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Fish viscera are produced in large quantities in the fishing industry and represent a waste disposal and environmental pollution problem. However, this material is a rich source of trypsins that may have some unique properties, such as high molecular activity at low processing temperature, low thermostability, and high pH optimum/pH stability, for both basic research and industrial applications. The main objectives of this project were to extract, purify and characterize trypsin from the pyloric ceca of hoki (Macruronus novaezealandiae ), which is by far the most important commercial fish in New Zealand. / Trypsin was purified from the pyloric ceca of hoki by ammonium sulfate fractionation, followed by acetone fractionation and affinity chromatography on SBTI-Sepharose 4B. The purified extract was simultaneously desalted and concentrated by ultrafiltration, and then characterized using N-alpha-benzoyl-DL-arginine-p-nitroanilide (BAPNA) as substrate. The affinity fraction migrated as a signal band in SDS-PAGE gels as well as in isoelectric focusing gels. The molecular weight of the isolated trypsin was determined by SDS-PAGE to be approximately 26,000 Da, whereas the MALDI-TOF MS method of analysis indicated a molecular weight of 23,791 Da. The isoelectric point was determined as 6.5. / The kinetic properties, temperature, pH and inhibition effects on the activity of the purified trypsin were verified. On the basis of the kinetic properties, hoki trypsin showed better amidase activity than bovine trypsin. The hoki trypsin had alkaline pH optimum (pH 9.0) and was stable at a high pH. Hoki trypsin had a higher optimum temperature (60°C) and still had relative higher activity at lower temperature. On the other hand, hoki trypsin was unstable at higher temperature. The enzyme was inhibited by well known trypsin inhibitors (SBTI, aprotinin, benzamidine and PMSF). The N-terminal residues of hoki trypsin, IVGGQECVPNSQPFMASLNY, displayed considerable homology with other fish trypsins. Based on the above characteristics, it is suggested that the hoki enzyme is authentic trypsin with potential for use in food industry and related applications.

Thesis (Ph. D.)--Columbia University, 1925. / Vita. Bibliography: p. 28-29.

Thesis (Ph. D.)--Columbia University, 1924. / Vita. Bibliography: p. 24.

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Thesis (M.S.)--University of Puerto Rico, Mayagüez Campus, 2004. / Printout. Tables. Includes bibliographical references (leaves 27-29).

Typescript (photocopy). / Digitized by Kansas Correctional Industries

Thesis (M.A.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / Enzymes are proteins which have the ability to catalyze chemical reactions and which do so with a high degree of specificity. Since trypsin has a sharp specificity for two positively charged substrates, arginine and lysine, an attempt was made to assess the role of the positive charge in determining this specificity. Both Pressman and Wilson did similar studies (Pressman on hapten-antibody reactions and Wilson on acetylcholinesterase reactions) in which they compared the hydrolysis of a substrate possessing a charged quaternary ammonium group with that of the same substrate in which the charged group had been replaced by a tertiary butyl group. In both cases, Ko/K+ was approximately eight where Ko is the dissociation constant for the uncharged substrate and K+ is the dissociation constant for the charged species. The free energy change associated with this change in K is about 1.5 kilocalories per mole and was attributed by both authors to the electrostatic contribution to the binding energy. Attempts to relate these changes in free energy to physical force expressions are discussed. Webb attempted to calculate group separation distances from the free energy changes using the expression for the potential energy of two separated charges corrected for ion atmosphere interactions. He tentatively concluded that for both hapten-antibody and cholinesterase-substrate interactions the groups involved are in direct contact without an intervening water molecule. [TRUNCATED] / 2031-01-01

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