Thesis (M.S.)--University of Michigan, 1970.

Thesis (M.S.)--University of Michigan, 1970.

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2006. / Includes bibliographical references (leaves 107-118). Also available in electronic version.

Twenty different macrolide and ketolide antibiotics were analyzed by reversed phase high performance liquid chromatography on an ODS-2 cartridge column. Each of these compounds was uniquely separated and purified by varying the flow rate. Retention times of the individual drugs were proportional to the flow rate of the mobile phase. Recovery of antimicrobial activity for most of the drugs was greater than 90% based on a microbiological assay of material recovered from the column. Retention times were related to structural differences between these antimicrobial agents.

No description available.

Supplementation of the diet with low concentrations of antibiotics stimulates the growth of poultry by affecting the intestinal flora. The bacterial flora of the small intestine of turkey poults was extensively analyzed in an attempt to correlate changes in populations with growth response. Lactobacillus species comprised almost 100% of the duodenal flora of two-week-old poults but there was no difference in species associated with antibiotic (zinc bacitracin, 55 ppm) treatment. The ileal flora also was predominantly lactobacilli (average 75% of the flora). The most common lactobacilli from the turkey intestinal tract were several previously undescribed Lactobacillus species followed by L. acidophilus, L. salivarius subsp. salivarius, L. fermentum, and L. plantarum. Antibiotic treatment resulted in a shift in the proportions of several of the unnamed Lactobacillus sp. Preliminary feed trials using two strains of lactobacilli that belonged to species that increased in numbers with antibiotic treatment did not stimulate growth when one-day-old birds were colonized with the strains. A probable explanation for the increase in growth is the effect of antibiotic treatment on the multiplication of bacteria in the small intestine. As the digesta move from the gizzard to lower ileum an average 16-fold increase in bacteria occurs in untreated birds. In antibiotic-treated birds the increase was only 2-fold. This inhibition of growth is not due strictly to cell lysis because there are no significant differences in microscopic counts, but the viable counts do decrease. As a corollary there is significantly less lactic acid in the lower ileum of antibiotic-fed birds. Antibiotics did not affect total microscopic or viable counts in the crop or ceca. The above experiments were all done with zinc bacitracin; however, the inhibition of bacterial multiplication was also observed with procaine penicillin. The conclusion from my data is that zinc bacitracin, and probably procaine penicillin, stimulate the growth of turkey poults by a general suppression of the small intestinal flora rather than by an effect on any individual bacterial species. / Ph. D.

Clostridium sordellii cause gas gangrene in man and animals, and more recently it has been implicated as a causal agent of diarrhea and enterotoxemia in domestic animals. This organism was once believed to cause pseudomembranous colitis (PMC) in humans, however, Clostridium difficile, not C. sordellii, was found to be the causative agent of this disease. It is now known that C. difficile produces two toxins, designated A and B, that are implicated in the pathogenesis of the disease. C. sordellii produces two toxins, designated HT (Hemorrhagic Toxin) and LT (Lethal Toxin), that are similar to toxins A and B of C. difficile. The goal of my research was to purify and characterize the two toxins of C. sordellii, and compare their properties to those of C. difficile. Toxin HT was purified from C. sordellii (VPI strain 9048) culture filtrate by ultrafiltration through an XM-100 membrane filter and immunoaffinity chromatography using a monoclonal antibody to toxin A of C. difficile as the ligand. Toxin LT was purified to 80% homogeneity by ultrafiltration on an XM-100 membrane filter and ion-exchange chromatography. Toxin HT migrated as a major band with molecular weight of 525,000 and a minor band at 450,000 on non-denaturing PAGE. By SDS-PAGE the molecular weight was estimated at 300,000. Isoelectric focusing indicated a pI of 6.1. Like toxin A, toxin HT was cytotoxic to cultured cells, lethal for mice, and elicited an accumulation of hemorrhagic fluid in rabbit ileal loops. Toxin LT exhibited properties similar to toxin B, although LT was about a 1000-fold less cytotoxic than toxin B. By SDS-PAGE the molecular weight was estimated at 260,000. Immunodiffusion analysis revealed a reaction of partial identity between these toxins and their amino-terminal sequences were very similar. Toxins HT and LT of C. sordellii have retained remarkable immunological similarities as well as physicochemical and biological properties with toxins A and B of Q. difficult however the toxins are not identical. / Ph. D.

