The nuclease gene of Vibrio vulnificus, vvn, is 696 bp long encoding a protein¡]Vvn¡^of 232 amino acids. Vvn is a periplasmic protein and is active in the oxidized form. DNA shuffling is a powerful method for in vitro mutational mechanism by homologous recombination with a low level of point mutation . DNA shuffling consists of four steps¡G¡]1¡^preparation of genes to be shuffled, ¡]2¡^random fragmentation with DNase I, ¡]3¡^fragment reassembly by primerless polymerase chain reaction¡]PCR¡^, and¡]4¡^amplification of reassembled products by a conventional PCR. The advantage of this process is that it can be used to rapidly evolve any protein, without any knowledge of its structure. The goal of this work was using DNA shuffling to generate a diversity of mutation in vvn within a short time. Followed by analyzing the DNase activity of periplasmic protein or in vivo, the mutants were divided into three groups for increase, decrease or no change in DNase activity. Randomly DNA sequencing vvn gene of fourteen transformed clones from the three groups showed only one clone has one base change with comparison to wild-type sequence. The mutation is at amino acid 22 of the N-terminus of Vvn, the change is from serine to isoleucine. The relative activity of mutant Vvn was 82 % in DNase and 59 % in RNase. The effect of a single amino acid change on the DNase and RNase activity of Vvn is different. It supports the postulation that there are two distinct but overlapping active sites exist in Vvn.

The interactions of glycoconjugates with carbohydrate binding proteins are responsible for a wide range of recognition events in vivo; including immune response, cell adhesion and signal transduction. Glycoconjugates have already found many medicinal uses as therapeutic and diagnostic agents, but their full potential is yet to be realised. Access to a variety of homogeneously glycosylated glycoproteins is essential for the study of these important carbohydrate binding events. This requires the chemical synthesis and attachment of biologically relevant glycans to unglycosylated protein scaffolds in a site selective manner. Here we describe the use of a range of glycosyl iodoacetamides to glycosylate proteins selectively via their cysteine residues. We have chosen the green fluorescent protein mutant GFPuv for use as a protein scaffold due its known tolerance of two cysteine mutations (E6C and I229C) and the previous successful derivatisation of these cysteines with iodoacetamides.1 The inherent fluorescence of GFPuv also makes it a useful candidate for fluorescence based binding assays or cell labelling studies.16 active, mutants of GFPuv were created using a mixture of site directed mutagenesis and DNA shuffling (including one mutant containing six reactive cysteine residues). This was achieved by producing random combinations of two synthetic variants of GFPuv, one of which contained 33 surface cysteines. 94 bacterial colonies expressing active GFPuv were then sequenced and the new chimeric genes analysed. Four monosaccharides and one trisaccharide (N-glycan core mimic) suitable for the chemical glycosylation via cysteines were synthesised and successfully used to create a selection of homogeneous neoglycoproteins. These neoglycoproteins were demonstrated to interact differently with different lectins (including ConA, GNL and Jacalin) in a qualitative fluorescence based assay. Interactions were shown to vary with glycan structure, position of glycosylation sites and the number of glycosylation sites.

Cyclodextrin glucanotransferase (EC 2.4.1.19) from the alkaliphilic Bacillus sp. G-825-6 converts starch mainly to c-cyclodextrin (CD8). A combination of error-prone PCR and DNA shuffling was used to obtain variants of this enzyme with higher product specificity for CD8 and a broad pH activity range. The variant S54 with seven amino acid substitutions showed a 1.2-fold increase in CD8-synthesizing activity and the product ratio of CD7:CD8 was shifted to 1:7 compared to 1:3 of the wild-type enzyme. Nine amino acid substitutions of the cyclodextrin glucanotransferase were performed to generate the variant S35 active in a pH range 4.0–10.0. Compared to the wild-type enzyme which is inactive below pH 6.0, S35 retained 70% of its CD8-synthesizing activity at pH 4.0.

