Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Dissertation (PhD)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: The moderately thermophilic (45 to 50DC), highly acidophilic (pH 1.5 to 2.5), chemolithoautotrophic Acidithiobaci/lus caldus strain "f' was isolated from a biooxidation process used to treat nickel ore concentrates. Trans-Alternating Field Electrophoresis (TAFE) analysis of total DNA from the At. caldus cells revealed two plasmids of approximately 14 and 45-kb. The 14-kb plasmid, designated pTC-F14, was cloned and shown by replacement of the cloning vector with a kanamycin resistance gene to be capable of autonomous replication in Escherichia coli. Autonomous replication was also demonstrated in Pseudomonas putida and Agrobacterium tumefaciens LBA 4404 which suggested that pTC-F14 was a broad host-range plasmid. Sequence analysis of the pTC-F14 replicon region revealed five open-reading frames, and a replicon organization like that of the broad host-range IncQ plasmids. Three of the open-reading frames encoded replication proteins with amino acid sequence identities similar to that of the IncQ-like plasmid pTF-FC2 (RepA, 81%; RepB, 78%; RepC, 74%). This high level of relatedness suggested that the two replicons had evolved from a common ancestor. Since closely related replicons are usually incompatible, the compatible replicons of pTC-F14 and pTFFC2 raised the question of how the replicons of the two sister plasmids had evolved such that they can now co-exist in the same host cell line. Further incompatibility testing with the IncQ-like plasmid pIEll08 and the IncQ prototype plasmid RSF10101R11621R300B determined that pTC-F14 was compatible with pIEI108, but incompatible with the IncQ prototype plasmid. It was found that the RepB and RepA replication proteins ofpTF-FC2 and pIEll08 were able to complement the pTC-FI4 orthologs only ifpTC-F14 RepC was present in trans. The RepC protein ofpTC-F14 was thus plasmid-template specific, while the RepA and RepB proteins were less plasmid-template specific. A five nucleotide possible iteron-discriminating region in the direct repeats of IncQ-like plasmid oriV regions has been identified (Tietze, E. (1998) Plasmid 39: 165-181). The iteron sequence ofpTC-F14 differs from pTF-FC2 and pIE 1108 by three nucleotides in this iteron-discriminating region. It was therefore proposed that co-evolution of the iterons and the RepC protein to a point where the RepC protein no longer recognizes the iteron sequence of a closely related sister plasmid is the mechanism by which replicons evolve to become compatible in the same host cell. The incompatibility determinant of the IncQ prototype plasmid RSFlOlOIR11621R300B was also sought, and subsequently localized to the region encoding the IncQ prototype plasmid's repAC genes. Interference with the initiation of pTC-F14 replication by the IncQ prototype plasmid was demonstrated by growth inhibition of a replication-deficient M13 bacteriophage into which oriVpTC-F14 had been cloned. Secondly, the IncQ prototype derivative pKE462 displaced a ColEloriVpTC- F14 construct in complementation assays, and a construct containing only the pTC-F14 repBAC genes similarly displaced the pKE462 plasmid. As the oriVRSFIOIO region was not incompatible with a pTC-F14 replicon, this suggested that it was not the oriV region which was expressing incompatibility, but the products of the IncQ prototype plasmid repAC genes. It is proposed that incompatibility between pTC-F14 and the IncQ prototype plasmid was the consequence of the repAC gene products binding to the iterons of the related rep licon, and that these products are unable to initiate replication. The compatible phenotypes expressed by members of the IncQ plasmid family indicates the inadequacy of using plasmid incompatibility as a classification system. Alignment of the amino acid sequences of the three replication protein orthologs clearly showed that the IncQ plasmid family was divided into two groups. To account for replication protein relatedness and the incompatibility phenotype expressed, it is now proposed that that members of the IncQ family be classified into subdivisions that reflect the different IncQ-like replicons identified in this study. Investigation of pTC-F14 replicon regulation identified a putative promoter sequence which is believed to regulate the initiation of a 5.l-5.7-kb polycistronic transcript that encodes all the replication proteins of the pTC-F14 replicon and the MobB and MobA proteins of the IncP-type mobilization module. The large polycistronic transcript appears to regulated by the RepB protein of the pTC-F14 replicon, and is not subject to cross-regulation by related IncQ plasmids. This suggested that the RepB primase function was not plasmid specific, but that its regulatory function was replicon