The bacteriophage lambda tail protein gpK is required for tail assembly. The activity of the protein can be found at the assembling tail tip and is believed to be localized to this structure. GpK is a 27 kDa protein that has sequence identity to two families of proteins: the Mov34 family of peptidases and the NlpC/P60 family of peptidoglycan endopeptidases. Point substitutions and complementation data confirm that gpK possesses each of these domains and that they can function in trans. When the Mov34 domain is inactivated tail assembly is disrupted whereas when the NlpC/P60 domain is inactivated tails assemble but are inactive. Evidence is presented here that the C-terminal domain possesses lytic activity in isolation but not when part of the full-length protein.

Hemicellulases are key components in the degradation of plant biomass and carbon flow in nature. Thermotoga hypogea is a bacterium that can grow anaerobically at 90°C. It utilizes carbohydrates and peptides as energy and carbon sources. Three hemicellulytic enzymes: β-xylosidase, α-L-arabinofuranosidase and acetylesterase were investigated. Xylan and xylose were the best substrates for the growth as well as for yielding high activity for all three enzymes in the cells. Glucose grown cells possessed the least amount of enzyme activity for all three enzymes. More than 87% ± 3.0 of β-xylosidase and α-L-arabinofuranosidase activities and 34% ± 11 of acetylesterase activity were associated with the cells. Arabinofuranosidase and acetylesterase were partially purified but β-xylosidase was purified to homogeneity using the Fast Performance Liquid Chromatography system. The latter enzyme has an apparent molecular mass of 75 kDa demonstrated through sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a nondenatured weight of 130 kDa estimated by Gel-filtration. Its optimal temperature and pH-value for activity were 70°C and 6.0, respectively. The purified enzyme had a half life of 22 min at 70°C and pH 6.0. Among all tested substrates, the purified enzyme had specific activities of 44, 32, 4.5, 1.71 U/mg on p-nitrophenyl-β-xylopyranoside (pNβxp), 4-nitrophenyl-β-D-glucopyranoside (pNβgp), 4-nitrophenyl-α-L-arabinofuranoside (pNαLaf) and 4-nitrophenyl-α-D-xylopyranoside (pNαxp) respectively. The apparent Km of the xylosidase with pNβxp, was 2.6 mM and Vmax was 196 U/mg and for pNβgp the Km and Vmax values were 0.31 mM and 24 U/mg respectively. Based on N-terminal analysis, xylosidase showed high homology with Family 3 β-glucosidases.

Hemicellulases are key components in the degradation of plant biomass and carbon flow in nature. Thermotoga hypogea is a bacterium that can grow anaerobically at 90°C. It utilizes carbohydrates and peptides as energy and carbon sources. Three hemicellulytic enzymes: β-xylosidase, α-L-arabinofuranosidase and acetylesterase were investigated. Xylan and xylose were the best substrates for the growth as well as for yielding high activity for all three enzymes in the cells. Glucose grown cells possessed the least amount of enzyme activity for all three enzymes. More than 87% ± 3.0 of β-xylosidase and α-L-arabinofuranosidase activities and 34% ± 11 of acetylesterase activity were associated with the cells. Arabinofuranosidase and acetylesterase were partially purified but β-xylosidase was purified to homogeneity using the Fast Performance Liquid Chromatography system. The latter enzyme has an apparent molecular mass of 75 kDa demonstrated through sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a nondenatured weight of 130 kDa estimated by Gel-filtration. Its optimal temperature and pH-value for activity were 70°C and 6.0, respectively. The purified enzyme had a half life of 22 min at 70°C and pH 6.0. Among all tested substrates, the purified enzyme had specific activities of 44, 32, 4.5, 1.71 U/mg on p-nitrophenyl-β-xylopyranoside (pNβxp), 4-nitrophenyl-β-D-glucopyranoside (pNβgp), 4-nitrophenyl-α-L-arabinofuranoside (pNαLaf) and 4-nitrophenyl-α-D-xylopyranoside (pNαxp) respectively. The apparent Km of the xylosidase with pNβxp, was 2.6 mM and Vmax was 196 U/mg and for pNβgp the Km and Vmax values were 0.31 mM and 24 U/mg respectively. Based on N-terminal analysis, xylosidase showed high homology with Family 3 β-glucosidases.

