Thesis (M.Sc.)--Memorial University of Newfoundland. / Bibliography: leaves 104-109. Also available online.

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.

No description available.

Many proteins with special properties have been identified in aquatic organisms. Due to their peculiarity, some of these biomolecules could be used advantageously for some industrial and health care applications. However, the preparation of these biochemicals from its original source is highly impractical and generally unfeasible for commercial purposes. Nevertheless, the advances in DNA manipulation open the possibility of producing the recombinant proteins in different organisms to allow a viable production and extraction on a commercial scale. Among such particular proteins, the cunner-fish trypsin (CFT) is an enzyme with a high potential for exploitation by the food processing industry. The CFT is a cold adapted protease that has a proven ability to inactivate undesirable endogenous enzymes during the processing of some foodstuffs. Thus, the CFT gene was characterized and cloned in E. coli and Pichia pastoris for over-expression. A cDNA library from pancreatic mRNA was screened using a salmon trypsin cDNA probe. Positive clones harboring a cDNA insert of the predicted size were sequenced and a full-length cDNA was obtained that contained an ORF encoding the zymogen and a signal peptide. Multiple alignment of the gene sequence showed 85 to 90 per cent identities with other fish trypsins and the deduced amino acid sequence for the mature enzyme exhibited the entire characteristic features observed in trypsins. E. coli expressed the recombinant CFT (rCFT) at levels higher than 20% in the form of insoluble aggregates, while P. pastoris showed expression levels below 1%. Despite the low expression, the yeast rCFT appeared to be correctly folded and could be partially purified by immobilized nickel affinity chromatography. Trypsin-specific activity was confirmed in the purified rCFT after digestion with bovine enterokinase.

No description available.

Thesis (M.Sc.)--Memorial University of Newfoundland, 2002. / Restricted until October 2003. Bibliography: leaves 93-103.

Digestive proteases were isolated from the pancreas of the stomachless cunner fish (Tautogolabrus adspersus) and characterized in terms of their physicochemical properties, their ability to hydrolyze native pectin methylesterase (PME) from orange and polyphenol oxidases (PPO) from mushroom and the ability of the cunner enzyme(s) to maintain the stability of orange juice cloud. / The cunner trypsin fraction exhibited exceptional capacity to hydrolyze native proteins versus the bovine trypsin. Incubation of native PME with cunner or bovine trypsin resulted in a loss of 75% or 35% in PME activity respectively. Similarly, a 75% or 55% loss in PPO activity was observed after treatment with cunner and bovine trypsin respectively. Bovine trypsin, however, hydrolyzed the heat-denatured PME and PPO better than the cunner trypsin. Also, there was no reactivation of both PME and PPO activity after treatment with either the cunner or bovine enzyme during storage at 4$ sp circ$C for 3 weeks. However, PPO retained up to 20% or 50% of the initial activity after treatment with cunner or bovine trypsin, respectively. / A 3 x 3 factorial design involving the factors of temperature, enzyme concentration and incubation time carried out gave an r$ sp2$ of 0.92 and 0.95 for cunner and bovine trypsin treated PME respectively. On the other hand, an r$ sp2$ of 0.91 and 0.94 was obtained for the combined effects using cunner and bovine trypsin for PPO inactivation. Validation of the model of PME inactivation measured as the % cloud remaining revealed that the cunner trypsin fraction upheld the cloud stability of cloud juice better than bovine trypsin, with cunner trypsin retaining more than 90% of the cloud whereas the juice treated with bovine trypsin only resulted in a 70% retention of the juice cloud. (Abstract shortened by UMI.)

No description available.