specific. A second putative promoter sequence identified upstream of the pTC-F14 pasAB operon was, however, cross-regulated by the closely related pTF-FC2 plasmid. The pTC-F14 pas operon encodes two proteins with high amino acid sequence identity (PasA, 81 %; PasB, 72 %) to the plasmid addiction system ofpTF-FC2. This is the second time a plasmid addiction system of this type has been found on an IncQ-like plasmid. / AFRIKAANSE OPSOMMING: Die matig termofiliese (45 to 50°C), hoogs asidofiliese (pH 1.5 to 2.5), chemolitooutotrofiese Acidithiobaci/lus caldus ras "f' is geïsoleer vanaf 'n biooksiderende proses wat gebruik word om gekonsentreerde nikkel-erts te behandel. Trans- Afwisselende Veld Elektroforese (TAVE) analise van totale DNA vanaf die At. caldus selle, het twee plasmiede van ongeveer 14 en 45-kb. onthul. Die 14-kb plasmied, genaamd pTC-F14, is gekloneer en deur vervanging van die kloneringsvektor met 'n kanamisien weerstandsgeen is daar gewys dat hierdie plasmied in staat is tot outonome replikasie in Escherichia coli. Outonome replikasie is ook gedemonstreer in Pseudomonas putida en Agrobacterium tumefaciens LBA 4404 wat suggereer dat pTC-F14 'n wye gasheer-reeks plasmied is. Volgorde analise van die pTC-F14 replikon area het vyf oop leesrame onthul, en 'n replikon organisasie soortgelyk aan dié van die wye gasheer-reeks IncQ plasmiede. Drie van die oop leesrame kodeer vir replikasie proteïene met aminosuur volgordes ooreenstemmend met dié van die IncQ-tipe plasmied pTF-FC2 (RepA, 81%; RepB, 78%; RepC, 74%). Hierdie hoë vlak van verwantskap stel voor dat die twee replikons vanaf 'n gemeenskaplike voorouer ontwikkel het. Aangesien naby-verwante replikons gewoonlik onverenigbaar is, het die verenigbaarheid van die replikons van pTC-F14 en pTF-FC2 die vraag laat onstaan van hoe die replikons van twee susterplasmiede ontwikkel het, sodat hulle nou gelyktydig in dieselfde gasheer sellyn kan voortbestaan. Verdere onverenigbaarheid toetsing van die IncQ-tipe plasmied pIE1108 en die IncQ prototipe plasmied RSF10101R11621R300B, het bepaal dat pTCF14 verenigbaar is met pIE1108, maar onverenigbaar met die IncQ prototipe plasmied. Daar is gevind dat die RepB en RepA replikasie proteïene van pTF-FC2 en pIE1108 in staat was om die pTC-F14 ortoloë te komplementeer, slegs as pTC-F14 RepC in trans teenwoordig was. Die RepC proteïen van pTC-F14 is dus plasmiedtemplaat spesifiek, terwyl die RepA en RepB proteïene minder plasmied-templaat spesifiek is. 'n Moontlike iteron-onderskeidende vyf-nukleotied area in die direkte herhalings van die IncQ-tipe plasmied oril/ areas, is geïdentifiseer (Tietze, E. (1998) Plasmid 39: 165-181). Die iteron volgorde van pTC-F14 verskil van pTF-FC2 en pIEll08 met drie nukleotiedes in hierdie iteron-onderskeidende area. Om hierdie rede is daar voorgestel dat ko-evolusie van iterons en die RepC proteïen, tot by 'n punt waar die RepC proteïen nie meer die iteron volgorde van 'n naby-verwante susterplasmied herken nie, die meganisme is waardeur replikons ontwikkel om verenigbaar te word in dieselfde gasheersel. Die onverenigbaarheidsbepaler van die IneQ prototipe plasmied RSFIOIOIR11621R300B is ook ondersoek en gelokaliseer tot die area wat kodeer vir die IneQ prototipe plasmied se repAC gene. Inmenging met die inisiasie van pTC-F14 replikasie deur die IneQ prototipe plasmied is gedemonstreer deur groei vertraging van 'n replikasie-gebrekkige M13 bakteriofaag waarin die oriVpTC-F14 gekloneer is. Tweedens is die ColEl-oriVpTc-FI4 konstruk vervang deur die IneQ prototipe-afgeleide pKE462 in komplementasie proewe, en is die pKE462 plasmied op soortgelyke wyse vervang deur 'n konstruk wat slegs die pTC-F14 repBAC gene bevat. Aangesien die oriVRSF1010 area nie verenigbaar was met 'n pTC-F14 replikon nie, stel dit voor dat dit nie die oriV area is wat onverenigbaarheid uitdruk nie, maar die produkte van die IneQ prototipe plasmied se repAC gene. Dit is voorgestel dat onverenigbaarheid tussen pTC-F14 en die IneQ prototipe plasmied die gevolg is van die repAC geenprodukte wat bind aan die iterons van die verwante replikon en dat hierdie produkte nie in staat is om replikasie te inisieer nie. Die verenigbare fenotipes wat deur die lede van die IneQ plasmied familie uitgedruk word, dui aan op die ontoereikendheid van die gebruik van plasmied onverenigbaarheid as 'n klassifikasie sisteem. Vergelyking van die aminosuur volgordes van die drie replikasie proteïen ortoloë wys duidelik daarop dat die IneQ plasmied familie in twee groepe verdeel is. Om verantwoording te doen vir die replikasie proteïen verwantskap en die onverenigbare fenotipe wat uitgedruk is, word daar nou voorgestel dat die lede van die IneQ familie geklassifiseer word in subafdelings wat die verskillende IneQ-tipe replikons geïdentifiseer in hierdie studie, reflekteer. Ondersoek na die pTC-F14 replikon regulering het 'n moontlike promotor volgorde geïdentifiseer. Daar word gemeen dat hierdie promotor die inisiasie van 'n 5.l-5.7-kb polisistroniese transkrip reguleer, wat kodeer vir al die replikasie proteïene van die pTC-F14 replikon en die MobB en Mob A proteïene van die IneP-tipe mobilisasie module. Die groot polisistroniese transkrip blyk om gereguleer te word deur die RepB proteïen van die pTC-F14 replikon, en word nie gekruis-reguleer deur die IneQ plasmiede nie. Dit stel voor dat die RepB primase se funksie nie plasmiedspesifiek is nie, maar dat die reguleerbare funksie replikon-spesifiek is. 