Investigations into the nitrogen fixing apparatus in cyanobacterium Nostoc commune revealed a gene encoding for a hemoprotein, known as cyanoglobin. The cyanoglobin gene was isolated and subcloned into Escherichia coli previously. The study presented here encompasses the optimization of growth conditions for the transformed F. coli, with subsequent induction of cyanoglobin synthesis. These conditions were applied to large-scale (24-1) fermentor culture, permitting purification of approximately 200 mg cyanoglobin. Structural analyses, including absorption spectroscopy and circular dichroism, are presented. These studies indicate that cyanoglobin is a cytoplasmic hemoglobin with properties quite unlike those of leghemoglobin a and sperm whale myoglobin, which are used as references of comparison. For example, the optical spectral properties of oxycyanoglobin are different from those of leghemoglobin α and sperm whale myoglobin. In addition, the met-form of cyanoglobin has characteristics of a low-spin hemoglobin, in contrast to the high-spin met-forms of sperm whale myoglobin and leghemoglobin α. Unusually, the met- form of cyanoglobin fails to coordinate the strong-field ligands, cyanide and azide, at pH 7 and pH 9. The Soret region circular dichroism (CD) spectrum of cyanoglobin is unlike that of sperm whale myoglobin, yet is very similar to leghemoglobin α, suggesting a similar heme environment in these two hemoproteins. Far-UV CD of cyanoglobin revealed alphahelical character comparable to that of sperm whale myoglobin and leghemoglobin α. Cyanoglobin is the first monomeric hemoglobin detected in a prokaryote, raising questions concerning a possible role of cyanoglobin in early globin gene evolution. / Master of Science

Submitted in complete fulfillment for the Degree of Master of Technology (Biotechnology)in the Department of Biotechnology and Food Technology, Faculty of Applied Sciences, Durban University of Technology, Durban, South Africa, 2009. / Protein engineering has been a valuable tool in creating enzyme variants that are capable of withstanding the extreme environments of industrial processes. Xylanases are a family of hemicellulolytic enzymes that are used in the biobleaching of pulp. Using directed evolution, a thermostable and alkaline stabl xylanase variant (S340) was created from the thermophilic fungus, Thermomyces lanuginosus. However, a host that was capable of rapid growth and high-level expression of the enzyme in large amounts was required. The insert containing the xylanase gene was cloned into a series a pET vectors in Escherichia coli BL21 (DE3) pLysS and trimmed from 786 bp to 692 bp to remove excess fungal DNA upstream and downstream of the open reading frame (ORF). The gene was then re-inserted back into the pET vectors. Using optimized growth conditions and lactose induction, a 14.9% increase in xylanase activity from 784.3 nkat/ml to 921.8 nkat/ml was recorded in one of the clones. The increase in expression was most probably due to the removal of fungal DNA between the vector promoter and the start codon. The distribution of the xylanase in the extracellular, periplasmic and cytoplasmic fractions was 17.3%, 51.3% and 31.4%, respectively. The modified enzyme was then purified to electrophoretic homogeneity using affinity chromatography. The xylanase had optimal activity at pH 5.5 and 70°C. After 120 min at 90°C and pH 10, S340 still displayed 39% residual activity. This enzyme is therefore well suited for its application in the pulp and paper industry. / National Research Foundation

Thesis (MScAgric)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Cellulose, a ~-1,4-linked polymer of glucose, is the most abundant renewable carbon source on earth. It is well established that efficient degradation of cellulose requires the synergistic action of three categories of enzymes: endoglucanases (EG), cellobiohydrolases (CBH) and ~-glucosidases. ~-Glucosidases are a heterogenous group of enzymes that display broad substrate specificity with respect to hydrolysis of cellobiose and different aryl- and alkyl-ê-u-glucosides. They not only catalyse the final step in the saccharification of cellulose, but also stimulate the extent of cellulose hydrolysis by relieving the cellobiose mediated inhibition of EG and CBH. The ability to utilize cellobiose is widespread among gram-negative, gram-positive, and Archaea bacterial genera. Cellobiose phosphoenolpyruvate- dependent phosphotransferase systems (PTS) have been reported in various bacteria, including: Bacillus species. In this study, we have used a cellobiose chromophore analog, p-nitrophenyl- ~-D-glucopyranoside (pNPG), to screen a Bacillus pumilus genomic library for cellobiose utilization genes that are functionally expressed in Escherichia coli. Cloning and sequencing of the most active clone with subsequent sequence analysis allowed the identification of four adjacent