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Natural nitro compounds with diverse structures and biological functions are produced by bacteria, fungi, plants and animals. Little is known about the behavior of such compounds in natural ecosystems. The lack of accumulation in the biosphere implies that they are biodegraded. Microbial strategies for biodegradation of synthetic nitro compounds are well established; however only two pathways are known for degradation of natural nitro compounds. The research described here examines the genes that encode the key enzymes required for biodegradation of 3-nitrotyrosine (3NTyr) and 1-nitro-2-phenylethane (NPE). 3NTyr is a biological marker for disease and inflammation in plants and animals. A 3NTyr degrading microbe, Variovorax sp. JS669 was isolated from soil. We identified the JS669 denA, which encodes an enzyme that catalyzes denitration of 4-hydroxy-3-nitro-phenylacetate, the key step in metabolism of 3NTyr. The isolation of 3NTyr degraders and development of molecular probes specific to denA revealed that 3NTyr degradation is a widespread phenomena in natural habitats and the compound is metabolized by phylogenetically diverse bacteria. Phylogenetic analysis of the 4-hydroxy-3-nitro-phenylacetate denitrase from JS669 revealed it to be the first functionally annotated protein in a clade of unidentified Class A flavin monooxygenases. NPE has been identified from several plants, yet the biodegradation of the compound remained a mystery. Here we report the degradation of NPE and its analog 2-nitropropylbenzene. Discovery of the metabolic pathway revealed a novel microbial strategy to use a meta-ring fission degradation pathway to cleave an undesirable side chain from an aromatic compound and use the remainder of the compound as a carbon and energy source. Two genes that encode enzymes in the biodegradation pathway were identified and both are deeply branched within their respective phylogenetic trees, indicating that both represent highly specialized microbial enzymes. Furthermore, microbial degradation of NPE resulted in the production of 3-nitropropionic acid, a natural toxin that inhibits succinate dehydrogenase and is responsible for livestock illness and death. This is the first report of bacterial production of 3-nitropropionic acid, and might represent a significant source of 3-nitropropionic acid in natural habitats. The findings from these studies contribute to the overall understanding of microbial metabolism. Specifically, this research reveals genes that encode novel enzymes and strategies for the biodegradation of two natural nitro compounds. Furthermore, discovery of mechanisms for the biodegradation of such compounds reveals novel microbial metabolic diversity and provides insight into the evolution of degradation pathways for synthetic compounds.

Le but de mon projet de doctorat est la recherche de chitosanases thermostables qui peuvent mener la réaction d'hydrolyse du chitosane à de hautes températures. La procédure mise au point pour isoler ces chitosanases était planifiée pour moduler l'effet antimicrobien du chitosane qui augmente avec son poids moléculaire. Les objectifs spécifiques de ce projet sont, mettre au point un nouveau dosage de Csn, purifier, caractériser et cloner le gène des chitosanases les plus thermostables sélectionnées et mettre au point un milieu de production de chitosanase. La première étape du projet est la recherche de nouvelles chitosanases thermostables, via un criblage ciblé de bactéries productrices de chitosanases. En effet, une nouvelle méthode d'enrichissement était utilisée par l'ajout de chitosane de différents poids moléculaires à notre source bactérienne, soit les composts. La deuxième étape, est la réalisation d'un dosage de l'activité chitosanase en utilisant le soluble-dyed Remazol Brillant Bleu-Chitosane (sRBB-C) qui a été mise au point pour détecter à grande échelle une activité chitosanase de manière facile et rapide. Enfin, la troisième étape est un test de thermostabilité en présence de substrat, appliqué à des chitosanases choisies, pour sélectionner les plus performantes à l'étape de la purification. Parmi le lot de chitosanases testées, la chitosanase notée Csn1794 s'est distinguée par sa thermostabilité à 70 degrés C, ainsi elle a été retenue pour des études plus approfondies. Les études biochimiques réalisées sur la Csn1794 après purification ont révélé qu'elle a un poids moléculaire de 40 kDa, un pH optimal de 4.8 et des K[indice inférieur m] et k[indices inférieurs cat] de 0.042 mg/ml et 7588 min[indices supérieurs -1] respectivement. Le