'n Tweede moontlike promotor volgorde wat stroom-op van die pTC-F14 pasAB operon geïdentifiseer is, is egter gekruis-reguleer deur die pTF-FC2 plasmied. Die pTC-F14 pas operon kodeer vir twee proteïene met hoë aminosuur volgorde verwantskappe (PasA, 81 %; PasB, 72 %) aan die plasmied-verslaafde sisteem van pTF-FC2. Dit is die tweede keer dat hierdie tipe plasmied-verslaafde sisteem in 'n IncQ-tipe plasmied gevind is.

No description available.

No description available.

<p>Nitrile hydratases (NHases) are enzymes that catalyse the conversion of organocyanides to amides via a non-hydrolytic hydration reaction. They are industrially relevant enzymes, currently used in the manufacture of nicotinamide and acrylamide. The target of this study belongs to the thermophilic bacteria Geobacillus pallidus.</p>

<p>Alternative sources of fuel are required worldwide, and bio-ethanol is the leading candidate. Lignocellulosic biomass, a waste component of the agricultural industry, is a promising renewable source. Due to its complex structure it is highly recalcitrant, requiring the synergistic action of a battery of enzymes to achieve complete digestion. These enzymes include cellulases, hemicellulase and the accessory enzymes acetyl xylan esterase (AXE) and ferulic acid esterase (FAE). Thermpohilic Actinomyces isolates with the ability to hydrolyze xylan were screened for esterase activity. Two isolates (ORS10 and GSIV1), identified as Streptomyces spp, were positive for AXE activity. A cosmid library representative of isolate ORS10 was composed and screened for AXE activity using -naphthyl acetate as substrate. An 18 kb cosmid clone, 18D7, tested positive for AXE activity. Intracellular fractions extracted from ORS10 were precipitated with ammonium sulphate and partially purified 161-fold. Specific activity was measured after dialysis and ion-exchange chromatography. Overall yield of the partially purified enzyme was 34 %. Two protein bands of molecular masses 40 kDa and 60 kDa have been subjected to trypsin digestion and MALDI-TOF mass spectrometry analysis. The partially purified AXE displayed optimum activity at pH 9 and at 50&deg / C. AXE activity was stable for at least 1.5 hours between 30&deg / C and 40&deg / C, and for 24 hours between pH 6-9. The kM and Vmax values were 16.93 mg/ml and 1645 units/mg enzyme, respectively. The stability of the partially purified AXE at 30&deg / C-40&deg / C suggests potential for industrial applications that utilise mesophilic fermentations.</p>

The understanding of the phenomenon of thermophily requires investigations of both thermophilic prokaryotes and eukaryotes. In the eukaryotes, thermophily is exhibited only by a few species of fungi which can grow up to 60°C. A comparative study of homologous enzymes from thermophilic and mesophilic fungi and the analysis of the observed differences is a useful approach not only in discerning the mechanisms in thermophily but also in understanding the features of fungal growth and metabolism. Because of the availability of background information of invertase from some mesophilic sources and the convenience of assaying the enzyme, it was chosen for the projected study in a thermophilic fungus, Themomyces lanuginosus. The behaviour of invertase in the thermophilic fungus differed from invertases of mesophilic organisms in several respects, e.g., in the thermophilic fungus the enzyme was induced only in the presence of its substrate; it was intracellular and it was unstable both in mycelia and in cell-free extracts. The enzyme specific activity was maximum in 6 h-sucrose-grown mycelia following, which it progressively declined before maximal increase in biomass occurred and much of the inducer (sucrose) was still present in the growth medium. Further, invertase activity in cell-free extracts was unstable; it was completely inactivated during storage for 3 days at O°C. The enzyme activity was stabilised by the addition of thiol compounds, dithiothreitol (DTT) and glutathione (GSH) to cell-& extracts. In contrast, the addition of disulphides and thiol-modifying compounds rapidly inactivated the enzyme indicating the involvement of free sulphydryl group(s) in enzyme activity. The enzyme