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<p>ABSTRACT</p><p>Nontypable Haemophilus influenzae is a common cause of otitis, sinusitis and conjunctivitis. It is the most common bacterial pathogen associated with chronic obstructive pulmonary disease (COPD). Studies have shown that nonpathogenic Haemophilus haemolyticus are often mistaken for Haemophilus influenzae due to an absent hemolytic reaction on blood agar. Distinguishing H. haemolyticus from H. influenzae is important to prevent unnecessary antibiotic use, and to understand the role of H. influenzae in clinical infections. In this study, PCR-primers for amplifying 16S rDNA sequences were used to set up a method for distinguishing H. haemolyticus from H. influenzae. The aim was to use the method for analyzing apparent H. influenzae strains, to investigate if some strains were in fact H. haemolyticus. However, because of problems with unspecific primerannealing,no conclusions could be drawn regarding misclassification of H. haemolyticus.</p><p>Moraxella catarrhalis is the second most common bacterial pathogen associated with COPD. It also causes otitis and sinusitis. An important virulence factor of M. catarrhalis is the outer membrane protein Moraxella catarrhalis IgD-binding protein (MID). One part of the protein; MID764-913 , has been shown to function as an adhesin, and this part has been fragmented to further investigate its adhesive properties. The aim of this second, independent study, was to express some of these proteinfragments by cloning in E. coli. The time spent on this project was too short, and no proteins could be expressed duing this period.</p>

In this study, Scytalidium thermophilum laccase gene was first cloned into E. coli and then heterologously expressed in A. sojae. S. thermophilum is a thermophilic fungus with an important role in determining selectivity of compost produced for growing Agaricus bisporus. S. thermophilum laccase gene was first cloned by Novo Nordisk Bio Tech, Inc. in 1998. This laccase gene (lccS) has an open reading frame of 2092bp. It is composed of five exons punctuated by four small introns. The coding region, excluding intervening sequences is very GC-rich (60.8% G+C) and encodes a preproenzyme of 616 amino acids: a 21 amino acid signal peptide and a 24 amino acid predicted propeptide. lccS gene was amplified using specific primers to exclude the signal and pro-peptide coding regions and ligated to expression vector pAN52-4. The recombinant plasmid was used to transform Aspergillus sojae ATCC11906 (pyrG-). Heterologuos expression was observed in glucose-containing media, under the control of the glyceraldehydes 3-phosphate dehydnogenese promoter and the secretion signal of glucoamylase gene. Laccase gene is an important step towards the high level expression of this enzyme in a GRAS eucaryotic host and for further biotransformation and enzyme engineering studies. In this study also bioinformatic analysis of N-terminal and C-terminal propeptide cleavage sites of fungal proteins including laccases were studied.

