published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / abstract / toc / Medicine / Master / Master of Philosophy

published_or_final_version / abstract / toc / Surgery / Doctoral / Doctor of Philosophy

published_or_final_version / Pathology / Master / Master of Philosophy

In recent years, biocatalytical steps in chemical synthesis are becoming increasingly important for economical and environmental-friendly production. In order to evaluate the use of enzymes in a process at Cambrex Karlskoga AB, an expression system was developed for a dehydrogenase. A synthetic gene was cloned into Escherichia coli DH5a cells, using the pTZ19R expression vector, as previously described in the literature. Protein expression was carried out at 25°C, 30°C and 37°C and results were measured using SDS-PAGE and activity assays. To improve expression, the gene was modified in three ways using PCR, yielding eight clones: It was inserted into the pSE420 expression vector, shortened to avoid inclusion body formation and a missing nucleotide was inserted into the sequence. A protocol for inclusion body screening was also developed. Finally, an assay for determining the kinetic constants of dehydrogenase was designed. It is concluded that further experiments must be done to obtain expression of the dehydrogenase and recommendations for additional work are given. / Biokatalytiska processteg har de senaste åren blivit ett allt viktigare inslag i kemisk syntes för att åstadkomma ekonomisk och miljövänlig produktion. För att utvärdera användandet av enzymer i en process hos Cambrex Karlskoga AB utvecklades ett expressionssystem för ett dehydrogenas. En syntetisk gen klonades in i Escherichia coli DH5a och uttrycktes med hjälp av expressionsvektorn pTZ19R, som tidigare finns beskrivet i litteraturen. Proteinuttrycket utfördes vid 25°C, 30°C och 37°C och resultatet mättes med hjälp av SDS-PAGE och aktivitetsmätningar. Genen för dehydrogenaset modifierades på tre sätt, vilket gav upphov till åtta varianter. Genen fördes över till expressionsvektorn pSE420, kortades för att undvika bildning av inklusionskroppar och en nukleotid som fattades från gensekvensen återinfördes. Ett protokoll utarbetades även för undersökning av inklusionskroppar. Till sist sammanställdes en metod för att undersöka de kinetiska konstanterna hos dehydrogenaset. Slutsatsen av arbetet är att fortsatta studier måste utföras för att erhålla uttryck av dehydrogenaset och rekommendationer ges för framtida undersökningar.

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Thesis (M.Tech.: Biotechnology)-Dept. of Biotechnology, Durban University of Technology, 2007 vi, 92 leaves / Interest in xylanases from different microbial sources has increased markedly in the past decade, in part because of the application of these enzymes in a number of industries, the main area being the pulp and paper industry. While conventional methods will continue to be applied to enzyme production from micro-organisms, the application of recombinant DNA techniques is beginning to reveal important information on the molecular basis and this knowledge is now being applied both in the laboratory and commercially. In this study, a directed evolution strategy was used to select an enzyme variant with high thermostability. This study describes the use of error-prone PCR to modify the xylanase gene from Thermomyces lanuginosus DSM 5826, rendering it tolerant to temperatures in excess of 80°C. Mutagenesis comprised of different concentrations of nucleotides and manganese ions. The variants were generated in iterative steps and subsequent screening for the best mutant was evaluated using RBB-xylan agar plates. The optimum temperature for the activity of xylanases amongst all the enzyme variants was 72°C whilst the temperature optimum for the wild type enzyme was 70°C. Long term thermostability screening was therefore carried out at 80°C and 90°C. The screen yielded a variant which had a 38% improvement in thermostability compared to the wild type xylanase from pX3 (the unmutated gene). Successive rounds of error-prone PCR were carried out and in each round the progeny mutant displayed better thermostability than the parent. The most stable variant exhibited 71% residual activity after 90 minutes at 80˚C. Sequence analysis revealed four single amino acid residue changes that possibly enhanced their thermostabilities. This in vitro enzyme evolution technique therefore served as an effective tool in improving the thermostable property of this xylanase which is an important requirement in industry and has considerable potential for many industrial applications.

This dissertation presents the application of mass spectrometry to the detection and characterization of microorganisms based on biomarker identification and DNA analysis. Two major topics are covered: affinity capture mass spectrometry using immunoassay methods and methods involving insertion of membrane receptors into polymerized planar supported lipid bilayers; and the application of mass spectrometry for use in clinical microbiology for the identification of microorganisms causing bloodstream infections. Affinity capture mass spectrometry on immunoassay-based platforms studied the capture of Protein A from Staphylococcus aureus , demonstrating capture that is both selective and sensitive. Experiments illustrated successful capture from a purified source and cell lysates. Affinity capture using receptors inserted into polymerized lipid bilayers was also performed using GM1 and cholera toxin subunit B, demonstrating the enhanced stability offered by polymerizing the lipid bilayers such that direct ionization could be performed. Detection of protein binding was achieved with mass spectrometry at low molar ratios of receptor, and enzymatic digestion experiments on the protein retained at the surface illustrated the ability to characterize the protein ligand bound, lending support to using this technique for reverse pharmacological applications. Lastly, experiments demonstrated that affinity capture of surface-bound proteins can also be used to extract cells from complex mixture prior to the polymerase chain reaction, illustrating utility as a pre-treatment for detecting microorganisms in blood samples. Mass spectrometry was applied to detection of microorganisms from blood culture bottles collected from patients with bloodstream infections. Polymerase chain reaction electrospray ionization and whole cell matrix-assisted laser desorption/ionization mass spectrometry were used to characterize hematopathogens. High diagnostic accuracy was demonstrated with respect to culture-based testing and these two platforms were compared considering accuracy in identification, time to result, and cost benefit analysis. The experiments presented here cover a broad range of detection strategies for identifying proteins and microorganisms. The affinity capture techniques describe the first application of peptide capture and polymerized bilayers for mass spectrometric analysis, and the clinical mass spectrometry work demonstrates validation of two emerging techniques and the first comparative study on both platforms simultaneously. All research presented here demonstrates promise for application of mass spectrometry in diagnostic biology.

Amblyomma americanum, the lone star tick, is an indigenous tick species in southern Indiana that harbors a diverse group of pathogenic and nonpathogenic microorganisms, including Borrelia lonestari, the putative agent for the southern tick associated rash illness (START) and a spotted fever group rickettsial endosymbiont. The purpose of this study was to implement the real-time polymerase chain reaction (real-time PCR) as a molecular technique to examine the microbial diversity in A. americanum ticks by estimating abundances of different microorgansisms. A SYBR Green real-time PCR assay was designed to detect and quantify B. lonestari in A. americanum ticks, and a previously published TaqMan real-time PCR assay, designed to detect (not quantify) Rickettsia species in ticks, was validated for the detection and quantification of the spotted fever group rickettsial endosymbiont in A. americanum ticks. Many pitfalls associated with real-time PCR were experienced in this study, such as difficulties in assay design and problems with contamination, and appropriate modifications are recommended to laboratories routinely performing real-time PCR. / Department of Biology