Made available in DSpace on 2016-06-02T19:02:39Z (GMT). No. of bitstreams: 1 2372.pdf: 3456814 bytes, checksum: 7894f1e8062bb948621e2d222d01e3b0 (MD5) Previous issue date: 2008-11-06 / Financiadora de Estudos e Projetos / The Molecular Evolution of the living organisms is a slow process that occurs over the years producing mutations and recombinations at the genetic material, i.e. at the DNA. The mutations can occur as nucleotide remotion, insertion and/or substitution at the DNA chain. The Directed Molecular Evolution is an in vitro process that tries to improve biological functions of specific molecules producing mutations at the molecule s genetic material, mimicking the natural process of evolution. Many technics that simulate in vitro molecular evolution, among them the DNA shuffling, have been used aiming to improve specific properties of a variety of commercially important products as pharmaceutical proteins, vaccines and enzymes used in industries. The original DNA shuffling methodology can be sumarized by the following steps: 1) selection of the parental sequences; 2) random fragmentation of the parental sequences by an enzyme; 3) repeated cycles of PCR (Polymerase Chain Reaction), in order to reassemble the DNA fragments produced in the previous step; 4) PCR amplification of the reassembled sequences obtained in step 3). The DNA shuffling technic success can be measured by the number of recombinat molecules found at the DNA shuffling library obtained, since these recombinant molecules potentially have improved functionalities in relation to their parent since their sequence may accumulate beneficial mutations originated from distinct parent sequences. Nowadays some few models can be found in the literature whose purpose is to suggest optimization to this process aiming the increase of the genetic diversity of the DNA shuffling library obtained. This research work presents a comparative study of four models used to predict/estimate the DNA shuffling results. In addition a computational tool for simulating the DNA shuffling proccess is proposed and implemented in an environment where other functionalities related to the analyses of the parental sequences and the resulting sequences from the DNA shuffling library is also implemented. / A Evolução Molecular dos organismos vivos é um processo lento que ocorre ao longo dos anos e diz respeito às mutações e recombinações sofridas por um determinado organismo em seu material genético, ou seja, em seu DNA. As mutações ocorrem na forma de remoções, inserções e/ou substituições de nucleotídeos ao logo da cadeia de DNA. A Evolução Molecular Direta é um processo laboratorial, ou seja, in vitro, que visa melhorar funções biológicas específicas de moléculas por meio de mutações/recombinações em seu material genético, imitando o processo natural de evolução. Diversas técnicas que simulam a evolução molecular em laboratório, entre elas a técnica de DNA shuffling, têm sido amplamente utilizadas na tentativa de melhorar determinadas propriedades de uma variedade de produtos comercialmente importantes como vacinas, enzimas industriais e substâncias de interesse famacológico. A metodologia original de DNA shuffling pode ser sumarizada pelas seguintes etapas: 1) seleção dos genes de interesse, dito parentais; 2) fragmentação enzimática dos genes; 3) ciclos de PCR (Polymerase Chain Reaction), para que ocorra a remontagem dos fragmentos; 4) amplificação das seqüências remontadas cujo tamanho é igual a dos parentais. O sucesso ou não da técnica de DNA shuffling pode ser medido pelo número de moléculas recombinantes encontradas na biblioteca de DNA shuffling obtida, uma vez que estas podem apresentar melhorias funcionais em relação aos parentais pelo fato de, possivelmente, acumularem em sua seqüência mutações benéficas presentes em parentais distintos. Atualmente podem ser encontradas na literatura algumas poucas modelagens computacionais capazes de sugerir otimizações para o processo, com vistas em aumentar a diversidade genética da biblioteca resultante. O presente trabalho apresenta um estudo comparativo de quatros modelos para predição/estimativa de resultados de experimentos de DNA shuffling encontrados na literatura bem como a proposta e implementação de uma ferramenta computacional de simulação para o processo de DNA shuffling. A ferramenta de simulação foi implementada em um ambiente que disponibiliza outras funcionalidades referentes à análise das seqüências a serem submetidas ao shuffling bem como ferramentas para análise das seqüências resultantes do processo.