temps de demi-vie de la Csn1794 en présence de chitosane est plus de 20 heures à 70 degrés C. L'activité de la Csn1794 varie légèrement avec le degré d'acétylation du chitosane, elle hydrolyse la carboxyméthyl-cellulose, mais pas la chitine. Le clonage du gène de la Csn1794 par génétique inverse a permis de déterminer sa séquence. Ce gène codé pour une protéine de 441 acides aminés. La Csn1794 appartient à la famille 8 des glycosides hydrolases (GH8). Le rang taxonomique de l'isolat produisant la Csn1794 a été déterminé par des méthodes classiques ainsi que par des tests de biologie moléculaire. Les résultats obtenus indiquent qu'il s'agit d'un isolat appartenant à une espèce non caractérisée appartenant au genre Paenibacillus qu'on a appelé Paenibacillus sp. 1794. Enfin, la méthode de plan d'expériences était utilisée pour mettre au point le milieu de production de la Csn1794. Les essais réalisés par les plans d'expériences Plackett-Burman ont permis non seulement de définir un milieu de base pour la production de la Csn1794, mais aussi les oligosaccharides et le sucrose se sont distingués comme facteurs à effet nettement positif sur la production de la Csn1794. Les essais par plans d'expérience Box-Hunter ont permis l'étude d'interactions entre les différents facteurs dont le niveau était déterminé par les plans Taguchi. Les résultats obtenus indiquent qu'en plus de milieu de base, l'ajout de 10g/l de glucosamine, 7g/l d'oligosaccharide et 4g/l de sucrose constitue la meilleure combinaison pour un milieu qui permet de produire une moyenne de 7U/ml de Csn1794 d'une manière constante. En conclusion, nous disposons d'une nouvelle chitosanase thermostable, facile à produire et à purifier, qui sera un outil adéquat pour l'application au niveau industriel. Ceci va non seulement permettre de mener le processus d'hydrolyse de chitosane à haute température, mais aussi d'utiliser de grandes concentrations de substrat sans que la viscosité ne devienne excessive. Au niveau de la recherche fondamentale, la Csn1794 peut nous apporter plus d'informations d'une part, sur la thermostabilité des enzymes et d'autre part, sur les enzymes de la famille GH8, notamment les chitosanases.

Ready-to-eat meat products such as dry-fermented sausages have been associated with foodborne outbreaks despite the multiple hurdles used in the manufacturing process to prevent growth of pathogens. As a result, new strategies such as natural products with antimicrobial activity are being used to control pathogens of importance like Escherichia coli O157:H7. This study investigated how different concentrations and sources of mustard can influence its antimicrobial activity against E. coli O157:H7 in dry-fermented sausage, as well as the contribution of residual myrosinase enzyme in mustard to this process. The genetic basis for the degradation of mustard glucosinolate by E. coli O157:H7, which is associated with the antimicrobial action of mustard, was also characterized. The ability of E. coli O157:H7 to withstand inhibitory allyl isothiocyanate (AITC) concentrations and the role of the two-component BaeSR system as a defense mechanism against AITC was also investigated. Results showed that 4% (w/w) deodorized yellow mustard powder was effective to control E. coli O157:H7 in dry-fermented sausage at 28 d. The presence of endogenous plant myrosinase in the mustard powder or meal enhanced E. coli O157:H7 reduction rates. Fully-deodorized, deoiled, yellow mustard meal as low as 2% (w/w) containing either 0.1% or 0.2% of residual plant myrosinase achieved the same results as 4% (w/w) mustard powder also containing similar residual myrosinase. Regardless of the type of mustard, the antimicrobial activity of yellow mustard derivatives were more pronounced than those of Oriental mustard. The initial genetic assessment through in silico analysis found similarity between plant myrosinase and enzymes encoded by genes (bglA, ascB, and chbF) from β-glucosidase families in E. coli O157:H7 strains. After disruption of these genes using lambda-red replacement, single (∆bglA, ∆ascB, ∆chbF) and double (∆bglAascB, ∆chbFascB, ∆chbFbglA) mutant strains were created and assessed for glucosinolate degradation. The comparison of the gene expression profiles and changes in the extent of sinigrin degradation by different mutants suggested that ascB have a prominent role in the degradation of this β-glucoside by E. coli O157:H7. E. coli O157:H7 did not develop resistance to AITC, the essential oil formed from sinigrin degradation that is responsible for the antimicrobial activity of Oriental mustard.