open reading frames. An operon of four genes (celBACH), encoding a cellobiose phosphotransferase system (PTS): enzyme II (encoded by celB, celA and celC) and a ó-phospho-f-glucosidase (encoded by celH) was derived from the sequence data. The amino acid sequence of the celH gene displayed good homology with ~-glucosidases from Bacillus halodurans (74.2%), B. subtilis (72.7%) and Listeria monocytogenes (62.2%). .As implied by sequence alignments, the celH gene product belongs to family 1 of the glycosyl hydrolases, which employ a retaining mechanism of enzymatic bond hydrolysis. In vivo PTS activity assays concluded that the optimal temperature and pH at which the recombinant E. coli strain hydrolysed pNPG were pH 7.5 and 45°C, respectively. Unfortunately, at 45°C the CelBACH-associated activity of the recombinant strain was only stable for 20 minutes. It was also shown that the enzyme complex is very sensitive to glucose. Since active growing cells metabolise glucose very rapidly this feature is not a significant problem. Constitutive expression of the B. pumilus celBACH genes in E. coli enabled the host to efficiently metabolise cellobiose as a carbon source. However, cellobiose utilization was only achievable in the presence ofO.01% glucose. This phenomenon could be explained by the critical role of phosphoenolpyruvate (PEP) as the phosphate donor in PTS-mediated transport. Glucose supplementation induced the glycolytic pathway and subsequently the availability of PEP. Furthermore, it could be concluded that the general PTS components . (enzyme I and HPr) of E. coli must have complemented the CelBACH system from B. pumilus to allow functionality of the celBACH operon, in the recombinant E. coli host. / AFRIKAANSE OPSOMMING: Sellulose (' n polimeer van p-l,4-gekoppelde glukose) is die volopste bron van hernubare koostof in die natuur. Effektiewe afbraak van sellulose word deur die sinnergistiese werking van drie ensiernklasse bewerkstellig: endoglukanases (EG), sellobiohidrolases (CBH) en P-glukosidases. p-Glukosidases behoort tot 'n heterogene groep ensieme met 'n wye substraatspesifisiteit m.b.t. sellobiose en verskeie ariel- and alkiel-ê-n-glukosidiesc verbindings. Alhoewel hierdie ensieme primêr as kataliste vir die omskakeling van sellulose afbraak-produkte funksioneer, stimuleer hulle ook die mate waartoe sellulose hidroliese plaasvind deur eindprodukinhibisie van EG en CBH op te hef. Sellobiose word algemeen deur verskeie genera van die gram-negatiewe, gram-positiewe en Archae bakterieë gemetaboliseer. Die sellobiose-spesifieke fosfoenolpirovaatfosfotransportsisteem (PTS) is reeds is in verskeie bakterië, insluitende die Bacillus spesies, beskryf. In hierdie studie word die sifting van 'n Bacillus pumilus genoombiblioteek m.b.V. 'n chromofoor analoog van sellobiose, p-nitrofeniel-p-o-glukopiranosied (pNPG), vir die teenwoordigheid van gene wat moontlike sellobiose-benutting in Escherichia coli kan bewerkstellig, beskryf. Die DNA-volgorde van die mees aktiewe kloon is bepaal en daaropvolgende analiese van die DNA-volgorde het vier aangrensende oopleesrame geïdentifiseer. 'n Operon (celBACH), bestaande uit vier gene, wat onderskeidelik vir die ensiem II (gekodeer deur celB, celA en celC) en fosfo-B-glukosidase (gekodeer deur celH) van die sellobiose-spesifieke PTS van B. pumilus kodeer, is vanaf die DNA-volgorde afgelei. Die aminosuuropeenvolging van die celH-geen het goeie homologie met P-glukosidases van Bacillus halodurans (74.2%), B. subtilis (72.7%) en Listeria monocytogenes (62.2%) getoon. Belyning van die DNA-volgordes het aangedui dat die celH geenproduk saam met die familie 1 glikosielhidrolases gegroepeer kan word. Hierdie familie gebruik 'n hidrolitiese meganisme waartydens die stoigiometriese posisie van die anomeriese koolstof behou word. PTS-aktiwiteit van die rekombinante E. coli ras, wat die celBACH gene uitdruk, is in vivo bepaal. Die optimale temperatuur en pH waarby die rekombinante ras pNPG hidroliseer, is onderskeidelik pH 7.5 en 45°C. Alhoewel die ensiernkompleks baie sensitief is vir glukose, is dit nie 'n wesenlike probleem nie, omdat aktief groeiende E. coli selle glukose teen 'n baie vinnige tempo benut. Die celBACH operon het onder beheer van 'n konstitiewe promotor in E coli die rekombinante gasheer in staat gestelom sellobiose as 'n koolstofbron te benut. Die benutting van sellobiose word egter aan die teenwoordigheid van 'n lae konsentrasie glukose (0.01 %) gekoppel. Hierdie verskynsel dui op die kritiese rol van fosfoenolpirovaat (PEP) as die fosfaatdonor gedurende PTS-gebaseerde transport. Glukose speel waarskynlik 'n rol in die indusering van glikoliese, en sodoende die produksie van PEP as tussenproduk. Verder kan afgelei word dat die algemene PTS komponente (ensiem I en HPr) van E. coli die B. pumilis CelBACH-sisteem komplementeer en derhalwe funksionering van die celBACH operon in E. coli toelaat.

