A chitinolytic bacterium of the genus Pseudomonas, isolated from a pond, was grown in liquid culture. Culture filtrates were subjected to purification procedures. The extracellular material was found to contain chitinase and chitobiase activity. The chitinase activity was isolated from contaminating proteins by ammonium sulfate fractionation, affinity chromatography, and molecular weight exclusion chromatography. Overall purification was found to be three-fold, with a recovery of fourteen percent of the `original activity. Polyacrylamide gel electrophoresis showed the presence of two proteins, both exhibiting chitinase activity. / Department of Biology

The main objective of this work was to develop a method whereby chitosan could be modified to give synthetic analogues of branched polysaccharides. To this end, a variety of allyl glycosides were prepared (99-102, 105, 108,109 and 111) and reductively ozonolyzed, to give the acetaldehydo glycosides 112-119. These aldehydes were then reductively alkylated to chitosan (1), to give branched chitosan derivatives (120-127) of the general structure 157. Pendant residues of α and β-glucopyranose, α and β-D-galactopyranose, 2-acetamido-2-deoxy-D-α and β-glucopyranose, glucopyranuronic acid and β-D-lactose were incorporated by this method, at various levels of substitution. Rheological evaluations of these derivatives by steady-shear viscometry demonstrated a relationship between the degree of substitution and rheological properties, as well as the effect of branch size and functionality on aqueous solution properties. Importantly, many of the trends seen in this study are similar to established explanations for the aqueous solution properties of seed galactomannans. It was also shown that intrinsic viscosities of the derivatives were supportive of observations based on concentrated solution properties. Also, it was demonstrated that these water soluble chitosan derivatives interacted, sometimes in a synergistic manner, with xanthan gum solutions. A similar route, involving the synthesis of 10-undece- nyl-β-D-glycopyranosides (134-136), reductive ozonolysis and reductive amination to chitosan, provided combined hydrophobic/hydrophilic branched chitosan derivatives (140-142) of the general structure 158. This methodology was demonstrated with the 10-undecenyl β-D-glycosides of glucopyranose, gal-actopyranose and lactose. Compounds 140a and 141a. bearing glucose and galactose pendant residues, showed uncommon thermally induced gelation properties in dilute aqueous acid solution. This property was studied by ¹H-nmr relaxation measurements and ¹³C-nmr spectroscopy. It was found that a high degree of substitution was necessary for gel formation, and that the pendant sugar was required, but excess hydrophilicity (such as the disaccharide branch, lactose) precluded gelation. In addition, a derivative (151) was prepared, which contained a metal-chelating moiety and a hydrophilic spacer group. This compound had substantial copper (II) binding capacity, and useful ion-exchange ability. Finally, a chitosan derivative (156) was synthesized, bearing a pendant 1-thio-β-D-glucopyranose moiety, and was shown to be useful for the affinity chromatographic purification of the enzyme β-glucosidase. [Formula Omitted] / Science, Faculty of / Chemistry, Department of / Graduate

No description available.

The main aim of this project was to determine the potential of increased chitin content as a mechanism of resistance to caspofungin in different fungal pathogens. <i>C. albicans</i> wild-type cells were pre-grown with a combination of CaCl₂ and CFW prior to caspofungin treatment. This result sin a three-fold increase in cell wall chitin. Wild-type cells, which had elevated chitin content, were less susceptible to caspofungin. Priming cells to activated chitin synthesis was also able to compensate for the loss of the normally essential <i>CaCHS1</i>, through formation of three novel forms of salvage septa. In the absence of both <i>Ca</i>Chs1 and <i>Ca</i>Chs3, which are typically involved in septum formation, the class I chitin synthases, <i>Ca</i>Chs2 and <i>Ca</i>Chs8, could be stimulated to synthesise a proximally offset salvage septum. When <i>Ca</i>Chs3 was the only remaining chitin synthase, treatment with CaCl₂ and CFW, led to the formation of thick chitin-rich salvage septa. <i>Ca</i>Chs2<i> </i>and <i>Ca</i>Chs3 could be stimulated by treatment with CaCl₂ and CFW to synthesise a thin salvage septum similar to the septum of wild-type cells. All three salvage septa were capable of restoring viability and cell division in <i>C. albicans.</i> The compensatory increase in chitin content in response to caspofungin treatment was not specific to <i>C. albicans</i> because clinical isolates of <i>C. tropicalis, C. parapsilosis </i>and <i>C. guilliermondii</i> and the filamentous fungus, <i>A. fumigatus</i>, also demonstrated an increase in chitin content after treatment with caspofungin. Isolates of <i>C. glabrata</i> and <i>C. krusei</i> showed no change in chitin content when exposed to caspofungin. The results of this thesis highlight the potential for using chitin synthase inhibitors in combination therapy with the echinocandins.