Although carbohydrates are a principal component of the human diet, the mechanism of the final stages of starch digestion is not fully understood. One approach to treating metabolic diseases such as type II diabetes, obesity, and congenital sucrase isomaltase deficiency is inhibition of intestinal α-glucosidases and pancreatic α-amylases. Intestinal α-glucosidases, sucrase isomaltase (SI) and maltase glucoamylase (MGAM), are responsible for the final step of starch hydrolysis in mammals: the release of free glucose. MGAM and SI consist of two catalytic subunits: N-terminal and C-terminal, with overlapping, but variant substrate specificities. The objective of this thesis is to increase the understanding of the differential substrate specificity seen in the catalytic subunits of SI and MGAM. Through inhibitor studies, the structural and biochemical differences between the enzymatic subunits are explored, illustrating that each individual catalytic subunit can be selectively inhibited. In Chapter 3, homology models of ctSI and ctMGAM-N20 are presented, giving insight into the residues hypothesized to impact substrate specificity, enhancing our understanding of the functionality of these enzymatic subunits and overlapping substrate specificity. The structural implications of mutations seen in ntSI in CSID patients and the potential functional and structural implications are discussed in Chapter 4 in addition to the prevalence of SNPs in the SI gene in different populations. The mammalian α-glucosidases are compared to the 3 Å structure of CfXyl31, a Family 31 glycoside hydrolase from Cellulomonas fimi. Comparison to Family 31 glycoside hydrolases of known structure gives rise to possible mutations proposed to mimic ntMGAM α-glucosidase activity. Through inhibitor studies, homology models, examining mutations found in disease states such as congenital sucrase isomaltase deficiency, and investigating a bacterial family 31 glycoside hydrolase from Cellulomonas fimi, the active site characteristics and substrate specificities of SI and MGAM are better understood.

Biocatalysis and enzyme technology represent significant research topics of contemporary biotechnology. The immobilisation of these catalysts on or in static supports serves the purpose of transforming the catalyst into a particle that can be handled through effortless mechanical operations, while the entrapment within a membrane or capsule leads to the restraint of the enzyme to a distinct space. This confinement leads to a catalyst with a superior stability, and cell durability under reaction conditions. Epoxide hydrolase is a widely available co-factor independent enzyme, which is known to have remarkable chemio-, regio- and stereoselectivity for a wide range of substrates. Recently it was found that certain yeasts, including Rhodosporidium toruloides, contain this enzyme and are able to enantioselectively catalyse certain hydrolysis reactions. The objective of this project was four-sided: a) to immobilise Rhodospridium toruloides in an optimised immobilisation matrix (calcium alginate beads), for the kinetic resolution of 1.2- epoxyoctane in order to obtain an optically pure epoxide and its corresponding vicinal diol, b) to determine the effect of immobilisation on activity as well as stability of the enzyme and gain better understanding of the parameters that influence enzyme activity in a support, c) to determine the effect of formulation parameters on some of the bead characteristics and, d) to gain some insight in the distribution of epoxide and diol in the water and bead phases and the formulation parameters that have an effect thereon. Rhodospridium toruloides was immobilised in calcium alginate beads consisting of different combinations of alginate and CaCl2 concentrations. Best results were obtained with a combination of 0,5 % (m/v) alginate and 0,2 M CaC12. The immobilised cells exhibited lower initial activity. but more than 40 times the residual activity of that of the free cells after a 12-hour storage period. Both the immobilised and free cells exhibited an increase in reaction rate (V) with an increase in substrate concentration. An increase in the alginate concentration lead to the formation of smaller beads, but a decrease in enzume activity, while