Enzyme immobilization as a technique attaches free forms of enzyme molecules to stationary support materials to permit enzymes to be reused several times. Bovine trypsin as a model enzyme was immobilized onto controlled pore glass (CPG) beads to investigate the optimum conditions for immobilization, as well as the physico-chemical properties of the immobilized enzyme versus the free form of the enzyme. At pH 9, about 60% of the enzyme protein incubated with CPG was immobilized onto the CPG, and immobilized bovine trypsin activity was determined as 0.265 BAPNA U/g CPG beads. The immobilized bovine trypsin showed lower affinity for its substrate, lower susceptibility to inhibition by soybean trypsin inhibitor and higher thermal stability, while the optimum pH and optimum temperature values were shifted to higher values compared to those of the free enzyme. The immobilized enzyme was evaluated for its capacity to extract carotenoproteins from shrimp shell. After 11 re-uses, the immobilized enzyme retained about 77% of its initial activity, and the total yield of the product from the same immobilized trypsin was 4.3 times higher than a single use of the same amount of the free enzyme. Cunner fish is a cold water adapted, stomachless teleost fish. Cunner fish trypsin possesses some unique properties compared with homologous trypsins from (i) species acclimated to warm temperature regimes, and (ii) species with functionally distinct-stomachs. Cunner fish trypsin was extracted from pancreatic tissue, and immobilized onto CPG beads using glutaraldehyde as cross-linking reagent. The influence of enzyme loading, the properties of the immobilized enzyme in terms of specific activities, and responses to pH and temperature were investigated. The kinetic properties and operational stability of the immobilized cunner trypsin were studied as well. The pH optimum of the immobilized fish trypsin shifted from pH 8.5 to pH 9, and the temperature optimum also increased from 45ºC to 50ºC versus the free form of the cunner enzyme. The catalytic efficiencies (Vmax/Km) of the immobilized fish trypsin were determined for both amidase and esterase reactions, using BAPNA and TAME as substrates and were found to be greater than those of immobilized bovine trypsin. Thus, the immobilized cunner fish trypsin had a higher catalytic capacity for the hydrolysis of both the amide and ester substrates. The operational stability of immobilized fish trypsin was studied by extracting carotenoprotein from shrimp shell. The immobilized fish trypsin retained 75% of its initial hydrolytic capacity after 11 re-uses, and the yield obtained was over 20% higher than that of immobilized bovine trypsin. When the immobilized cunner fish trypsin was applied to digest native pectin methylesterase (PME), it exhibited greater capacity to inactivate the PME than immobilized bovine trypsin. The inactivation efficiency of the immobilized fish trypsin was 20% higher than that of the immobilized bovine trypsin. The inactivation of PME was influenced by PME concentration, incubation time and temperature. In general, higher temperature, longer incubation period, and lower initial PME concentration produced more PME inactivation. PME inactivated by immobilized fish trypsin and bovine trypsin regained part of its activity during storage at 4ºC. The initial PME concentration affected the reactivation period. The kinetic studies indicated that the inactivation rate constants increased and D-values (time to inactivate 90% of the enzyme) decreased with increasing temperature for both immobilized fish trypsin and bovine trypsin. The activation energy (Ea) of PME inactivation by the immobilized fish trypsin was lower than that by the immobilized bovine trypsin, which explains why the immobilized fish trypsin had higher catalytic capacity at various temperatures than immobilized bovine trypsin.

Amyloid fibrils are an insoluble, highly ordered, fibrous protein structure, which have increasingly been recognised as having bionanotechnology applications. Their ability to selfassemble allows a bottom-up approach to material design. Their nanometre dimensions affords them a high surface-to-volume ratio and their proteinaceous building blocks from which they are assembled allow for decoration with biomolecules and chemicals through amino acid residues. Amyloid fibrils are therefore a potential nanoscaffold for immobilisation of biomolecules. Immobilisation offers a solution to the problems associated with the use of enzymes in in vitro applications, by increasing their stability, reusability, and in some cases, enhancing catalytic activity. Nanosupports offer a high surface-to-volume ratio compared to classical planar 2-D supports, potentially affording them dramatic increases in immobilisation capacity. To investigate the potential of amyloid fibrils as a novel nanoscaffold, organophosphate hydrolase (OPH), cytochrome P450BM3 (P450BM3), green fluorescent protein (GFP), tobacco etch virus protease (TEV), and glucose oxidase (GOD) were immobilised in solution to the model amyloid fibril forming protein, bovine insulin. Covalently immobilised OPH was found to have a ~300 % increase in relative thermostability at 40 and 50 °C. P450BM3 was not successfully immobilised in its active state, most likely due to unfolding of the enzyme on the amyloid fibril surface. Covalently immobilised GFP retained full fluorescence and acted as a fluorescent protein tag. TEV was shown to have a physical interaction with the nanoscaffold and retain activity. GOD was immobilised and retained activity. Although not all proteins retained activity, a range of different protein structures were successfully immobilised onto the insulin amyloid fibril nanoscaffold. Attachment to the crystallin amyloid fibril nanoscaffold remains a work in progress due to the complexities associated with post-translational modifications of these fibrils. Crystallin amyloid fibrils were assembled on a surface for the first time. Their surface assembled structure was found to resemble spherulites, not previously seen before with crystallin amyloid fibrils. Bovine insulin amyloid fibrils were assembled on the surface of glass beads to increase the available surface area for biomolecule immobilisation. The surface assembled bovine insulin nanoscaffold was first functionalised with GOD, demonstrating that the nanoscaffold provides more surface area for biomolecule immobilisation, although in this case the increase was limited due to high non-specific binding of GOD to the unmodified glass surface. GFP was successfully employed as a fluorescent protein tag to assess the degree of nanoscaffold coverage, confirming the nanoscaffold affords the glass bead a greater surface area. Moreover, a reusable immobilised TEV protease-bead system was developed that was able to sequentially cleave the poly-histidine tags of three different proteins. In conclusion, bovine insulin amyloid fibrils have been shown to be a versatile nanoscaffold for the immobilisation of a range of biomolecules. The surface characteristics of the nanoscaffold allows for both covalent and physical immobilisation of biomolecules. Thus, amyloid fibrils have exciting potential in the creation of novel bionanotechnologies.

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Serum lactic dehydrogenase, serum glutamic oxaloacetic transaminase, and lactic dehydrogenase isoenzyme (LDH5) levels were studied on fifteen Ball State University athletes who sustained contusions, hematomas, and muscle strains.Each individual's injury was classified as mild, moderate, or severe. There were seven subjects sustaining injuries classified as mild, seven receiving moderate injuries, and one individual with a severe injury.Serum enzyme levels of all subjects were elevated above controls after injury and then proceeded to decline unless re-injury occurred.The enzyme levels for the moderate group, as demonstrated by the graphs, revealed more marked elevations for LDH and GOT than did the ones in the mild group.A significant statistical difference was found to exist between the mild and moderate groups involving the total LDH. There were no statistical differences between these groups Sand the GOT or LDH5 isoenzyme levels.

Two direct methyltransferase systems in winter wheat have been reported using partially purified enzyme extracts. In order to gain further understanding of these enzymes, such classical enzyme investigations as pH range and optimum pH, the calculation of average activation energies, and inhibition investigations need to be undertaken using more highly purified enzymes. The purpose of this investigation was to develop procedures for further purification of S-methyl-Lmethionine:homocysteine methyltransferase in order that future researchers might undertake such studies.

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