The occurrence of chitinolytic microbes and the colonisation and degradation of a native chitin substrate (squid pen) was studied in riverine, estuarine and marine aquatic environments m the Aberdeen area. Chitinolytic microbes were prevalent at all the sites (means: aerobic water 616ml-1, 2% of heterotrophs; anaerobic sediment 11560ml-1 0.8% of anaerobic heterotrophs). It was proposed that the chitinolytic numbers were directly influenced by the presence of chitinous material and indirectly influenced due to a heterotrophic response by organic/suspended matter levels. Hence chitinolytic numbers in the river and were largely influenced by allochthonous inputs while numbers in the sea were influenced by autochthonous production of organic and chitinous matter. From the results of a chitin assay of seawater it was extrapolated that 6.65x1013 metric tons of particulate chitin exist in the world's oceans. Chitin was found to degrade in all the sites studied. The annual rate ranged between 0.905% (river) to 0.074% (marine) squid pen day-1(approx 50mg seeded). The maximum rate recorded was 1.39% day at the river. At most of the sites the rate was positively correlated (P 0.05) with temperature. It was proposed that the marine annual rate would not cope with the annual chitin production and that degradation in the sea is mediated by the synergistic action of the chitinolytic microflora and fauna. The colonisation studies indicated that chitin was rapidly colonised at all the sites studied. The microbial numbers and biomass increased up to about 14 days after exposure, then levelled off and remained relatively constant for the remainder of each exposure period. It was proposed that the numbers of chitinolytic microbial colonisers remained relatively constant at each site studied throughout the year (i.e. changes in the degradation rate were due to variation in activity and not numbers), but the numbers and biomass of chitinolytic microbial colonisers was different between sites and this accounted for some of the variation in degradation rate recorded between sites. Samples with dense colonising biofilms and extensive filamentous growth were also characteristic of sites of relatively high chitin degradation.

Chitin biosynthesis in the fungal cell wall is essential for cell viability. Chitin play roles in maintaining cellular integrity and up regulation of chitin synthesis helps protect the cell from cell wall stresses or cell wall damaging antifungal agents such as caspofungin. In Candida albicans chitin is synthesised by four chitin synthases (Chs), Chs1, Chs2, Chs3, and Chs8. The biological function of Chs2 and Chs8 (class I chitin synthases) is less well understood. The major objective of this thesis was to understand the biological function of the class I chitin synthases. Chs2-YFP and Chs8-YFP showed a dynamic localisation at septation sites, which were first visualised as a bar which then contracted to a spot. This spot then separated into two spots, one on each sides of the septum. These two spots remained there until yeast cell separation, and remained at this location throughout several subsequent hyphal cell cycles. Chs2-YFP also localised to hyphal tips. The phenotype of a chs2Δchs8Δ double mutant was re-investigated using the propidium iodide. Intact dead germ tubes and hyphal tip lysis was observed in a chs2Δchs8Δ mutant cells. This suggested that Chs2 and Chs8 play a major role in the maintenance of hyphal tip integrity and polarised growth and perhaps a minor role in septum formation. Studies were also performed to assess whether phosphorylation regulated the localisation of Chs2-YFP. It was shown that the localisation of Chs2-YFP to septation sites was regulated by phosphorylation on S222. A version of Chs2-YFP that could not be phosphorylated (Chs2S222A-YFP) localised at the septa in lower amounts than the Chs2-YFP, and a version of Chs2-YFP that mimicked constitutive phosphorylation (Chs2S222E-YFP) localised normally. This suggested that phosphorylation of Chs2 on S222 facilitates the localisation of Chs2-YFP at septation sites, and that dephosphorylation is not required for this cellular localisation. In the presence of cell wall stresses (CaCl2/CFW) and caspofungin, more Chs2-YFP was observed and the average intensity of fluorescence of Chs2-YFP was higher in the presence of these stresses than in untreated cells.

