Browsing by Subject "Enzyme activity"
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Item A rapid quantitative assay for bacterial protease activity(2006-08) Hien, Nguyen Thi Phuoc; Shelly, Dennis C.; Weber, JoachimA rapid quantitative stained hide powder assay was studied as a possible replacement for the gelatin film test, used to detect bacterial enzymes within hide juice of delayed cured hides. The ability of the method was investigated by employing different kinds of substrates and enzymes. Substrates from dogchew and rawhide were hydrolyzed by Proleather FG-F, Protex 6L and Alcalase enzyme under optimum conditions. Reproducibility of the dogchew substrate was higher but hydrolysis efficiency was less than for the rawhide substrate. To improve reproducibility and hydrolysis efficiency, a stable, neutralized stained hide powder was produced and served as a substrate for calibration. Analytical figures of merit are described. The relations between the efficiency of hydrolysis and enzyme concentration or enzyme activity were linear. The effect of salt shifted the calibration toward lower hydrolysis efficiency. The slopes did not change. The method could detect the Proleather FG-F enzyme concentration as low as 0.01 ìg/ml or enzyme activity of 1.01x10-3 LVU/ml without salt and 0.05 ìg/ml or enzyme activity of 5.05x10-3 LVU/ml with salt.Item Activity and kinetics of microbial extracellular enzymes in organic-poor sands of a south Texas estuary(2009-08) Souza, Afonso Cesar Rezende de, 1968-; Gardner, Wayne Stanley, 1941-; Pease, Tamara KayeThe respective kinetics of bacterial leucine aminopeptidase and [beta]-glucosidase activities were investigated to improve understanding of factors controlling activity and hydrolytic capacity in estuarine organic-poor sands. Depth distributions of enzyme activity and bulk organic matter content were determined in sediments of Aransas Bay and Copano Bay Texas, to investigate enzyme dynamics as related to the geochemical properties of the sediment. Vertical profiles of activity in sediment showed that the enzymes were more active at the surface and that the potential hydrolysis rate of leucine aminopeptidase was higher than that of [beta]-glucosidase. Vertical patterns of enzyme activity correlated (weakly) with variations in sediment organic matter (TOC, TN, and carbohydrates) content. Enrichments of sediment samples with monomeric organic compounds and inorganic nutrients did not affect leucine aminopeptidase and [beta]-glucosidase activities in short- and long-term incubations. Enzyme activity was independent of nutrient availability and suggested that microbial communities were not nutrient-limited. Time-course assays of bacterial hydrolysis of TOC, TN, and carbohydrates provided information about how substrate limitation may affect enzyme activity. Positive correlations between bulk TOC and TN content and enzyme activity indicated enzyme dependence on polymeric substrate content. Induction of enzyme activity after sediment enrichments with specific labile compounds confirmed the importance of available organic substrate to enzyme hydrolysis efficiency. A kinetic approach established the occurrence of enzyme inhibition and its effects on enzyme hydrolytic capacity. The addition of a specific-enzyme substrate to sediment samples modified enzyme parameters and indicated that a substrate-reversible type of inhibitor could reduce enzyme hydrolytic capacity. The addition of polyphenol, as a natural inhibitor of enzyme activity, to the sediment resulted in a concomitant reduction of leucine aminopeptidase activity and ammonium regeneration rate, and thus demonstrated a close coupling between enzyme activity and sediment ammonium regeneration. These research results demonstrate the dynamic nature of the hydrolytic enzymes, provide information about the mechanisms of induction and inhibition of activity, and demonstrate some implications of reducing the hydrolytic capacity to organic matter decomposition and nutrient regeneration rates.Item Mucoadhesive films for the buccal delivery of insulin(2012-12) Morales, Javier Octavio; Williams, Robert O., 1956-; McConville, Jason Thomas; Smyth, Hugh D; Cui, Zhengrong; Roy, KrishnenduTo address the need of a patient friendly and therapeutically effective method of administration of insulin (Ins) we sought to develop mucoadhesive films for delivery through the buccal mucosa. Ins is a labile molecule exhibiting limited activity and stability in solid solutions in films and other solid delivery devices. Early investigations outlined in Chapter 3 revealed the need for a certain particle size (below the one micrometer) for the addition of particulate material in films. In Chapter 4 a novel method for the manufacture of protein-coated nanoparticles (PCNP) is depicted. Successful particle batches were achieved in terms of size, uniformity, stability and activity and these particles were further investigated for their inclusion on films for buccal delivery. The method of manufacture of particles was based on an antisolvent co-precipitation process that immobilized macromolecules to the surface of crystalline core particles resulting in high yields and highly active protein loaded particles. Films loaded with PCNP were developed and characterized in Chapter 5. Lysozyme was utilized as a model macromolecule and high yields and activity were obtained after manufacture, demonstrating that after all the processing the protein is subjected to, activity is preserved. Using Eudragit® RLPO (ERL) as the matrix forming polymer, films with excellent mucoadhesion were developed. Here is described a high mucoadhesion for ERL that was even further increased by the addition of the water soluble PCNP. This occurred by the water movement into the ERL matrix that the solubilizing particles generate. Finally, films containing Ins were developed and assayed for permeation through buccal mucosa. By adapting the method of manufacture, Ins-coated nanoparticles were obtained and embedded in films. ERL films corroborated previous findings by exhibiting excellent performance. Investigations on the permeation of Ins through buccal mucosa revealed that the inclusion of Ins in films enhanced its permeation in comparison with a control Ins solution. Thus here is described the successful development of mucoadhesive films for the buccal delivery of Ins.