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Understanding of a global biological network structure by studying its simple local properties through the well-developed field of graph theory is of interest. In particular, in this research an observed biological network was explored through a simulation study. However, one difficulty in such exploration lies on the fitting of graphical models on biological network data. An Exponential Random Graph Model (ERGM) was considered to determine estimations of the several network attributes of complex biological network data. We also compared the estimates of observed network to our random simulated network for both Markov Chain Monte Carlo Maximum Likelihood Estimation (MCMCMLE) and Maximum Pseudo Likelihood Estimation (MPLE) methods under ERGM. The motivation behind this was to determine how different the observed network could be from a randomly simulated network if the physical numbers of attributes were approximately same. Cut-off points of some common attributes of interest for different order of nodes were determined through simulations. We implemented our method to a known regulatory network database of E. coli. / Department of Mathematical Sciences

Pseudomonas fluorescens does not appear to regulate the enzymes of de novo pyrimidine biosynthesis at the level of gene expression. Little or no apparent repression of pyr gene expression is observed upon addition of exogenous pyrimidines to the growth medium. The Escherichia coli pyrBI genes for aspartate transcarbamoylase (ATCase) were sized down and cloned into the broad host range plasmid, pKT230. Upon introduction into a P.fluorescenspyrB mutant strain, ATCase showed repression in response to exogenously fed pyrimidine compounds. Thus, it was possible to bring about changes in pyrimidine nucleotide pool levels and in transcriptional regulation of gene expression at the same time.

Cassette systems for creating intergeneric hybrid ATCases were constructed. An MluI restriction enzyme site was introduced at the carbamoylphosphate binding site within the pyrB genes of both Pseudomonas putida and Escherichia coli. Two hybrids, E. coli pyrB polar domain fused with P. putida pyrB equatorial domain and P. putida pyrB polar domain fused with E. coli pyrB equatorial domain, are possible. The intergeneric E. coli-P. putida hybrid pyrB gene was constructed and found to encode an active ATCase which complemented an E. coli Pyr- strain. These hybrids are useful for kinetic and expression studies of ATCase in E. coli.

Chi-shing Lo. / Thesis submitted in: November 1999. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 119-138). / Abstracts in English and Chinese. / Statement --- p.iii / Acknowledgments --- p.iv / Abbreviations --- p.v / Abstract --- p.vii / Abstract (Chinese version) --- p.ix / Table of contents --- p.xi / List of Figure --- p.xvii / List of Table --- p.xix / Chapter Chapter 1: --- General Introduction --- p.1 / Chapter 1.1 --- BIOLOGY OF TOXOPLASMA GONDII --- p.1 / Chapter 1.1.1 --- Life cycle of Toxoplasma gondii --- p.2 / Chapter (a) --- Tachyzoite --- p.3 / Chapter (b) --- Bradyzoite --- p.3 / Chapter 1.1.2 --- Genetics of Toxoplasma gondii --- p.4 / Chapter (a) --- Population genetics --- p.4 / Chapter (b) --- Molecular genetics --- p.5 / Chapter (c) --- Genome analysis --- p.7 / Chapter 1.1.3 --- Invasion --- p.8 / Chapter 1.1.4 --- Surface of Toxoplasma gondii --- p.9 / Chapter (a) --- Tachyzoite surface --- p.9 / Chapter (b) --- Bradyzoite surface --- p.11 / Chapter (c) --- Sporozite surface --- p.11 / Chapter (d) --- Glycoprotein antigens --- p.12 / Chapter 1.2 --- TREATMENT OF TOXOPLASMOSIS --- p.13 / Chapter 1.2.1 --- Chemotherapy --- p.13 / Chapter (a) --- Drug against metabolism and protein synthesis on nuclear genome --- p.13 / Chapter (b) --- Drug against other organelles --- p.14 / Chapter (c) --- Drug resistance --- p.15 / Chapter 1.2.2 --- Toxoplasma vaccine --- p.16 / Chapter (a) --- Mutant strains of Toxoplasma gondii as vaccine --- p.17 / Chapter (b) --- Subunit vaccine --- p.19 / Chapter (c) --- P30 as subunit vaccine --- p.20 / Chapter 1.3 --- AIM OF THE STUDY --- p.22 / Chapter Chapter 2 --- : Expression of P30 in Escherichia coli --- p.23 / Chapter 2.1 --- INTRODUCTION --- p.23 / Chapter 2.1.1 --- Why Escherichia coli? --- p.23 / Chapter 2.1.2 --- protein folding --- p.24 / Chapter 2.1.3 --- T7-based gene expression system --- p.25 / Chapter (a) --- Biology of T7 RNA polymerase --- p.26 / Chapter (b) --- pET translational vector --- p.26 / Chapter (c) --- Hislidine-tagged protein --- p.27 / Chapter (d) --- Host strain for expression --- p.28 / Chapter 2.2 --- MATERIALS --- p.29 / Chapter 2.2.1 --- Bactcrial strains --- p.29 / Chapter 2.2.2 --- Mouse strain --- p.29 / Chapter 2.2.3 --- Chemicals --- p.29 / Chapter 2.2.4 --- Nucleic acids --- p.30 / Chapter 2.2.5 --- Kit and reagents --- p.31 / Chapter 2.2.6 --- Antibodies --- p.31 / Chapter 2.2.7 --- Solutions --- p.32 / Chapter 2.2.8 --- Enzymes --- p.33 / Chapter 2.2.9 --- Sequencing primers --- p.33 / Chapter 2.3 --- METHODS --- p.34 / Chapter 2.3.1 --- Modification of P30 gene --- p.34 / Chapter (a) --- Preparation of recombinant plasmids，pBV220-ASP30PI and pBV220- SP30hisAPI --- p.36 / Chapter (b) --- Digestion of pBV220-ASP30PI and pBV220-SP30hisAPI with DraII and EcoRI --- p.37 / Chapter (c) --- Purification of DNA fragments from agarose gel --- p.37 / Chapter (d) --- Ligation of fragments of pBV220-ΔSP30PI and pBV220-SP30hisAPI --- p.38 / Chapter (e) --- Preparation of DH5α competent cells --- p.38 / Chapter (f) --- Transformation of recombinant pBV220-ΔSP30hisAPI --- p.38 / Chapter (g) --- Plasmid preparation of putative pBV220-ΔSP30API --- p.39 / Chapter (h) --- Plasmid preparation of pET-ΔSP30API --- p.39 / Chapter (i) --- Cycle sequencing reaction on putative plasmid pET-ASP30API --- p.40 / Chapter 2.3.2 --- Expression and Purification of his-tag P30 --- p.41 / Chapter (a) --- Expression profile of his-tag P30 production by IPTG induction --- p.41 / Chapter (b) --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.41 / Chapter (c) --- Purification of his-tag P30 --- p.43 / Chapter (d) --- Bradford Protein Microassay (Bio-Rad) --- p.43 / Chapter 2.3.3 --- Characterization of his-tag P30 --- p.44 / Chapter (a) --- Western blot of induced bacterial lysate by monoclonal anti-his-tag antibody --- p.44 / Chapter (b) --- Western blot of his-tag with seropositive sera of mice，rabbit and human --- p.46 / Chapter (c) --- Enterokinase digestion of his-tag P30 --- p.46 / Chapter (d) --- N'terminal amino acid sequencing of pure and enterokinase-cut his-tag --- p.47 / Chapter (e) --- Western blot of T. gondii lysate with antiserum against his-tag P30 --- p.47 / Chapter 2.4 --- RESULTS --- p.49 / Chapter 2.4.1 --- Modification of P30 gene --- p.49 / Chapter 2.4.2 --- "Expression, purification and characteriziation of his-tag P30 in bacteria" --- p.54 / Chapter 2.5 --- DISCUSSIONS --- p.64 / Chapter 2.5.1 --- Modification of P30 gene --- p.64 / Chapter 2.5.2 --- Expression and purification of his-tag P30 --- p.66 / Chapter 2.5.3 --- Characterization of his-tag P30 --- p.67 / Chapter Chapter 3 --- : Expression of P30 in Arabidopsis thalina --- p.69 / Chapter 3.1 --- INTRODUCTION --- p.69 / Chapter 3.1.1 --- Why Arabidopsis thalina? --- p.69 / Chapter 3.1.2 --- In planta transformation --- p.70 / Chapter 3.1.3 --- Transgenic plants as vacine