Cyclodextrin glucanotransferase (EC 2.4.1.19) from the alkaliphilic Bacillus sp. G-825-6 converts starch mainly to c-cyclodextrin (CD8). A combination of error-prone PCR and DNA shuffling was used to obtain variants of this enzyme with higher product specificity for CD8 and a broad pH activity range. The variant S54 with seven amino acid substitutions showed a 1.2-fold increase in CD8-synthesizing activity and the product ratio of CD7:CD8 was shifted to 1:7 compared to 1:3 of the wild-type enzyme. Nine amino acid substitutions of the cyclodextrin glucanotransferase were performed to generate the variant S35 active in a pH range 4.0–10.0. Compared to the wild-type enzyme which is inactive below pH 6.0, S35 retained 70% of its CD8-synthesizing activity at pH 4.0.

<p>Evolution of enzymes with novel functional properties has gained much attention in recent years. Naturally evolved enzymes are adapted to work in living cells under physiological conditions, circumstances that are not always available for industrial processes calling for novel and better catalysts. Furthermore, altering enzyme function also affords insight into how enzymes work and how natural evolution operates. </p><p>Previous investigations have explored catalytic properties in the directed evolution of mutant libraries with high sequence variation. Before this study was initiated, functional analysis of mutant libraries was, to a large extent, restricted to uni- or bivariate methods. Consequently, there was a need to apply multivariate data analysis (MVA) techniques in this context. Directed evolution was approached by DNA shuffling of glutathione transferases (GSTs) in this thesis. GSTs are multifarious enzymes that have detoxication of both exo- and endogenous compounds as their primary function. They catalyze the nucleophilic attack by the tripeptide glutathione on many different electrophilic substrates. </p><p>Several multivariate analysis tools, <i>e.g.</i> principal component (PC), hierarchical cluster, and K-means cluster analyses, were applied to large mutant libraries assayed with a battery of GST substrates. By this approach, evolvable units (quasi-species) fit for further evolution were identified. It was clear that different substrates undergoing different kinds of chemical transformation can group together in a multi-dimensional substrate-activity space, thus being responsible for a certain quasi-species cluster. Furthermore, the importance of the chemical environment, or substrate matrix, in enzyme evolution was recognized. Diverging substrate selectivity profiles among homologous enzymes acting on substrates performing the same kind of chemistry were identified by MVA. Important structure-function activity relationships with the prodrug azathioprine were elucidated by segment analysis of a shuffled GST mutant library. Together, these results illustrate important methods applied to molecular enzyme evolution.</p>

Evolution of enzymes with novel functional properties has gained much attention in recent years. Naturally evolved enzymes are adapted to work in living cells under physiological conditions, circumstances that are not always available for industrial processes calling for novel and better catalysts. Furthermore, altering enzyme function also affords insight into how enzymes work and how natural evolution operates. Previous investigations have explored catalytic properties in the directed evolution of mutant libraries with high sequence variation. Before this study was initiated, functional analysis of mutant libraries was, to a large extent, restricted to uni- or bivariate methods. Consequently, there was a need to apply multivariate data analysis (MVA) techniques in this context. Directed evolution was approached by DNA shuffling of glutathione transferases (GSTs) in this thesis. GSTs are multifarious enzymes that have detoxication of both exo- and endogenous compounds as their primary function. They catalyze the nucleophilic attack by the tripeptide glutathione on many different electrophilic substrates. Several multivariate analysis tools, e.g. principal component (PC), hierarchical cluster, and K-means cluster analyses, were applied to large mutant libraries assayed with a battery of GST substrates. By this approach, evolvable units (quasi-species) fit for further evolution were identified. It was clear that different substrates undergoing different kinds of chemical transformation can group together in a multi-dimensional substrate-activity space, thus being responsible for a certain quasi-species cluster. Furthermore, the importance of the chemical environment, or substrate matrix, in enzyme evolution was recognized. Diverging substrate selectivity profiles among homologous enzymes acting on substrates performing the same kind of chemistry were identified by MVA. Important structure-function activity relationships with the prodrug azathioprine were elucidated by segment analysis of a shuffled GST mutant library. Together, these results illustrate important methods applied to molecular enzyme evolution.

