Strains of the Gram-negative bacterium Klebsiella pneumoniae secrete pullulanase, a maltose-inducible starch debranching enzyme that exists as a cell surface bound intermediate. Three classes of secretion mutants were obtained by transposon In 10 mutagenesis. Class I and class III mutants carry Tn10 insertions in pullulanase secretion genes. Class II mutants carry insertions in regulatory loci that are required for the high level expression of pullulanase and other maltose-inducible genes (the maltose regulon). One such locus appears to correspond to a previously unknown locus. The phenotypes of the secretion mutants and the analysis of E.coli expressing pullulanase and/or cloned pullulanase secretion genes suggest that pullulanase secretion functions are involved in translocating pullulanase across the outer membrane and in releasing it from the cell surface. Most if not all pullulanase secretion genes are located to both sides of the structural gene for pullulanase (pu1A). Pullulanase was found to be a lipoprotein. Surprisingly, secreted pullulanase also carried lipid. However, strain K21 differed from other strains of Klebsiella pneumoniae by its ability to secrete not only acylated pullulanase but also a second, unacylated form of pullulanase. Strain K21 is also unusual because of its ability to secrete most pullulanase during logarithmic growth. This pullulanase corresponds to the unacylated pullulanase, with the remaining secreted pullulanase being acylated and secreted during stationary phase, as is the case for most or all pullulanase of other strains of Klebsiella pneumoniae. Strain K21 is also unusual because of the high level expression of the maltose regulon, including pulA, in the absence of maltose. This property, including the unusual features described above, may be a consequence of the selection of strain K21 for high level commercial production of pullulanase. Models for pullulanase secretion are discussed and approaches towards increasing the efficiency of commercial pullulanase production by strain K21 are outlined.

A recombinant phage that produced a polypeptide possessing the characteristics of pneumolysin, the membrane damaging toxin of the pneumococcus, was isolated from a bank of pneumococcal sequences in ?gt10. Subclones carrying the pneumolysin gene in various plasmids were haemolytic regardless of the orientation of the insert. The nucleotide sequence of a 5 kb fragment carrying the pneumolysin gene was determined. An open reading frame 1413 bp long was identified that when translated encoded a polypeptide with 471 amino acids and a molecular weight 52.8 kD. The N-terminal amino acid sequence of the predicted protein was identical to that of native pneumolysin. A single cysteine residue was present at position 428 in the amino acid sequence. Comparison of the DNA and amino acid sequences of pneumolysin with streptolysin O (SLO) revealed extensive homology in the amino acid sequence. The longest region of identity was a sequence of 12 amino acids surrounding the unique cysteine. A hybrid gene consisting of the 5' region of the pneumolysin gene and the 3' end of the SLO gene was constructed. The fusion polypeptide was made in E. coli, but possessed a very low haemolytic activity. Using the technique of oligonucleotide-mediated site-directed mutagenesis, two mutant genes were constructed in which the cysteine codon was changed to either a glycine or serine codon. Modified toxins when purified from E. coli had a specific activity of about 1-2 % that of wild type pneumolysin.

Orientador: Sergio Roberto Peres Line / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-19T23:00:58Z (GMT). No. of bitstreams: 1 Aidar_Marisi_D.pdf: 1551109 bytes, checksum: 0124186b772962c7d027715999e971c5 (MD5) Previous issue date: 2012 / Resumo: As anidrases carbônicas mantém o PH fisiológico catalisando a hidratação do dióxido de carbono na reação...Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital / Abstract: The carbonic anhydrases maintain the physiological pH catalyzing the hydration of carbon dioxide in the reaction ...Note: The complete abstract is available with the full electronic document / Doutorado / Histologia e Embriologia / Doutor em Biologia Buco-Dental

