Dissertation (PhD)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: The production, purification and functional characterisation of feruloyl esterase from Aureobasidium pullulans were set as the primary objectives of this study. A further objective was to investigate a possible co-operative effect with other selected lignocellulolytic enzymes on substrates relevant to industry. In a comprehensive review, feruloyl esterases from various micro-organisms were compared both functionally and with regard to their primary structure, where applicable. Feruloyl esterases show intriguing differences in substrate specificity and sequence structure. Enzymes that are closely related regarding their amino acid sequence exhibit different substrate specificities. Sequence similarities can be found with a range of other enzyme families, including serine esterases, acetyl xylan esterases, lipases, tannases, glycosyl hydrolases and xylanases. More data on the three dimensional structure of feruloyl esterases as well as an examination of all available feruloyl esterases with the same substrates is necessary before structure-function relationships can be established and before the feruloyl esterases can be organized into discrete families based on ancestral origins. The highest production levels of feruloyl esterase by A. pullulans are achieved when grown on birchwood xylan. Expression was not repressed when glucose or xylose was present in the medium. However, free ferulic acid supplemented to the medium affected fungal growth and therefore did not increase feruloyl esterase activity. It is also suggested that the synthesis of feruloyl esterase is independently regulated from xylanase synthesis. Feruloyl esterase from A. pullulans acts on a- and l3-naphthyl acetate, as well as naphthol AS-D chloroacetate as substrates. Feruloyl esterase from A. pullulans was purified to homogeneity using ultrafiltration with high molecular weight cut-off, anion exchange, hydrophobic interaction and ultimately gel filtration chromatography. With a molecular weight of 210 kDa, the enzyme is the largest of the feruloyl esterases reported to date. Kinetic data was produced using both synthetic and natural substrates. A. pullulans feruloyl esterase shows properties similar to other fungal feruloyl esterases, especially from Aspergillus niger cinnamic acid esterase and Penicillium funiculosum feruloyl esterase B. The N-terminal sequence of A. pullulans feruloyl esterase was identified, but no similarities to known enzyme families were found. Peptide mass mapping did not reveal structural information. In an effort to evaluate the significance of feruloyl esterase from A. pullulans in the degradation of lignocellulose, dissolving pulp and sugar cane bagasse were selectively treated using feruloyl esterase and hemicellulolytic enzymes. The enzymatic degradation reaction was monitored using microdialysis sampling, anion exchange chromatography, online desalting and mass spectrometry. It has been shown, that feruloyl esterase activity together with xylanase activity releases monosaccharides from both substrates. Sugars of higher degree of polymerisation were not released, giving evidence for the recalcitrance of the material. The fibre architecture of the substrates was apparently not accessible to the enzymes and therefore complete hydrolysis was hindered. / AFRIKAANSE OPSOMMING: Die produksie, suiwering en funksionele karakterisering van feruloïel esterase afkomstig van Aureobasidium pullulans was die primêre doelwitte van hierdie studie. 