The work presented in this thesis is aimed to design efficient reactive crystallization operations that could potentially be implemented in the manufacture of enantiomerically pure compounds and β-lactam antibiotics. Multiple aspects of solution thermodynamics, reaction engineering and crystallization from complex solutions are involved and will be discussed in detail through the following chapters. The first piece of this work utilizes reactive crystallization for the manufacture of enantiomerically pure amino acids. Chemo-enzymatic stereoiversion reactions are used to enrich saturated or supersaturated solutions to favor the selection of a desired enantiomer. L-Methionine and L-Phenylalanine were resolve successfully from racemic mixtures by cyclic stereoinversion. r D-amino acids were oxidized by D-amino acid oxidase (D-AAO) and the resulting ketoacid was subsequently reduced by ammonia borane producing a racemic-mixture After the necessary enantiomeric enrichment was reached, system conditions were changed to induce supersaturation and promote crystal formation. In each case crystals with chemical and enantiomeric purities greater than 99% wt. were recovered. experimental information about reaction and crystallization kinetics was used to developed models. Such models were used to design model-based optimizations in which the productivity of the operation was enhanced by selecting an optimal temperature profile. The second example is a reactive crystallization towards the manufacture of β-lactam antibiotics. One of the major drawbacks of the utilization of enzymes towards the manufacture of β-lactam antibiotics is the fact that the same enzyme that catalyzes the synthesis of the antibiotic also catalyzes its hydrolysis and thus, its degradation. The reaction scheme is a kinetically controlled synthesis in which the desired product is an intermediate within the network. Hence, the focus of this work is to design an efficient reactive crystallization in which the product is crystallized before it is consumed by hydrolysis. In order to accomplish this goal we have study solution equilibria, reaction kinetics, and crystallization kinetics. Even though crystallization kinetics of ampicillin has been previously reported; the reported models are not applicable to a reactive crystallization scheme for a variety of reasons. In this work, we have developed a robust model that can be applied to multiple crystallization protocols that are consistent with the conditions at which the enzymatic reaction can be performed. Finally, a reactive-crystallization scheme in which ampicillin was successfully recovered from solution was developed. In this work, crystal seeds were used to promote crystallization of the desired product from the complex media. The results indicated that is possible to perform the reaction and crystallization in parallel, and still recover crystals with high purity. This work is the first example in which ampicillin was produced and recovered with high purity in a single stage. Previous work on reaction crystallization of antibiotics reported ampicillin crystallization; however, this was accompanied by precipitation of by-products which greatly reduces the applicability of the operation as product purification is required after the reaction.

No description available.

Small molecules containing a 7-deazapurine moiety are ubiquitous in nature. They comprise a broad range of structurally diverse antibiotics produced by terrestrial and marine microorganisms that possess demonstrated antibiotic and antineoplastic activity. In addition, queuosine, a hypermodified nucleoside located in the wobble position of select tRNAs that is almost universally conserved throughout biology, contains a 7-deazapurine functional group. The since their initial identification over 50 years ago, the chemical transformations underlying the biosynthesis of 7-deazapurines have remained elusive. This work describes the identification of a cluster of co-localized genes in the Streptomyces rimosus chromosome that are responsible for the biosynthesis of the 7-deazapurine containing antibiotics toyocamycin and sangivamycin. Further, the in vitro conversion of GTP to the previously identified queuosine biosynthetic intermediate 7-cyano-7-deazaguanine (preQ₀) is demonstrated using purified, recombinant enzymes. Also included herein is a kinetic, spectroscopic, and mechanistic characterization of QueE, an enzyme that catalyzes the third step in the biosynthesis of 7-deazapurines using a radical-mediated rearrangement. A possible mechanism for the reaction catalyzed by QueD, the second step in the deazapurine biosynthetic pathway, is explored based on X-ray crystallographic data of site directed QueD mutants containing bound substrate. Finally, hitherto unrecognized gene clusters that are likely devoted to the biosynthesis of 7-deazapurines other than queuosine are described.