No description available.

Biologically active peptides are found in the amino acid sequence of bacterial, fungal, plant and animal proteins. They are among the most potent pharmacologically active agents. Examples are venom toxin (mellitin), hormones (oxytoxin), opioids (beta-endorphin) and enzyme inhibitors (hirudin). These bioactive peptides are released from proteins by enzymatic proteolysis by processes such as gastrointestinal digestion or food processing. / Soybeans were fermented with Bacillus subtilis ATCC 41332 and Rhizopus oligosporus NRRL 2710 to produce tempeh and natto, respectively. Samples were taken throughout the fermentation and analysed for biochemical changes. Protease activity and ammonia production detected in the early stage of the tempeh preparation suggested that protein was used as a carbon source during that period and contributed to a rise in pH. Bacillus subtilis did not produce ammonia and maintained a constant pH throughout the fermentation. The total peptides produced were at a maximum at the end of the fermentation cycles. Angiotensin-converting enzyme inhibitory activity increased throughout both fermentations. A method was developed to monitor the production of biogenic amines throughout the fermentation. The levels of biogenic amines increased dramatically as the fermentation proceeded until maturity was achieved. In the tempeh fermentation, the polyamines levels rose from the initial 19 ppm to final concentration of 862 ppm, with the largest increase in histamine (616 ppm) followed by putrescine (204 ppm). These compounds also contributed to the pH rise from 3.8 to 6.8 in 24 h. In the Bacillus fermentation, the total polyamines at the end of the fermentation was 110 ppm with the largest increase in putrescine, followed by cadaverine. / Soy hydrolysate and the soy fermented foods, natto and tempeh, were deglycosylated and treated with proteolytic enzymes (plasma proteases, kidney homogenate, pronase, pepsin, thermolysin, trypsin, chymotrypsin and proteinase K) to produce oligopeptides. Several peptides were isolated, purified and characterized. The peptides had a range of biological activities---angiotensin converting enzyme inhibitory, antithrombotic, surface tension, antibacterial, anti-oxidant and insulin-modulating activities. Three potent ACE inhibitors, three thrombin inhibitors, five peptides with surface-active properties and one peptide with antibacterial activity were identified. They were all derived from glycinin and were found in the plasma protease digest, the kidney homogenate digest and the pronase digest of fermented foods. Another sequence ELLVYLL possessed good surface active properties but its precursor could not be identified. However, it was analogous to a peptide produced by Bacillus subtilis , and was probably synthesized during fermentation. Peptide analogs were synthesized and evaluated. They showed similar activities. Other sequences of known inhibitors, TPKDFEEIPEE, FPRGGG and DFEEIPEEL, were found to be competitive substrates for ACE.

To optimize conditions to prepare a good quality of enzyme-modified cheese (EMC), EMC samples were prepared by using combinations of Neutrase RTM/Lactobacillus casei enzymes, Neutrase RTM/Debitrase(TM), NeutraseRTM/Flavorzyme(TM) and NeutraseRTM/PalataseRTM. Based on the results obtained from sensory and RP-HPLC analysis, the optimal combinations to prepare a good quality of EMC were found to be: NeutraseRTM with (I) L. casei enzymes (aminopeptidase activity 86.4 LAPU/g and esterase activity 110.0 U/g), (II) Debitrase(TM) (aminopeptidase activity 22.0 LAPU/g), (III) Flavorzyme(TM) (aminopeptidase activity 6.5 LAPU/g), and (IV) PalataseRTM M (lipase activity 200 LU/g). / The water-soluble fractions of EMCs prepared with different enzyme combinations were subjected to RP-HPLC on a Delta Pack C18 column, and selected peaks were purified on the same column using a binary gradient. One peak from NeutraseRTM digest, five peaks from NeutraseRTM /Debitrase(TM) digest, and two peaks from NeutraseRTM /L. casei enzyme digest were purified and identified by API mass spectrometry. All the purified peptides contained active sites within their sequences. / The volatile compounds in a series of EMCs prepared by L. casei and commercial enzymes as well as Cheddar cheese (mild, old, extra old) were also identified by using Pyrolysis/GC/MS and dynamic headspace techniques. Overall, 5 ketones, 8 fatty acids, 3 alcohols and 2 aldehydes were detected in most of the samples using Py/GC/MS. Propanoic, hexanoic, octanoic, decanoic, dodecanoic and tetradecanoic acids were found to be the major fatty acids present in EMC prepared by L. casei enzymes. Dynamic headspace analysis revealed the presence of 17 compounds including fatty acids, ketones, alcohols, aldehydes, and hydrocarbon in most of the sample analyzed. The flavor of EMC seems to depend not on any particular key component, but rather on a critical balance of all components present.