'n Verdere doelwit was om vas te stelof daar 'n kooperatiewe effek met ander geselekteerde lignosellulitiese ensieme op substrate wat industrierelevant is, bestaan. Die feruloïel esterase van verskillende mikro-organismes is vanuit die oogpunt van funksie en primêre struktuur omvattend met mekaar vergelyk, waar toepaslik. Interessante verskille tussen die substraat spesifisiteit en volgordestruktuur van feruloïel esterase kan waargeneem word. Ensieme wat nou aanmekaar verwant is wat hul aminosuurvolgorde betref, het duidelik verskillende substraatspesifiteite. Volgordeverwantskap kan in 'n reeks van ander ensiemfamilies, insluitende serienesterase, asetielxilaanesterase, lipases, tannases, glikosielhidrolases en xilanases vasgestel word. Meer inligting oor die driedimensionele struktuur van feruloïel esterase asook 'n analise van al die beskikbare feruloïel esterase met dieselfde substrate is nodig voordat struktuur-funksie verwantskappe vasgestel kan word en voordat die feruloïel esterases in eie families op die grond van huloorsprong georganiseer kan word. Die hoogste produksie vlakke deur feruloïel esterase van A. pullulans word bekom deur dit op berkhoutxilaan te groei. Ekspressie was nie onderdruk wanneer glukose of xilose in die medium aanwesig was nie. Wanneer vrye feruliensuur by die medium bygevoeg is, is die fungale groei beïnloed en het die feruloïel esterase aktiwiteit nie vermeerder nie. Dit word ook voorgestel dat die sintese van feruloïel esterase onafhanklik deur xilanase sintese gereguleer word. Feruloïel esterase van A. pullulans reageer op a- en f3- naftolasetaat, asook naftol AS-D chloroasetaat as substrate. Feruloïel esterase van A. pullulans is tot homogeniteit deur ultrafiltrering met .n hoë molekulêre gewiggrens, anioonuitruiling, hidrofobiese interaksie en eindelik gelfiltrasie-chromatografie gesuiwer. Met 'n molekulêre gewig van 210 kDa, is die ensiem die grootste van die feruloïel esterases tot dusver beskryf. Kinetiese data is met behulp van sintetiese en natuurlike substrate geproduseer. A. pullulans feruloïel esterase het eienskappe wat vergelykbaar is aan die van ander fungal feruloïel esterases, veral die wat afkomstig is van Aspergillus niger sinnamiensuur esterase en Penicillium funiculosum feruloïel esterase B. Die N-terminale volgorde van A. pullulans feruloïel esterase is identifiseer maar geen ooreenkoms aan bekende ensiemfamilies kon vasgestel word nie. Peptiedmassakaartering kon ook geen strukturele inligting gee nie. Oplosbare pulp en suikerrietbagasse is geselekteerd met behulp van feruloïel esterase en lignosellulitiese ensieme behandel om die belang van feruloïel esterase van A. pullulans in die afbraak van lignosellulose vas te stel. Die hidroliese-reaksie is deur mikrodialise monsterneming, anioonuitruilingschromatografie, oplyn ontsouting en massaspektrometrie gemonitor. Wanneer die aktiwiteit van feruloïel esterase met die van xilanase gekombineer is, is monosakkariede deur albei substrate afgeskei. Suikers met 'n hoër graad van polimerisering is nie afgeskei nie, wat 'n bewys van die materiaal se weerstandbiedendheid is. Dit het geblyk asof die vesel-argitektuur van die verbruikte substraat nie toeganklik was vir ensieme nie en dus is algehele hidroliese verhinder.

