published_or_final_version / Ecology and Biodiversity / Doctoral / Doctor of Philosophy

The creation of small molecule libraries for binding into the NS1A protein of influenza A viruses and the development of an indicator displacement assay for the differentiation of fatty acids are reported herein. Using Mitsunobu chemistry, a variety of structures based on hydroquinone, resorcinol and 2,7-dihydroxynaphthalene cores were synthesized. Both polar and non-polar functional groups were added to diversify the cores to help understand which molecule binds best to the protein. Because of poor protein binding, the focus of the project moved to a new lead compound, epigallocatechin-3-gallate (EGCG). EGCG showed promise in computational studies and efforts towards the synthesis of the epigallocatechin core were undertaken. Using a fluorescent indicator displacement assay (IDA), a sensing system for fatty acids was developed. The system consisted of bovine, rabbit, and human serum albumins as host molecules, while the fluorescent indicators were fluorescein, 2-anthracene carboxylic acid, and 1-anilino-8-naphthalene sulfonic acid. Fatty acids were able to be differentiated from one another based on their carbon chain length and the degree of unsaturation. The IDA was then subjected to a complex mixture of fatty acids, in the form of edible oils. The oils (extra virgin olive, hazelnut, peanut, sunflower and canola) with different fatty acid profiles were able to be differentiated from each other using principal component analysis. / text

The traditional filter paper assay for saccharifying cellulase originally described by M. Mandels et al (1976) has been modified to make possible low activity determinations of Trichoderma cellulases. The enzymatic activity appears to decline during a prolonged incubation period if no precautions have been taken. By means of adding bovine serum albumin and potassium chloride as protein stabilizers and sodium azide as an antimicrobial agent filter paper activities in the range from 0.02 to 0.37 (IUPAC assay, 1987) can be estimated by extending the incubation time up to 20 hours. Filter paper activity values obtained by this method may be compared to those obtained by the IUPAC assay by using a conversion factor from 1.4 to 1.7. Acid whey and sweet whey permeate have been investigated as media for growth and metabolite production by Trichoderma reesei QM 9414 using shake flask cultures and spore inocula. In the case of acid whey the mycelial growth after 2 weeks is 13 mg dry weight /ml substrate. The specific growth rate is 0.29/day. The fungus appears to metabolize the whey protein the first 2 weeks. The alkalinity of acid whey rises continuously over a three week period up to a pH of 8.5. In the case of whey permeate the maximal mycelial weight gain is 4.4 mg/ml which appears after 8 days. A rise in net soluble protein level comes after 3-5 days and reaches a maximum value of 0.23 mg/ml after 2 weeks. The pH of whey permeate rises continuously to 7.5 after 3 days and then slowly declines. The net production of cellulases is low on both media. Dilution 1:6 of the acid whey, supplementation with ammonium sulfate and pHadjustments did not enhance the production of cellulases. Acid whey supports a significant growth and sweet whey permeate shows potential for extracellular protein production. A literature review surveys the composition and uses of acid whey, environmental aspects of whey wastes, the fungus Trichoderma reesei, the mode of action of the Trichoderma reesei cellulase system and the structure of cellulose in cotton and wood. / Graduation date: 1994

A casein agar diffusion method was developed to detect and quantify pertinent levels of proteinases produced in raw milk supplies by heat resistant Bacillus sporeformers. In order to optimize the required heat treatment conditions of raw milk samples, trials that involved a combination of different temperatures and times were evaluated. A heat treatment of 75°C for 20 min was the most effective for recovering the highest number of surviving spores. A sporulation broth containing five different minerals and supplemented with 0.2% nonfat dry milk was used to maximize spore production in all heat-treated samples. A β-casein based assay detected proteinase activity from raw milk samples that ranged from 0.093 to 4.034 units/mg which corresponded to zones of β-casein precipitation in the β-casein agar of 5.0 and 15.0 mm respectively, and was compared to Protease Type VIII (from B. licheniformis). This assay correlated well with the fluorescein isothiocyanate casein-labeled assay (FITC), R=0.995 (Protease Type VIII). Proteases of Bacillus origin such as Protease Type IX, X, XV and XXXI were also evaluated but were rejected in favor of a broader range of activity expressed by Protease Type VIII. For an initial set of 370 raw milk samples, no quality deterioration, such as coagulation or bitter taste was observed in heat-treated (75°C for 20 min) and incubated samples (7.2°C for 10 days). However, during the winter season, 18 of 75 incubated samples (7.2°C for 10 days) tasted slightly bitter and exhibited a slight degree of casein precipitation. One sample coagulated but exhibited no proteinase activity on the β-casein agar gel, hence it was considered a false negative. The positive results for proteinase activity from raw Grade A samples tested by the β-casein agar diffusion method did not correlate either with fresh spore counts (R=0.21) or post-heat treatment incubation counts (R=0.03) or with psychrotrophic sporeformer counts (R=0.06). The β-casein agar diffusion method is simple, rapid and sensitive to Bacillus spp. proteinases, but was unreliable in projecting results related to the psychrotrophic sporeformer count. Consequently, further research is required to establish optimum conditions (time and/or temperature) and inoculum volumes into sporulation broth for attainment of a more positive correlation between β-casein agar precipitation zones and psychrotrophic sporeformer populations of either raw or processed milk samples. / Graduation date: 1991

The components involved in an immunoassay were investigated in order to improve the detection limits of the ELISA and to make the assay adaptable to a flow injection analysis (FIA) configuration. The goal being the total automation of the ELISA procedure which is long, tedious and has high standard deviation. The antibody purification and cleavage methods were studied with special emphasis on obtaining products with highest immunological activity. The antibody-enzyme coupling reactions using homobifunctional reagents and heterobifunctional reagents were studied in order to attempt the preparation of highly characterized reagents. The fragments of IgG were coupled to polymeric supports via the hinge thiol groups to retain the maximum immunological activity. This method was found to be superior to those methods involving coupling via amino group. These reagents were used in the development of a sandwich ELISA for bovine IgG. The range of assay was in the 20-1000 femtomole range with a linear dynamic range of 2 orders of magnitude and an accuracy of 2-5%. A competitive ELISA based on the use of immobilized anti-human IgG Fab' fragments was developed. The linear dynamic range for this assay was found to be less than one order of magnitude. The detection limit was in the low picomole range with an accuracy of 2-5%. Based on the principle used in the two assays an enzyme immobilization scheme was developed for the reversible immobilization of these enzymes. Which was subsequently utilized in the determination of substrate in the picomole range in a reagent less FIA technique. The goals of this research project were realized in that the FIA system utilized in this work was capable of carrying out totally automated ELISA assays with an accuracy far surpassing the conventional plate ELISA assays.

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