production systems --- p.72 / Chapter (a) --- Stable expression of E. coli heat-liable enterotoxin B subunit and cholera-toxin B subunit --- p.73 / Chapter (b) --- Stable expression of Hepatitis B surface antigen (HBsAg) --- p.74 / Chapter (c) --- Stable expression of Norwalk virus capsid protein --- p.75 / Chapter (d) --- Transient expression by tobacco mosaic virus --- p.75 / Chapter (e) --- Transient expression by Cowpea mosaic virus capsid protein fusion --- p.76 / Chapter 3.2 --- MATERIALS --- p.77 / Chapter 3.2.1 --- Bacterial strains --- p.77 / Chapter 3.2.2 --- Arabidopsis strains --- p.77 / Chapter 3.2.3 --- Chemicals --- p.77 / Chapter 3.2.4 --- Nucleic acids --- p.78 / Chapter 3.2.5 --- Kit and reagents --- p.78 / Chapter 3.2.6 --- Solutions --- p.79 / Chapter 3.2.7 --- Enzymes and buffers --- p.81 / Chapter 3.2.8 --- PCR and Sequencing primers --- p.81 / Chapter 3.3 --- METHODS --- p.82 / Chapter 3.3.1 --- Construction of V7-ASP30API --- p.82 / Chapter 3.3.2 --- Agrobacterium-mediated transformation of Arabidopsis by vacuum infiltration --- p.83 / Chapter (a) --- Preparation of electro-competent Agrobacterium --- p.83 / Chapter (b) --- Transformation of electro-competent Agrobacterium with V7- ASP30API --- p.84 / Chapter (c) --- Plasmid preparation of V7-ASP30API from transformed Agrobacterium --- p.84 / Chapter (d) --- Vacuum infiltration --- p.85 / Chapter 3.3.3 --- Screening of homozygous transgenic plants --- p.86 / Chapter 3.3.4 --- Detecton of transgene P30 in genomic DNA of transgenic plants --- p.87 / Chapter (a) --- Preparation of DIG-labelled probe --- p.87 / Chapter (b) --- Estimation the yield of DIG-labelled probe --- p.88 / Chapter (c) --- Extraction of genomic DNA from transgenic plants --- p.88 / Chapter (d) --- Restriction digestion of genomic DNA with EcoRI and HindIII --- p.89 / Chapter (e) --- DNA transfer from gel to nylon membrane --- p.89 / Chapter (f) --- Detection of hybridized DIG-labelled probe on membrane/ blot --- p.90 / Chapter (g) --- PCR on genomic DNA of transgenic plants with specific primers --- p.91 / Chapter 3.3.5 --- Analysis of transgene RNA expression in transgenic plants --- p.91 / Chapter (a) --- Extraction of total RNA from plants --- p.91 / Chapter (b) --- Northern blot on RNA of F2 transgenic plants --- p.92 / Chapter (c) --- RT-PCR on RNA of F3 transgenic plants --- p.93 / Chapter 3.3.6 --- Detection of his-tag P30 protein in F3 transgenic plants --- p.93 / Chapter 3.4 --- RESULTS --- p.95 / Chapter 3.4.1 --- Construction of V7-ASP30API --- p.95 / Chapter 3.4.2 --- Screening of homozygous transgenic plants --- p.99 / Chapter 3.4.3 --- Molecular analysis of transgene P30 in transgenic plants --- p.101 / Chapter 3.5 --- DISCUSSIONS --- p.108 / Chapter 3.5.1 --- Construction and optimization of expression construct --- p.108 / Chapter 3.5.2 --- Screening and selection of homozyous transgenic plants --- p.109 / Chapter 3.5.3 --- Analysis of transgenic plants --- p.110 / Chapter Chapter 4 : --- General Discussions --- p.112 / Chapter 4.1 --- Significances of studying Toxoplasma gondii --- p.112 / Chapter 4.2 --- Expression of recombinant P30 in prokaryotic systems --- p.113 / Chapter 4.2 --- Expression of recombinant P30 in eukaryotic systems --- p.115 / Reference --- p.119

Bibliography: leaves 267-287.

Bibliography: leaves 144-174. / ix, 176, [66] leaves, [22] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Isolates Escherichia coli from Adelaide children and screens for the presence of Shiga-like toxins genes using the polymerase chain reaction and by hybridization with specific DNA and oligodeoxynucleotide probes. Four SLT-producing strains were isolated. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1994