an increase in the CaCl2 solution concentration had no effect on bead diameter or enzyme activity. Epoxide diffused preferentially into the beads (± 96 %), and the diol into the water phase, which leads to the natural separation of the epoxide and the diol. The CaCl2 concentration affected epoxide diffusion with no effect on diol diffusion, which opens up the possibility to regulate the diffusion of epoxide into the beads. Although only a very small fraction of the epoxide inside the beads could be extracted, the alginate proved to be chirally selective for the (R)-epoxide, improving the reaction efficiency by increasing the % ee, of the epoxide extracted from the beads between 26 % and 43 %. The possibility to develop a system where the product is formed, purified and concentrated in a one-step reaction by extracting the product from the bead phase was clearly demonstrated. / Thesis (M.Sc. (Pharm.) (Pharmaceutical Chemistry))--Potchefstroom University for Christian Higher Education, 2003.

Fat facets in Mouse (FAM) or mUSP9x is a deubiquitylating enzyme of the USP class. Knockdown of FAM protein levels in mouse pre-implantation embryos by antisense oligonucleotides is known to prevent embryos from progressing to the blastocyst stage indicating an important role for FAM in early mammalian development. In mammals, the Fam gene is located on the X-chromosome. In mice, the Y homologue, Dffry or usp9y, is expressed exclusively in the testes and maps to the Sxrb deletion (Brown et al., 1998). Sxrb is associated with an early post-natal blockage of spermatogonial proliferation and differentiation leading to absence of germ cells (Bishop et al., 1988; Mardon et al., 1989). The human Y homologue of Fam is closely associated with oligozoospermia (Sargent et al., 1999; Sun et al., 1999) and the human X homologue has been linked to the failure of oocytes to pass through the first meitoc prophase in Turner syndrome (Cockwell et al., 1991; Speed, 1986) Despite these associations, the substrates and precise role of Fam and its homologues in these processes have not yet been defined. Due to the complex nature of Fam expression and the lack of data tying FAM to specific cellular functions, much attention has been paid in identifying interacting partners and cellular targets of FAM activity to aid in the definition of its role in the cell and development. Three common molecular biology techniques were applied here in an attempt to further characterise known interactions of FAM, including interactions with the cell adhesion molecule β-catenin and the protein trafficking pathway proteins epsin-1 and itch. The aim of these investigations was to generate FAM mutants that could abolish individual interactions, enabling investigation of individual interactions in cellular function and development. These experiments failed to identify the amino acids of FAM that were critical for its interactions with β-catenin, epsin-1, or itch. Experiments aimed at characterising a novel ubiquitin-like domain located in the N-terminal half of the FAM protein, did however identify novel interactions of FAM with the three Golgi associated adaptor proteins GGA1, GGA2, and GGA3. Further investigations prompted by this interaction, examined the role of FAM in the trafficking of proteins from the Golgi apparatus. Cellular FAM protein levels were altered either by exogenous expression of FAM protein or knockdown of endogenous FAM using FAM specific shRNA triggers. The cellular protein levels and extent of post-translational modification of eleven lysosomal proteins were monitored in each case. It was found that increased FAM protein levels resulted in decreased cellular protein levels of five of the eleven lysosomal proteins studied. In contrast, a reduction in FAM protein levels was found to result in an increase in the cellular protein levels of eight of the eleven lysosomal proteins. This study provides the first evidence of a deubiquitylating enzyme that is able to interact with the GGA proteins. It is also the first to describe a deubiquitylating enzyme that can affect the biosynthesis of lysosomal proteins and provides valuable new insight into the cellular function of FAM/USP9X. / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Sciences, 2008