No description available.

Graduation date: 1994

Plant natriuretic peptides (PNP) are novel peptides which, like in vertebrates, have been shown to have a function associated with water and salt homeostasis. Two PNP-encoding genes have been identified and isolated from Arabidopsis thaliana, namely; AtPNP-A and AtPNP-B. In this study, the focus was on PNP-B, which has not been extensively studied. Bioinformatic analysis was done on the AtPNP-B gene. This included the bioinformatic study of its primary structure, secondary structure, tertiary structure, transcription factor binding sites (TFBS) and its relation to other known proteins. The AtPNP-B gene was shown to be a 510 bp long, including a predicted 138 bp intron. AtPNP-B was also shown to have some sequence similarity with AtPNP-A and CjBAp12. The TFBS for AtPNP-B and OsJPNP-B were compared and they comprised of TFBS that are related to water homeostasis and pathogenesis. This suggested two possible functions; water stress and homeostasis and a pathogenesis related function for PNP-B. Following bioinformatic analysis, the heterologous expression of the AtPNP-B was attempted to investigate whether the AtPNP-B gene encoded a functional protein and to determine the functional role of PNP-B. However, expression was unsuccessful. An evolutionary study was then carried out which revealed that there were some plants without the intron such as, rice, leafy spurge, oilseed rape, onion, poplar, sugar cane, sunflower and tobacco. These plants would therefore be used for expression and functional studies in the future. The evolutionary studies also revealed that PNP-B had a relationship with expansins and the endoglucanase family 45. Other PNP-B related molecules were also obtained from other plant genomes and therefore used in the construction of a phylogenetic tree. The phylogenetic tree revealed that AtPNP-B clustered in the same group as CjBAp12 while AtPNP-A had its own cluster group. There were also other PNP-B like molecules that clustered in the same group as expansins (α- and β-). Thus, we postulate that, like PNP-A, PNP-B also has a possible function in water and salt homeostasis. However, due to the clustering iii of AtPNP-B into the same group as CjBAp12, a possible role of PNP-B in pathogenesis-related response is also postulated.

Indiana University-Purdue University Indianapolis (IUPUI) / This thesis describes separations performed by Pressurized Planar Electrochromatography (PPEC), which is a chromatographic method developed at IUPUI. In PPEC the mobile phase is driven by electroosmotic flow, while the system is pressurized to allow temperature control. This results in a highly efficient chromatographic system that has several attractive attributes including the ability to separate multiple samples simultaneously. The first three chapters of the thesis describe the relationship of PPEC to other forms of chromatography, the theoretical background of PPEC, the PPEC apparatus, including the plate holders used, and the different manipulations involved in preparing a plate for a PPEC run. The fourth chapter describes two short studies. The first demonstrates that a very fast separation of steroids on a high efficiency sorbent layer can be effected by PPEC. This is illustrated by the separation of six steroids in three minutes on a Superspher layer, with an efficiency of over 100,000 plates per meter. The second study attempted to improve the efficiency of separation by imposing a temperature gradient. The study was not successful, possibly due to Joule heating within the layer overriding the temperature gradient. The final chapter of the thesis describes two different studies on separating peptides by PPEC. The first study was performed on a bonded C18 sorbent layer that was treated with Brij-35, which is a non-ionic surfactant that prevents irreversible adsorption of the peptides to the sorbent surface while allowing electroosmotic flow. The variables involved in preparing the plates by soaking in a Brij-35 solution were investigated as well as the variables for PPEC (temperature, pressure, electrical potential, and mobile phase composition and pH). It was possible to separate six peptides in eight minutes using this approach. The second study used monolithic sorbent layers prepared by Dr. Frantisek Svec of Lawrence Berkeley National Laboratory. Separations were by conventional PPEC on charged monoliths and by electrophoresis on neutral monoliths. The same variables for PPEC, listed in the above paragraph, were investigated for the monolith study. It was possible to separate six peptides in two minutes on neutral monoliths and in one minute on negatively charged monoliths.