A procedure was developed for the rapid isolation of human phosphoglucose isomerase by substrate-induced elution from cellulose phosphate. The high degree of selectivity of the elution provided homogenous enzyme from erythrocytes after a purification of approximately 30,000-fold with a recovery of approximately 70%. The enzyme was also isolated from other human tissues by a similar procedure.

We rely on oral drugs to treat several diseases and infections. Yet, the gut microbiome modifies oral drugs within the human gut by using enzymes, facilitating efficient chemical reactions. These drug modifications impact effective doses and outcomes for individuals. The gut microbiome can convert drugs destined for excretion back to active drugs, and the converse is also true, the microbiome can inactivate active drugs, and both may lead to toxic effects. There is no resource for cataloging bacterial drug-metabolizing genes within the human gut microbiome with analytical tools to annotate these genes in sequenced gut microbiomes. I created a resource called the Human Microbiome Drug Metabolism (HMDM) database. I analyzed 1,196 unpublished sequenced gut bacterial genomes from 8 healthy adult donors to predict genes that encode enzymes capable of metabolizing drugs using two in silico methods I developed, namely MAGIS and AutoPhylo. I reviewed the scientific literature and built an ontology-centric database, the HMDM, to catalog the bacterial drug-metabolizing genes and drugs they modify. I developed DME software to predict bacterial genes capable of metabolizing host-directed drugs using the HMDM data. We experimentally validated four novel AMR gene homologs predicted from the genomes. The HMDM is curated with 50 genes reported to metabolize drugs and 45 gene variants of the β-glucuronidase (uidA) gene. MAGIS was used to predict 246 putative bacterial drug-metabolizing genes. I predicted the three novel AMR gene homologs that resemble fosfomycin thiol transferase enzymes using AutoPhylo. The MIC experiment shows that fosD1, fosD2, and fosD3 have MIC of 8μg/mL, 8μg/mL, and >512μg/mL, respectively. The genes fosD1 and fosD2 are of unknown function, and FosD3 converts fosfomycin. The HMDM database is limited to bacterial genes. The in silico methods are critical for studying bacterial drug metabolism to predict drug fate and patient outcomes. / Thesis / Master of Science (MSc) / We use medications in our everyday life to treat infections and manage diseases. Yet, bacteria residing within the human gut can interact with these medications, which can cause undesirable outcomes. Many bacteria in the human gut produce biological catalysts known as enzymes that break down chemicals, including drugs. Medication is measured and given to an individual, called a dose, and the oral route is preferred. Enzymes break down oral and biliary system drugs, reducing the effective dose. As a result, medication becomes ineffective or toxic to the body. As such, we must study how each drug is affected by bacterial enzymes. I built a resource, the Human Microbiome Drug Metabolism (HMDM) database, to catalog all the bacterial genes that code for the enzymes reported in scientific papers to break down oral drugs. We can use the HMDM database to study bacterial enzymes that lead to poor drug efficacy.

Currently, feed enzyme supplementation into commercial broiler diets is a routine practice due to hydrolysis of antinutrients, such as non-starch polysaccharides (NSP) and phytate (IP6); ultimately leading to optimized broiler production. As a result, the market has grown rapidly, and a multitude of feed enzymesare commercially available. Complete removal of antinutrients has not been achieved and research has primarily focused on the use of singular enzyme inclusion. Due to biochemical differences among enzyme characteristics, there may be potential for synergistic action to remove antinutrients and further maximize broiler performance. Therefore, the overall objective of this dissertation was to investigate enzyme strategies to determine optimal inclusion for broilers. Chapter 2 investigated the interactive effects of phytase dose (PD; 250 to 1500 FTU/kg) and carbohydrase enzyme (CE) of xylanase (XAN), β-mannanase (MAN), or their combination (XM). These data demonstrated an interaction between CE and PD for d 0-14 FCR, but this significance was lost during the remainder of the study. Overall benefits for performance and processing were demonstrated with feeding a PD of 1500 FTU/kg and XAN. Chapter 3 took a preliminary approach (0-14 d) to investigate the potential synergy among 3 phytase enzymes of varying biochemical properties when fed alone or in combination with low PD (120 or 240 FTU/kg). These data demonstrated potential synergy with the supplementation of 2 phytases combined at a higher PD (240 FTU/kg), as identified from ileal IP (inositol phosphate) lower ester concentration and tibia ash. Chapter 4 built upon the previous chapter, choosing 2 phytases (A or B) and addressing previous limitations by including a broader/more practical range of phytase activity (250 or 1500 FTU/kg); providing 3 diets varying in calcium (Ca) and non-phytate phosphorus (nPP); and using an entire grow-out. Feeding diets lowest in Ca and nPP along with 1500 FTU/kg of A, B, or A+B resulted in improved broiler performance. Additionally, the use of A+B at 250 FTU/kg within diets of medium Ca and nPP levels demonstrated synergism through improved broiler performance and tibia ash. Overall, data suggest that the strategic utilization of enzyme combinations may provide additional benefits towards broiler production.

Over the past decade the poultry industry has made tremendous improvements in production and efficiency that can be attributed to areas of nutrition. Feed additives are one area of nutrition that the poultry industry utilizes to improve performance and efficiency. Experiment 1 investigated the effects of two nutrient densities diets and three enzyme inclusion strategies on 59 d Ross x Ross 708 male broiler performance under a natural disease challenge. These data suggest that when supplementing EIS2 broiler performance and processing characteristics are improved. Additionally, HD also improved broiler performance. The objective of the second experiment was to examine the interactive effects of probiotic and copper inclusion on 42 d Ross x Ross 708 male broiler performance. The resulting data suggests that inclusion of Cu-H at 250 ppm enhances broiler performance while lowering E.coli presence. These data also suggests that BL and Cu-H interact, ultimately reducing the efficacy of BL.

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