Made available in DSpace on 2016-06-02T19:02:40Z (GMT). No. of bitstreams: 1 3427.pdf: 6177352 bytes, checksum: f9f6df521c16815add3a5b1dba3f6753 (MD5) Previous issue date: 2009-04-30 / Financiadora de Estudos e Projetos / Cystatins are proteins that inhibit specifically cysteine peptidases. The Canecystatin 1 gene which codifies a protein containing 106 amino acids residues was identified in sugarcane and possesses significant similarity with Oryzacystatin, a cystatin from rice. In order to obtain a cystatin with improved activity, direct evolution experiments were carried out. A DNA shuffling library was constructed using these two cystatins. One clone named A10PL3 obtained from these shuffled cystatins was selected, expressed in E.coli, purified and an analyzed by activity assays. These results showed that the activity of hybrid protein A10PL3 increased, in particular regarding its inhibitory activity on cathepsin B compared with its two precursors. The present study aimed to revert the changes of individual clone A10PL3 through site-directed mutations, generating three mutant cystatins: Mutant I (Thr17Ile), Mutant II (Gln 84Leu) and Mutant III (Thr17Ile); (Gln84Leu). Assays for inhibitory activity against the human cathepsins B and L were performed. Structural studies were also made by means of molecular modeling of proteins by homology that were enabled to understand the molecular mechanisms related to improvement of the inhibitory activity of these cystatins. These studies therefore corroborate with the observed data previously which demonstrated the improvement of specific protein A10PL3 in cathepsin B inhibition (Ki 16 nM) in relation to their parents. The mutants I, II and III, did not present improvement in inhibitory activity against cathepsin B. The structural studies revealed that the mutations performed on cystatin A10PL3 destabilized the hydrophobic core making it more flexible, thus increasing the inhibitory activity on cathepsin B. The absence of interactions underlying the hydrophobic core resulted in a trend of lower solubility, probably due to their inability to adopt a compact formation, which resulted in the exposure of some residues which are part of that core, which can lead to aggregation and also contribute to increasing the flexibility of cystatin, influencing their inhibitory activity. / Cistatinas são proteínas que inibem especificamente cisteíno peptidases. A canacistatina 1, uma proteína com 106 resíduos de aminoácidos, apresenta grande similaridade com a proteína Orizacistatina 1, uma cistatina de arroz. Com o objetivo de se obter uma cistatina com a atividade inibitória melhorada, experimentos de evolução molecular direta de proteínas foram realizados, através da construção de uma biblioteca de DNA shuffling usando estas duas cistatinas. Um clone denominado A10PL3 foi selecionado, expresso, purificado e submetido a ensaios de inibição de atividade enzimática. Foi demonstrado que a proteína A10PL3 exibiu aumento da atividade inibitória contra catepsina B humana em comparação com seus dois precursores. O presente estudo teve como objetivo reverter as alterações do clone A10PL3 através de mutações sítio-dirigidas, gerando mais três cistatinas mutantes: Mutante I (Thr17Ile), Mutante II (Gln 84 Leu) e Mutante III (Thr17Ile); (Gln84Leu). Ensaios de atividade inibitória dos mutantes contra as catepsinas humanas B e L foram realizados. Além disso, foram desenvolvidos estudos estruturais por meio de modelagem molecular de proteínas por homologia que permitiram a compreensão dos determinantes moleculares relacionados à melhoria da atividade inibitória destas cistatinas. Os resultados aqui apresentados são importantes, pois corroboram com os dados observados anteriormente, demonstrando a melhora na especificidade da atividade inibitória da proteína A10PL3 contra a catepsina B (Ki = 16 nM) em relação aos seus parentais. Os mutantes I, II e III não foram capazes de inibir a catepsina B. Os estudos estruturais revelaram que as mutações na cistatina A10PL3 desestabilizaram o núcleo hidrofóbico provavelmente tornando a região N-terminal da proteína mais flexível, influenciando a atividade inibitória contra a catepsina B. A desestabilização do núcleo hidrofóbico resultou na tendência de uma menor solubilidade, provavelmente devido à sua tendência de expor resíduos que fazem parte desse núcleo, o que pode levar à agregação e também contribuir para o aumento da flexibilidade da cistatina.