activity was reciprocally modulated by reduced (GSH) and oxidized (GSSG) glutathione, suggesting that invertase may be regulated by thiol/disulphide exchange reaction. Such a modulation of invert- activity has not been reported hr any other invertase. This observation suggested that the enzyme in the thermopbilic fungus is different from invert- that have been studied from mesophilic sources, notably from yeast and Neurospora. To obtain more information on this unusual behaviour of invertase of T. lanuginosus, an attempt was made to purify the enzyme and study its physico-chemical properties. Invertase was purified by ammonium sulphate fractionation of cellular proteins, ion-exchange and thiol-affinity chromatography followed by preparative electrophoresis. The final preparation of invertase after the electroelution step gave a single band on a native PAGE. However, the same preparation of invertase resolved into five bands of different molecular mass. The heterogeneity of the enzyme preparation on SDS-PAGE raised two possibilities with respect to the purity of the enzyme: (1) the final preparation contained multiple invertases of different molecular mass, or (2) the invertase preparation was associated with contaminating proteins. To distinguish between these two possibilities, proteins from induced (sucrose-grown) and non-induced (glucose-grown) mycelia were compared after identical steps of purification. The rationale of this experiment was that if heterogeneity of invertase is due to multiple forms of the same enzyme, they would most likely be absent in the non-induced mycelia. When the final preparations of proteins from both the mycelia were analysed on SDS-PAGE, it was observed that certain proteins were present in both the induced and the non-induced mycelia, suggesting that they might be the contaminating proteins present in the invertase purified by the above procedures. Some physico-chemical properties of invertase were studied. The purified enzyme was unstable during storage, losing activity completely in five days at O°C. Addition of DTT or glutathione did not prevent this loss of enzyme activity. This response of purified invertase preparations to DTT was quite opposite to that in cell-free extracts where invertase activity was stabilised by thiol compounds. To elucidate the reason for this difference in the behaviour of invertase in cell-free extracts and in pursed preparations, the approach taken was to first inactivate the enzyme in both1 type of preparations and then attempt to reactivate it. Dialysis of cell-free extracts had been found to cause an accelerated and complete inadivation oft he enzyme. The same treatment also inactivated freshly purified invertase, but to a lesser extent (60%). Whereas addition of DTT completely reatored the enzyme activity in the dialysed cell-bee extracts, it caused only a marginal revival of activity in dialysed invertase. This change in the response of purified invertase to DTT suggested that some cellular proteins were required br the reactivation of the enzyme by DTT that had been removed during the purification of invertase. A cellular protein was identified that reactivated inactive invertase in the presence of DTT. This protein was given the acronym "PRIA" for 'protein which restores i nvertase activity'. The mechanism of reactivation involved the conversion of the inactive invertase molecules into an active form. A model has been proposed to explain the requirement of UPRIA" for the reactivation of invertase. The salient features of this model are : (i) invert= requires free sulphydryl group(s) for activity, (ii) inactivation of invertase involves the formation of intramolecular disulphide bond(s) in the enzyme, (iii) the disulphide bond(s) is inaccessible to reduction by DTT, (iv) interaction of invertase and "PRIA” results in a conformational change in the enzyme that exposes the disulphide bond(s), rendering it susceptible to reduction by DTT and converting inactive invertase into active enzyme molecules. A surprising observation was the resistance of purified invertase to inactivation by the disulphides, GSSG, CoASSCoA and cystine. This was in marked contrast to their effective inhibition of invertase in the cell-& extracts. The experimental analysis of this unexpected resistance of purified invertase to disulphides revealed that following thioldnity chromatography on a Afegeldol column, invertase became resistant to disulphide inactivation. Moreover, the purified invertase was more stable during storage and to dialysis treatments in contrast to invertase activity in the cell-free extracts. These obsemtions suggested that invertase was altered- presumably it underwent a conformational change during the -el-501 chromatography step; possibly, the interaction of invertase with the gel matrix resulted