Polyacrylamide gel electrophoresis was used to study B-esterases (EC 3.1.1.1) and C-esterases (EC 3.1.1.6) in five species of Collembola--; Folsomia candida, Proisotoma vesiculata, H,ypogastrura denticulata, and two undescribed species of Isotoma,. Ef values were computed for zymograms of 105 individuals and compared both within and among species. Certain bands-three to six in number--predominated on the zymograms of each species. The predominating bands differed among the five species resulting in a unique zymogram for each.It was also found that zymograms of normal and ecomorphic forms of Proisotoma vesiculata were dissimilar. Zymograms of specimens of Folsomia candida with access to food and those without food for 48 and 72 hours were similar. Zymograms from mascerated specimens frozen in buffer were not satisfactory.

No description available.

Nonspecific esterase, and in some cases a "Tween" lipase, were investigated histochemically in arteries and several other tissues of rats and rabbits under various conditions known to affect the development of atherosclerosis: age and sex differences, endocrine and metabolic factors (sex steroids, thyroid function, alloxan diabetes, and fasting), arterial injury, and acute and chronic lipemia. A study of the above enzymes was also made in atherosclerotic rabbit and human aortae. Of the several nonvascular tissues studied, only the enzymes of the male gonads and a male accessory sex organ (the prostate) were influenced by some of the experimental conditions tested: maturation greatly increased the activity of both enzymes in the Leydig cells of the testis, and stilbestrol markedly diminished the esterase activity in the prostate epithelium. Thus, in certain reproductive organs, the enzymes reflected the functional state of the cells under investigation. The following findings were made concerning the enzymes of the arterial wall: 1. Neither of the enzymes was influenced by ageing or sex difference in the rat, rabbit or human. 2. Neither of the enzymes was influenced by sex hormones, acute or chronic lipemia, or any of the metabolic conditions studied in the rat. 3. Esterase disappeared from the foci of acute arterial injury induced by Calciferol treatment in the rat and did not reappear six weeks after infliction of the injury. 4. Strong esterase activity appeared in the cytoplasm of lipo-phages within both human and experimental rabbit atherosclerotic lesions, in contrast to the normal arterial wall which exhibited no esterase activity at all in either species with the methods used. 5. Esterase was absent from the superficial fibrous cap of the human atherosclerotic lesion. It is concluded that: In the rat arteries, which normally exhibit appreciable esterolytic activity, this enzyme appears to be quite stable since it is not visibly modified by ageing or a series of endocrine and metabolic influences; it is, however, drastically diminished by acute vascular injury and this may account, at least in part, for the well known preferential accumulation of lipids in foci of acute arterial damage. In the rabbit arteries, which normally exhibit no histochemically demonstrable esterase, appreciable esterolytic activity appears only within the cytoplasm of cells that have taken up lipid after exposure to chromic lipemia (foam cells of atheromata). Similarly, in human atheromata esterase appeared only in foam cells and was absent from the fibrous cap of the atherosclerotic lesions; thus the absence of lipid from the cap cannot be attributed to increased enzyme activity in this part of the lesion. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

From its introduction in 1952 onwards, the clinical use of macrolides has been steadily increasing, both in human and veterinary medicine. Although initially designed to the treatment of Grampositive microorganisms, this antimicrobial family has also been used to treat specific Gram-negative bacteria. Some of them, as azithromycin, are considered in the armamentarium against Enterobacteriaceae infections. However, the facility that this bacterial genus has to gain or develop mechanisms of antibiotic resistance may compromise the future usefulness of these antibiotics to fight against Enterobacteriaceae infections. The present review is focused on the mechanisms of macrolide resistance, currently described in Enterobacteriaceae.

Carboxyl esterases, particularly arylesterases, were predicted from 16 microbial genomes, and then expressed in E. coli. Of the more than 175 cloned genes, 86 were expressed in soluble form. These were screened for activity using a range of both commercial and natural substrates. Forty-eight proteins were active on pNP-acetate at pH 8 whereas 38 proteins did not exhibit any activity towards any substrates. Among the 48 active proteins, 20 proteins showed arylesterase activity. To date, 8 bacterial esterases and 2 archaeal arylesterases were characterized in terms of pH stability and optima, thermal inactivation, solvent stability, and kinetics. To our knowledge there is only one other published report of arylesterases from archaea. The synthetic capability of arylesterases can transform phenolic acids to value-added chemicals. Accordingly, this project provides an arsenal of industrially significant activities that can extend the antioxidant properties of lignin-derived molecules in a broader range of renewable products.

Carboxyl esterases, particularly arylesterases, were predicted from 16 microbial genomes, and then expressed in E. coli. Of the more than 175 cloned genes, 86 were expressed in soluble form. These were screened for activity using a range of both commercial and natural substrates. Forty-eight proteins were active on pNP-acetate at pH 8 whereas 38 proteins did not exhibit any activity towards any substrates. Among the 48 active proteins, 20 proteins showed arylesterase activity. To date, 8 bacterial esterases and 2 archaeal arylesterases were characterized in terms of pH stability and optima, thermal inactivation, solvent stability, and kinetics. To our knowledge there is only one other published report of arylesterases from archaea. The synthetic capability of arylesterases can transform phenolic acids to value-added chemicals. Accordingly, this project provides an arsenal of industrially significant activities that can extend the antioxidant properties of lignin-derived molecules in a broader range of renewable products.

Thesis (Ph. D.)--Ohio State University, 2004. / Title from first page of PDF file. Document formatted into pages; contains xiii, 196 p.; also includes graphics (some col). Includes abstract and vita. Advisor: Robert W. Bruegemeier, Dept. of Pharmacy. Includes bibliographical references (p. 176-196).

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 83-102).

Thesis (Ph. D.)--Catholic University of America, 1941. / Reproduced from type-written copy. Description based on print version record. Bibliography: p. 76-81.