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Experiments facilitating the engineering of the PETNR active site to accommodate a range of non natural enone substrates with substituents localised on the α and β carbons of the unsaturated bond are described. In order to facilitate the high throughput purification of PETNR libraries poly histidine (PETNRHis) and biotin (PETNRBio) tagged PETNR variants were generated. High throughput protocols were developed for the automated generation, purification and screening of libraries in a 96 well format. Protocols were optimised and trialled using blocks consisting of PETNRHis WT only and characterised in terms of intra block variation. A range of single site saturation mutagenic libraries were generated at positions in the active site consisting of T26, Y68, W102, H181, H184, Y186, Q241 and Y351. Sequencing results indicated randomised libraries with the occasional instance of bias evident. Expression and purification in a 96 well format was monitored by SDS PAGE and protein quantitation. Library activity was quantified and demonstrated to retain varying degrees of activity with the model substrate 2-cyclohexenone. Following this verification of the experimental protocol libraries were screened against a range of substrates analogous to substrates demonstrated to be active with PETNRWT but incorporating substituents at the α and β carbons. 'Hits' generated from these screening reactions were studied further by the determination of the specific activity and quantitation of substrate/product from biotransformation reactions. From these screening experiments totalling 3,600 individual reactions, 35 were identified as potential hits, of these 8 proved to be genuinely improved variants. Substituents at the β carbon were demonstrated to compromise the activity of the WT enzyme most severely. Positions 68, 102, and 351 were demonstrated to play an important role in the accommodation of substituents at the α carbon whilst residues 26 and 351 are important for the β carbon. The best variants demonstrated up to 9 fold improvements in poor substrates which represented rates in excess of those observed for model substrates.

The need to high quality therapeutic proteins has grown significantly in the past 30 years. Recombinant proteins are often produced from vectors inserted into E. coli cell lines for large scale production. However, over-expression of the protein within the cell can lead to the formation of large, insoluble aggregates known as inclusion bodies. Native monomer protein can be isolated from inclusion bodies through a refolding process. This entails disruption of the aggregate structure with high concentrations of denaturant and renaturation in native-promoting solution. Our work characterises protein-protein interactions and aggregation between partially unfolded proteins during the refolding process. The protein-protein interactions are characterized in terms of the osmotic second virial coefficient (B22). A positive value indicates repulsive interactions while a negative value indicates attractive interactions. Measurements are carried out for lysozyme, ribonuclease A and preproinsulin as a function of pH, ionic strength and denaturant concentration, alongside a range of known refolding excipients. Past studies (Ho and Middelberg, 2004; Ho et al., 2003) have shown a link between higher B22 values in denaturant solutions and reduced aggregation during refolding. Our experiments have focused on the effects of urea and GdmHCl upon protein-protein interactions, alongside how ionic strength and refolding additives influence interactions between partially-folded states. At low ionic strength, solutions of urea increase net repulsive interactions compared to GdmHCl solutions through an attenuation of short-range attractive interactions. Electrostatic repulsive interactions are screened in solutions of GdmHCl due to the increased ionic strength of the solution; however short-range attractive interactions are also attenuated in a similar fashion to urea solutions. Protein-protein interactions in low and high concentration denaturant solutions have been shown to be highly sensitive to ionic strength and refolding experiments have shown that this correlates with increased aggregation during refolding. The solubilising additive Arg HCl has been shown to reduce short-range attraction between proteins in urea solutions, while the folding-promotor additives sucrose and hexylene glycol have been shown to have a more complex effect on protein-protein interactions in urea solutions dependent on denaturant concentration. Within the wider context of the field of protein aggregation and refolding, the work conducted here will contribute towards the understanding of how denaturants and solutes influence attractive protein-protein interactions and aggregation behaviour between unfolded or partially folded proteins.

Please read the abstract in the pdf. / Thesis (PhD)--University of Pretoria, 2013. / gm2013 / Food Science / unrestricted