in some cysteine residues in the enzyme becoming inaccessible to oxidation, thereby conferring resistance to inactivation by disulphides. The in vitro modulation of invertase activity by GSH and GSSG suggested the possibility that the enzyme may be regulated by a similar mechanism in the fungal mycelium. To substantiate this, GSH and GSSG levels in the mycelia were estimated. The GSH/GSSG ratio dememed in the mycelia between 6 and 18 h of growth and this was correlated with the decline in invertase activity. The fall in the GSHIGSSG ratio suggested that the intracellular environment waa becoming progressively oxidised during growth. Because NADPH participates in maintaining the cellular glutathione in a reduced state by the glutathione reduct- reaction, NADPH and NADPt levels were estimated. The NADPH/NADPt ratio declined by a factor of four between 6 and 36 h of growth and this decrease was positively correlated with the decrease in the flux of glucose through the pentose phosphate pathway. Incorporation of 'H-thymidine in mycelia indicated that with age of the culture, the number of growing hyphal tipslunit weight of mycelia declined. An attempt was made to integrate the changes in various biochemical parameters with the pattern of invertase development in T. lanuginosus when grown in a medium containing sucrose, i.e. invertase activity appeared rapidly as soon as perceptible growth occurred but it did not increase in parallel with the increase in biomass. Rather, the activity started to decline at approximately 6 h at which time growth was quantitatively mall. Since invertase activity in T. lanuginosus was induced by sucrose which is transported inside by a specific transporter, the development of invertase activity was linked to the uptake of sucrose by the fungal mycelia. It was considered likely that the sucrose transporter in T. lanuginosus, is localised at the hyphal tip where the entry of sucrose induces invertase. He enzyme is kept active in the hyphal tip because of a reductive environment due to a high GSHIGSSG ratio as a result of high NADPH levels. The latter serves to maintain GSH in a reduced state by the glutathione reductase reaction. In mature hyphae, lower generation of NADPH will result in lower GSHIGSSG ratio that will inactivate invertase by thiol oxidation. According to this model, the early burst of invertase activity in sucrose grown T. lanuginosus mycelia is due to the initiation of branch initials whereas the fall in enzyme activity is because of the decline in the proportion of hyphal tips per unit mass of mycelium as elongation growth and wall thickening occurs.

<p>Nitrile hydratases (NHases) are enzymes that catalyse the conversion of organocyanides to amides via a non-hydrolytic hydration reaction. They are industrially relevant enzymes, currently used in the manufacture of nicotinamide and acrylamide. The target of this study belongs to the thermophilic bacteria Geobacillus pallidus.</p>

<p>Alternative sources of fuel are required worldwide, and bio-ethanol is the leading candidate. Lignocellulosic biomass, a waste component of the agricultural industry, is a promising renewable source. Due to its complex structure it is highly recalcitrant, requiring the synergistic action of a battery of enzymes to achieve complete digestion. These enzymes include cellulases, hemicellulase and the accessory enzymes acetyl xylan esterase (AXE) and ferulic acid esterase (FAE). Thermpohilic Actinomyces isolates with the ability to hydrolyze xylan were screened for esterase activity. Two isolates (ORS10 and GSIV1), identified as Streptomyces spp, were positive for AXE activity. A cosmid library representative of isolate ORS10 was composed and screened for AXE activity using -naphthyl acetate as substrate. An 18 kb cosmid clone, 18D7, tested positive for AXE activity. Intracellular fractions extracted from ORS10 were precipitated with ammonium sulphate and partially purified 161-fold. Specific activity was measured after dialysis and ion-exchange chromatography. Overall yield of the partially purified enzyme was 34 %. Two protein bands of molecular masses 40 kDa and 60 kDa have been subjected to trypsin digestion and MALDI-TOF mass spectrometry analysis. The partially purified AXE displayed optimum activity at pH 9 and at 50&deg / C. AXE activity was stable for at least 1.5 hours between 30&deg / C and 40&deg / C, and for 24 hours between pH 6-9. The kM and Vmax values were 16.93 mg/ml and 1645 units/mg enzyme, respectively. The stability of the partially purified AXE at 30&deg / C-40&deg / C suggests potential for industrial applications that utilise mesophilic fermentations.</p>

Thesis (Ph.D.)--Montana State University--Bozeman, 2006. / Typescript. Chairperson, Graduate Committee: Mark Young. Includes bibliographical references (leaves 203-233).