely studied. Enzymes are perfect indicators because they combine selectivity with catalytic power. Enzymes play an important role in bioanalytical chemistry and biosensorics. There are many commercialized analytical testkits based on enzymatic reactions. With respect to improvements in speed, cost and on-line capabilities, enzyme sensors offer attractive alternatives to existing methods. Furthermore, portable enzyme sensors could be used for monitoring in sports, food and environmental monitoring. The aim of this thesis is to describe novel biosensors with enzymes as the biocomponent, and to apply them in the area of sports monitoring, food production, and environmental monitoring. Chapter 2 decribes the application of biosensors and biotests for fitness and muscle stress in sports, monitoring the plasma creatine kinase (CK), L-lactate, β-D-glucose and Fatty Acid-Binding Protein (FABP) concentration of racing horses and a group of junior rowers. Measurements of CK and FABP were performed with biosensors and biotests with sequential enzymatic reactions and immuno reactions, respectively. Quantification of L-lactate (or β-D-glucose) was undertaken by a biosensor with immobilized L-lactate oxidase (or glucose oxidase) on a Clark oxygen type electrode. The information obtained will be a base for further applications of biosensors and biotests to improve the performance of athletes. In Chapter 3, novel thick film biosensors are used for the determination of L-glutamate and L-lactate in foodstuffs. The sensors were prepared by immobilization of L-glutamate oxidase using polycarbamylsulfonate hydrogel on a thick-film sensor. L-Glutamate oxidases obtained from Streptomyces sp. with different degree of purification were compared with their characteristic response to L-glutamate at different conditions and for their specificity, inhibition behavior, and storage properties. These sensors were applied to determine monosodium glutamate in soy sauce samples that available in food market, and are correlated with calorimetric method. In Chapter 4, a simple and elegant method for determination of both phytate and phytase activity in animal feed will be introduced. Both phytate and phytase are important in animal feed. Sensors using immobilized phytase, acid phosphatase, glucose and pyruvate oxidases are investigated. These sensors can help to optimize the use of phytase and phytic acid as a feed additive, which can in turn reduce phosphorous pollution of the environment. Chapter 5 describes novel methods to increase the operation stability of enzyme sensors by using crystals of glucose oxidase and catalase. A combined method of crosslinking and hydrogel-entrapment of the enzyme crystals is described. Moreover, a novel technology of encapsulation of the enzyme crystals by polyelectrolytes is developed that keeps the crystal structure intact. In Chapter 6, a novel method of sample pretreatment by enzymatic hydrolysis is introduced for rapid Biochemical Oxygen Demand (sensorBOD) measurements. With this fast, easy-to-handle and safe procedure the sensorBOD values of macromolecules (e.g. starch, cellulose or milk powder) give a close correlation to the traditional 5-day BOD test (BOD5). The final chapter, Chapter 7, summarizes and interprets the major findings of the previous chapters and discusses the merits of the research for future development.