Porcine circovirus type 2 (PCV2) is a small, non-enveloped, single-stranded DNA virus that causes disease in pigs and is an economically important pathogen affecting pig populations worldwide. PCV2 contains two major open reading frames (ORF): ORF1 encodes two replicase proteins and ORF2 encodes the immunogenic capsid protein. There are three genotypes of PCV2 (PCV2a, PCV2b, and PCV2c), but vaccines available for PCV2 infection are only targeted against PCV2a. The objective of this thesis was to create viable chimeric PCV2 viruses with an ORF2 displaying genetic diversity from all PCV2 genotypes by synthetic DNA shuffling. Variation was identified at 55 amino acid positions in the ORF2 gene among 853 PCV2 capsid gene sequences available in the GenBank database. Degenerate oligonucleotide primers spanning ORF2 were synthesized to contain this naturally observed sequence diversity. Sets of overlapping oligonucleotide primers were fused together using overlap extension PCR to create full-length shuffled ORF2 sequences. The shuffled library of the ORF2 genes was subsequently cloned into the genomic backbone of a wildtype PCV2a infectious DNA clone and transfected into porcine kidney cells (PK-15). After transfection and infection of PK-15 cells, viability of chimeric viruses was screened by immunofluorescence assay (IFA) using anti-PCV2 Rep antibodies. PCR was used to amplify the genomes of viable shuffled viruses from infected cells. PCV2 viruses containing an ORF2 displaying genetic diversity from PCV2a, PCV2b, and PCV2c were isolated in vitro. These shuffled PCV2 viruses may be used as potential candidates for a broadly-protective PCV2 vaccine, although additional studies are warranted to determine in vivo infectivity and pathogenicity. / Master of Science

This thesis presents the application of different protein engineering methods on enzymes and non-catalytic proteins that act upon xyloglucans. Xyloglucans are polysaccharides found as storage polymers in seeds and tubers, and as cross-linking glucans in the cell wall of plants. Their structure is complex with intricate branching patterns, which contribute to the physical properties of the polysaccharide including its binding to and interaction with other glucans such as cellulose. One important group of xyloglucan-active enzymes is encoded by the GH16 XTH gene family in plants, including xyloglucan endo-transglycosylases (XET) and xyloglucan endo-hydrolases (XEH). The molecular determinants behind the different catalytic routes of these homologous enzymes are still not fully understood. By combining structural data and molecular dynamics (MD) simulations, interesting facts were revealed about enzyme-substrate interaction. Furthermore, a pilot study was performed using structure-guided recombination to generate a restricted library of XET/XEH chimeras. Glycosynthases are hydrolytically inactive mutant glycoside hydrolases (GH) that catalyse the formation of glycosidic linkages between glycosyl fluoride donors and glycoside acceptors. Different enzymes with xyloglucan hydrolase activity were engineered into glycosynthases, and characterised as tools for the synthesis of well-defined homogenous xyloglucan oligo- and polysaccharides with regular substitution patterns. Carbohydrate-binding modules (CBM) are non-catalytic protein domains that bind to polysaccharidic substrates. An important technical application involves their use as molecular probes to detect and localise specific carbohydrates in vivo. The three-dimensional structure of an evolved xyloglucan binding module (XGBM) was solved by X-ray diffraction. Affinity-guided directed evolution of this first generation XGBM resulted in highly specific probes that were used to localise non-fucosylated xyloglucans in plant tissue sections. / QC 20100902