Biobased polymers are a critical research topic as they may serve as replacement to traditional unsustainable petro-chemical polymers. It is vital to widen its range of applications by improving its physical and mechanical properties via light weighting and strength improvements. Light weighting can be accomplished by introducing foam morphology to the material while mechanical strength improvements can be achieved by inserting stiff filler material to the base polymer to form a composite. This study explores the physical, mechanical, thermal, rheological and morphological properties of blends, foams and composites between biobased PLA and PHBV matrix polymers and biobased chitin nanowhisker filler. It was found that foams produced from PLA and PHBV blends exhibits refined cellular morphology which leads to light weighting and good strength preservation while chitin nanowhiskers was determined to be a very effective filler for mechanical property improvements in both solid and porous materials.

The enzyme chitin deacetylase was extracted from the fungus Mucor rouxii. The fungus was grown aerobically at pH 4.5, and a temperature of 28 +/- 2°C, using a shaker speed of 100 rpm. The highest enzymatic activity was obtained at 36 h of Mucor rouxii's growth. The protein content of Mucor rouxii was a function of incubation time and it increased in the course of the fungal growth up to 3 days. / The enzyme was partial purified by ammonium sulfate fractionation, followed by ion exchange chromatography using a Fast Protein Liquid Chromatography (FPLC) System. The specific activity in the 60--85% ammonium sulfate fraction showed higher specific activity (51.44 U/mg) and a 2.2-fold purification as compared with that of the crude extract (24.27 U/mg). The fraction after ion exchange chromatography step showed a specific activity of 233.65 U/mg, representing about 7.2-fold purification compared to the crude extract. This ion exchange fraction showed protein bands with molecular weights ranging from 45 to 75 kDa on SDS-PAGE (12%). / The enzyme showed optimal activity at a temperature of 50°C, and more than 90% of its activity was retained after 30 min incubation at this temperature. The optimal pH activity of chitin deacetylase from Mucor rouxii ranged from 5.5--6. The enzyme retained more than 80% of its activity in the pH range of 4.5--8.5 after 1 h incubation at room temperature, suggesting that this enzyme is quite stable in the slightly acidic to alkaline pH range. However, a lower pH (<4) and a higher pH (>9) caused the enzyme to lose more than 60% of its activity. / It was shown that the chitin deacetylase did not require the following metal ions (Co+2 and Mn+2) for activity. However, Mn+2 slightly increased the enzyme activity. The activity of the enzyme was also inhibited by EDTA.

The optimization of isolation and characterization of chitin deacetylase (CDa) from Mucor rouxii was the focus of this study. The crude extract in the active form was partially purified by ammonium sulfate fractionation, followed by column chromatography by ion exchange in the Fast Protein Liquid Chromatography (FPLC) system. / Using a 10 mM concentration, the order of effectiveness for the inhibitors in achieving 35--40% was CaCl2, CuSO4, MgCl 2 and EDTA. At a 25 mM concentration, propionic acid and sodium acetate inhibited the enzyme up to 40% & 50%, respectively. M. rouxii CDa activity was greater in the mycelial extract, and was capable of efficiently hydrolyzing the substrate glycol chitin. / Two major bands were observed after native PAGE of the partially purified enzyme using Mono Q column (FPLC). The estimated molecular weight of CDa bands by SDS-PAGE containing 0.1% glycol chitin was 21, 23, 26 and 44 kDa when compared to the migration of molecular weight markers. (Abstract shortened by UMI.)

Biobased polymers are a critical research topic as they may serve as replacement to traditional unsustainable petro-chemical polymers. It is vital to widen its range of applications by improving its physical and mechanical properties via light weighting and strength improvements. Light weighting can be accomplished by introducing foam morphology to the material while mechanical strength improvements can be achieved by inserting stiff filler material to the base polymer to form a composite. This study explores the physical, mechanical, thermal, rheological and morphological properties of blends, foams and composites between biobased PLA and PHBV matrix polymers and biobased chitin nanowhisker filler. It was found that foams produced from PLA and PHBV blends exhibits refined cellular morphology which leads to light weighting and good strength preservation while chitin nanowhiskers was determined to be a very effective filler for mechanical property improvements in both solid and porous materials.