Submitted by Ana Damasceno (ana.damasceno@unipampa.edu.br) on 2017-05-12T16:58:20Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Clonagem e expressão Datransposase mos1 de Drosophila simulans e o desenho in silico de um vetor para expressão heteróloga de proteínas.pdf: 1982928 bytes, checksum: 704cbac648c3f7621a8bb8d5cf3116c7 (MD5) / Made available in DSpace on 2017-05-12T16:58:21Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Clonagem e expressão Datransposase mos1 de Drosophila simulans e o desenho in silico de um vetor para expressão heteróloga de proteínas.pdf: 1982928 bytes, checksum: 704cbac648c3f7621a8bb8d5cf3116c7 (MD5) Previous issue date: 2016-03-10 / A importância da expressão de proteínas de forma heteróloga tem crescido surpreendentemente, tendo em vista a progressiva necessidade de sua produção para aplicações na indústria farmacêutica, para uso terapêutico, ou uso comerciais, e estudo de estrutura e função. No entanto, a expressão das proteínas pode ser limitada por diversos fatores, podendo apresentar diversas dificuldades como a formação de corpos de inclusão ou proteínas com conformação incorreta, por exemplo. Estudos em hospedeiro para expressão de proteínas heterólogas tem sido desenvolvido, a fim de otimizar fatores como temperatura e concentração de indutor, com o intuito de melhorar a qualidade das proteínas obtidas. Neste estudo, inicialmente objetivou-se otimizar a expressão da transposase do elemento mariner mos1, em diferentes cepas de Escherichia coli BL21(DE3), E. coli BL21 (DE3) pLysS, E. coli BL21-CodonPlus (DE3) -RP; E. coli BL21-CodonPlus (DE3) -RIL; E. coli BL21(DE3) pT-GROE, usando para subclonagem o vetor plasmidial de expressão pGEX-4T1. Analisando o melhor sistema de expressão para este elemento em particular, comparando as temperaturas de 30°C e 37°C para expressão e utilizando concentrações de 0,1mM; 0,5mM e 1,0mM de IPTG. Mesmo obtendo sucesso na expressão, com todas as condições de IPTG e em ambas as temperaturas testadas, o sistema de expressão não foi eficaz para obter proteínas de forma solúvel. Frente a este problema procuramos desenvolver um vetor de expressão de proteínas heterólogas que afim de tornar este processo mais rápido e mais eficiente, utilizando apenas um evento de clonagem e de transformação da bactéria de expressão. O vetor para expressão foi desenhado in silico a partir de um promotor Lac responsivo a IPTG, seguido por um sinal de exportação celular (peptídeo-sinal), um fragmento Strep-tagII para purificação e um sítio para trombina, permitindo a clivagem da proteína de interesse dos tags fusionados. Foram inseridos dois sítios para endonucleases de restrição (BamHI e XbaI) que permitem a clivagem do gene clonado no constructo. Desta forma demonstramos que todas as cepas foram eficazes na expressão da transposase mos1, sendo a temperatura mais adequada de 37°C em todas as concentrações de IPTG, já que não houve nenhuma diferença quanto a mudança deste fator. Apresentamos um novo vetor de expressão de proteínas heterólogas, com a finalidade de facilitar a obtenção de proteínas heterólogas purificadas, tais problemas encontrados durante a tentativa de expressão da transposase. / The importance of heterologous protein expression has surprisingly grown in view of the progressive need for its production for applications in the pharmaceutical industry, for therapeutic use, or commercial use, and study of structure and function. However, the expression of the proteins may be limited by several factors, and may present several difficulties such as the formation of inclusion bodies or proteins with incorrect conformation, for example. Host studies for expression of heterologous proteins have been developed in order to optimize factors such as temperature and concentration of inducer in order to improve the quality of proteins obtained. In this study, we initially aimed to optimize transposase expression of the mariner element mos1 in different strains of Escherichia coli BL21 (DE3), E. coli BL21 (DE3) pLysS, E. coli BL21CodonPlus (DE3) -RP; E. coli BL21-CodonPlus (DE3) -RIL; E. coli BL21 (DE3) pTGROE, using for subcloning the pGEX-4T1 expression plasmid vector. Analyzing the best expression system for this particular element, comparing the temperatures of 30°C and 37°C for expression and using concentrations of 0.1 mM; 0.5mM and 1.0mM IPTG. Even with expression success, with all IPTG conditions and at both temperatures tested, the expression system was not effective in obtaining proteins in a soluble form. In view of this problem we have tried to develop an expression vector of heterologous proteins in order to make this process faster and more efficient, using only one event of cloning and transformation of the expression bacterium. The vector for expression was drawn in silico from an IPTG responsive Lac promoter, followed by a cell export signal (peptide-signal), a Strep-tagII fragment for purification and a site for thrombin, allowing cleavage of the protein from Interest of merged tags. Two sites for restriction endonucleases (BamHI and XbaI) were inserted which allow the cleavage of the cloned gene in the construct. In this way, we demonstrated that all strains were effective in the expression of the transposase mos1, with the most suitable temperature being 37 ° C in all concentrations of IPTG, since there was no difference in the change of this factor. We present a new expression vector of heterologous proteins, in order to facilitate the obtaining of purified heterologous proteins, such problems encountered during the attempt to express the transposase.

This study generated two cyanide dihydratase (CynD) mutants of Bacillus pumilus C1 with improved activity at higher pH by random mutagenesis. The purpose of this study was to create enzyme variants better suited to degrade cyanide under the harsh conditions of industrial applications. We employed error-prone PCR to construct a library of CynD mutants. A high throughput screening system was developed to screen the library for improved activity. Two mutants were identified that could degrade cyanide at pH10 whereas the wild-type enzyme was inactive at pH9 or higher. The mutants each had three amino acid substitutions compared to the wild-type enzyme. The mutants were also more stable than the wild-type enzyme at 42oC. E327G was identified as one of the key amino acids that are responsible for the improved activity. The goal of the second project was to convert substrate specificity of the Bacillus sp. OxB-1 nitrilase to that of a cyanidase by mutagenesis or construction of hybrid genes. The OxB-1 nitrilase of Bacillus sp. shows a high level of identity with the cyanide dihydratases from B. pumilus C1 and P. stutzeri AK61 but utilizes different substrate. This provides a valuable resource to study the substrate specificity determinants of cyanide degrading enzymes. One deletion mutant and four hybrid proteins were constructed based on the alignment information. The constructed proteins were all unable to degrade cyanide.

Scytalidium thermophilum is a thermophilic fungus with an important role in the composting process of mushroom cultivation. An extracellular phenol oxidase of Scytalidium thermophilum (STEP) with novel features was previously studied in our laboratory. This enzyme later turned out to be a catalase having phenol oxidase activity. The aim of this study was to clone Scytalidium thermophilum bifunctional catalase/phenol oxidase encoding gene and express the gene in Aspergillus sojae for future site directed mutagenesis studies. Scytalidium thermophilum catalase gene was first cloned into E. coli XL1 Blue MRF&rsquo / and then heterologously expressed in Aspergillus sojae ATCC11906. For that aim, the catalase gene was amplified using specific primers, excluding the signal and pro-peptide coding regions and amplified fragment was then cloned into E.coli XL1 Blue MRF&rsquo / and sequenced. It was observed that the cloned gene, named as catpo, was 10 amino acids different from the amino acid sequence of the S.thermophilum catalase gene formerly cloned by Novo Nordisk. The catpo gene encoding a mature protein of 681 amino acids was then ligated onto expression vector pAN52-4 and the recombinant plasmid was transformed into Aspergillus sojae ATCC11906. Heterologous expression was observed under the control of the glyceraldehydes 3-phosphate dehydrogenese promoter of Aspergillus nidulans and the secretion signal of the glucoamylase gene of Aspergillus niger. Catalase activity of the transformants reached at a level of 13206 U/g at the end of the fourth day of cultivation. However, this is still lower than the catalase activity of the gene donor strain of Scytalidium thermophilum.

In Streptomyces clavuligerus, the route to the biosynthesis of &amp / #945 / -aminoadipic acid (&amp / #945 / -AAA) represents an important primary metabolic pathway providing carbon flux to the synthetases of antibiotic formation. This carbon flow comes through the lysine-specific branch of the aspartate pathway and is rate limiting in the formation of cephamycin C, a second generation cephalosporin produced by this organism. In this study, the lysA gene which encodes for an important key enzyme of aspartate pathway / meso-diaminopimelic acid (DAP) decarboxylase (E.C.4.1.1.20) catalyzing the conversion of diaminopimelate to lysine was cloned and characterized for the first time from S. clavuligerus NRRL 3585. The attempts to clone the gene by constructing libraries of S. clavuligerus genomic DNA and screening of the libraries either by homologous probing or complementation approach gave no positive results. Then, PCR-based cloning was taken as the approach and the gene was amplified with PCR using the primers derived from the conserved sequences of lysA genes in two fragments (620 and 983 bp) which had overlapping regions. Fragments were then cloned and nucleotide sequencing revealed a complete open reading frame (ORF) encoding a protein of 463 aa (Mr 49, 907). The GC content of the gene was identified as 70.98 %. The gene sequence showed 83 % identity to the sequence of S. coelicolor lysA gene and 81 % identity to S. avermitilis lysA gene. By comparing the amino acid sequence of this protein to those available in database, the sites of the enzyme important for catalysis were identified.