<div lang="en" class="abs"> Abstract The increasing awareness of the causes and consequences of climate chance has led to actions to reduce the dependency on oil and other finite energy and raw material sources. Plant biomass is used in increasing amounts as a resource for biofuel, biochemical and fiber production. Carbohydrate enzymology has provided new ways to utilize and modify renewable carbon sources, especially the lignocellulolytic systems of fungi. Cellulolytic enzymes work in a synergistic manner on recalcitrant structure of cellulose, hydrolyzing it into soluble oligosaccharides, and eventually, glucose. Lytic polysaccharide monooxygenases (LPMOs) contribute to this system by oxidizing either C1- or C4-carbon from the carbohydrate chain on a crystalline cellulose with the help of copper-core induced radicals, thus creating available substrates for the other cellulolytic enzymes. Since their discovery in 2010, the research on their activities and specificities have increased rapidly, but the analytical methods to investigate this diverse group of enzymes is mostly limited to short and soluble products, which are only a fraction of the oxidation products. In addition, most of the methods require special equipment, wide range of standards and expertise to interpret the results. In this study, HPLC and HPAEC-PAD were tested, unsuccessfully, to quantify soluble products from LPMO-catalysis. A TTC-method, in which 2,3,5-triphenyl-2H-tetrazolium chloride is reduced into red and spectrophotometrically quantifiable formazan by reducing ends from insoluble LPMO-products, was successfully optimized and downscaled, and used to optimize reaction conditions for a type 3 LPMO from Trichoderma reesei, TrAA9A, with Whatman filter paper 1 as a substrate. Experiments were conducted to investigate the effects of pH, temperature, donor, time and the presence/absence of H₂O₂ to the accumulation of reducing ends. The results did not show any substantial differences in the accumulation of aldehydes in different reaction conditions. This study showed that cellulose degrades in the presence of TrAA9A and an electron donor. The greatest effects were observed with longer reaction times and the addition of H₂O₂, both increasing the amount of measured aldehydes in the insoluble products. The highest yield was recorded from the reactions with gallic acid as a donor at pH 6, and in the presence of 0.7 mM H₂O₂. The results from this study could lead to understanding the rate-limiting factors of the LPMOs and further improve the utilization of this enzyme in the degradation of lignocellulosic biomass.

Copper-containing nitrite reductase (NiR) is a homotrimer of two cupredoxin domains and catalyzes the single electron reduction of NO2- to NO during dissimilatory denitrification. To investigate the evolution of NiR, methods of mutagenic library generation and high-throughput variant screening from E. coli colonies were developed. These methods allow for facile screening of 105 mutants for folding efficiency or substrate specificity. Initial proof of principle studies yielded several variants that oxidized the artificial substrate ο-dianisidine up to 8 times faster than wild type NiR, suggesting that this methodology has the potential to engineer NiR to acquire other reductase functions. A crystal structure was solved for a putative multicopper oxidase (MCO) and NiR homologue from Arthrobacter sp. (AMMCO) to 1.8 Å resolution. The overall folds of AMMCO and NiR are very similar (r.m.s.d. of 2.0 Å over 250 Cα atoms); Like NiR, AMMCO is a trimer with type-1 Cu sites in the N-terminal domain of each monomer; however, the active site of AMMCO contains trinuclear Cu site characteristic of MCOs instead of a the mononuclear type-2 Cu site found in NiR. Detailed structural analysis supports the theory that two-domain MCOs similar to AMMCO were intermediaries in the evolution of NiR and the more common three-domain MCOs. The physiological function of AMMCO remains uncertain, but genomic, crystallographic and functional analysis suggests that the enzyme is involved in metal regulation. Considering the extensive similarity between AMMCO and NiR, particularly at the active site, engineering a trinuclear cluster into NiR appears feasible with a modest number of alterations to the polypeptide chain. With the aid of my newly developed high-throughput screening technique and site-directed mutagenesis, the mononuclear NiR active site was remodelled into a trinuclear Cu site similar to that of MCO. A crystal structure of this variant was solved to 2.0 Å and the presence of three copper atoms at the engineered cluster was confirmed by Cu-edge anomalous diffraction data. Although the trinuclear copper cluster is present and catalyzes the reduction of oxygen, achieving rates of catalysis seen in native MCOs has proven more difficult. With the framework provided, further engineering NiR into a robust MCO is likely to provide further insights into the structural basis of oxygen reduction by trinuclear copper sites. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Protein synthesis (mRNA) is tightly regulated under numerous conditions in cardiomyocytes. It can be activated by hormones such as insulin and also by other agents such as phenylephrine (PE) that activates hypertrophy in the heart. Cardiac hypertrophy involves an increase in the muscle mass of the heart, principally in the left ventricular muscle, and the increase is due to enlarged cell size, not increased cell number. A pivotal element of cardiac hypertrophy is an elevation in the rates of protein synthesis, which drives the increase in cell size causing hypertrophy. Unfortunately, we currently lack the understanding of the basic mechanisms that drives hyperactivated protein synthesis. Cardiac hypertrophy is clinically important because it is a major risk factor for heart failure. It initially serves as an adaptive response to increase cardiac output in response to higher demand, but ultimately leads to deterioration of contractility of the heart if hypertrophy is sustained. The main goal of this research project is to understand how hypertrophic agents, such as phenylephrine (PE), activate protein synthesis using adult rat ventricular cardiomyocytes as a model. Specifically, this study focuses on how the translational initiation is controlled by upstream signalling pathways. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate