Hajj, the annual Islamic pilgrimage to Makkah, Saudi Arabia, is a unique mass gathering event that brings more than 2 million individuals from around the world. Several public health considerations, such as the spread of infectious diseases, must be taken into account with this large temporary influx of people. Gastrointestinal diseases, such as diarrhea, are common at Hajj, yet little is known about the etiology. The human gut microbiome, collection of organisms residing within the intestinal tract, has been under intense study recently, since next generation DNA sequencing technologies allow for extensive surveying of genetic material found in complex biological samples, such as those containing many different organisms. Thus, using 16S rRNA and metagenomic shotgun sequencing, we have characterized the gut microbiome of over 612 pilgrims with and without diarrhea. Several metadata factors, such as hospitalization and different comorbidities, were found to have significant effects on the overall gut microbiome composition. Metagenomic shotgun sequencing efforts revealed the presence of antimicrobial resistance genes originating from disparate regions from around the world. This study provides a snapshot of information concerning the health status of the gut microbiome of Hajj pilgrims and provides more context to the investigation of how to best prepare for mass gathering events.

Le compostage est un procédé anthropique basé sur le processus naturel de décomposition de la biomasse qui exploite l'activité enzymatique des microorganismes sous le contrôle de plusieurs facteurs environnementaux. Les résidus lignocellulosiques de par leur composition et leur faible pourcentage d'humidité sont particulièrement adaptés au compostage dans lequel ils jouent le rôle d’élément structurant. Bien que majoritairement d’origine végétale, la matière organique dirigée vers les sites de compostages est très diversifiée, tout comme les types de contaminants qu’elle peut incidemment contenir et dont l’impact sur les processus de biodégradation, et de surcroit leur rémanence dans l’environnement, reste largement à investiguer. L’objectif de cette thèse vise ainsi à faire état de l’effet de la composition de la biomasse lignocellulosique et de la présence d’un contaminant fréquent tel que le glyphosate sur le compostage. Pour ce faire, le suivi de la transformation de la matière organique végétale et de la dégradation du glyphosate, l’évolution des paramètres physicochimiques et la dynamique de recrutement des populations bactériennes ont été effectués tout au long du processus. Deux expériences menées sur le terrain visaient dans un premier temps à mesurer l’effet de l’âge d’une plante ligneuse, dans ce cas-ci le saule arbustif (Salix), et d’une période d’entreposage hivernal sur la transformation de la biomasse, et dans un deuxième temps à étudier les dynamiques de succession bactériennes impliquées dans le cycle du carbone et de l’azote lors du compostage de résidus végétaux. Les résultats obtenus ont révélé une différence dans la composition de la biomasse des tiges âgées de 2 ans et de 3 ans. Alors que les premiers contenaient plus de composés extractibles, les seconds étaient plus riches en sucres structuraux. Ces différences expliquent une hausse des températures plus forte et plus rapide dans le tas de copeaux de tiges plus jeunes. La diminution des composés extractibles, la conservation des sucres structuraux et l’augmentation de la proportion de lignine démontrent l’importance de la source de carbone soluble pour l’initiation de la décomposition du bois et la récalcitrance des éléments lignocellulosiques durant l’entreposage hivernal. La seconde expérience a mis en évidence une très grande diversité de bactéries responsables de la décomposition de la cellulose, des hémicelluloses et de la lignine durant la phase thermophile du compostage. Cette phase qui était le théâtre d’une activité intense comptait moins d’espèces, mais ces dernières étaient très abondantes, une tendance qui s’est inversée avec la maturation de la matière organique. La dynamique observée traduit une redondance fonctionnelle des communautés qui semblent évoluer selon la température, le taux d’oxygène et la nature du substrat disponible. Une troisième expérience menée en milieu contrôlé a ensuite démontré l’impact négligeable du glyphosate sur l’activité microbienne et l’évolution des paramètres physicochimiques lors du compostage. Le glyphosate était presque ou entièrement dégradé à l’issue du compostage et la présence du principal produit de dégradation, l’acide aminométhylphosphonique (AMPA) n’a d’ailleurs même pas pu être quantifiée durant l’expérience. L’impact du glyphosate sur les communautés bactériennes était également négligeable. Seules quelques bactéries étaient différentiellement abondantes entre les deux traitements, la grande majorité étant moins abondante dans le traitement contenant du glyphosate. La richesse en espèces aux différents temps d’échantillonnage était la même entre le traitement témoin et le traitement contenant du glyphosate « pur » et l’analyse de la bêta-diversité n’a relevé aucune différence significative entre les communautés présentes dans le traitement témoin et le traitement glyphosate. Cette thèse a ainsi fait valoir l’importance de la nature initiale de la matière organique sur l’activité microbienne, le recrutement et la dynamique des communautés durant le compostage, tandis que la présence du contaminant glyphosate s’est présenté comme un facteur beaucoup moins déterminant sur les processus de décomposition et l’abondance des espèces bactériennes. Ces informations devraient non seulement permettre d’optimiser le traitement de la matière organique par compostage, mais aussi de mieux évaluer les risques potentiels associés au compostage de biomasse contaminé. / Composting is an anthropic process based on the natural decay of biomass that exploits the enzymatic activity of microorganisms under the control of several environmental factors. Due to their composition and low moisture content, lignocellulosic residues are particularly suitable for composting and serve as a structuring element, which confers them an important role in the process. Although mostly of plant origin, the organic matter (OM) directed towards composting sites is highly diversified, as are the types of contaminants it can contain. The impact of these contaminants, such as glyphosate, on the biodegradation process and their persistence in the environment remain to be investigated. The objective of this thesis is thus to report on the effect of the composition of the lignocellulosic biomass and the presence of glyphosate on the evolution of the physicochemical parameters and the recruitment of bacteria during composting, while ensuring the follow-up of the transformation of the vegetable organic matter and the degradation of glyphosate during the process. Two field studies were conducted to measure the effect of stem age and winter storage on the transformation of wood chips, and to study the dynamics of bacterial succession involved in the carbon and nitrogen cycle during the composting of plant residues. The results obtained revealed a difference in the composition of 2-year-old and 3-year-old stems from shrub willow (Salix sp.), with the younger ones containing more extractable compounds and the more mature ones richer in structural sugars. These differences were reflected in a higher and faster temperature rise in the younger chip pile. A decrease in extractives, retention of structural sugars, and an increase in the proportion of lignin demonstrate the importance of the soluble carbon source for the initiation of wood decomposition and recalcitrance of lignocellulosic elements. The second experiment revealed a very high diversity of bacteria responsible for the decomposition of cellulose, hemicelluloses and lignin during the thermophilic phase of composting. This phase, during which intense activity took place, had fewer species, but they were very abundant, a trend that reversed as the organic matter matured. The observed dynamics reflect a functional redundancy of the communities, which seems to evolve according to the temperature, oxygen level and nature of the available substrate. A third experiment conducted in a controlled environment demonstrated the negligible impact of glyphosate on microbial activity and the evolution of physicochemical parameters during composting. Glyphosate was almost or completely degraded after composting, while the main product of degradation, aminoethylphosphonic acid (AMPA), was not detected. The impact of glyphosate on bacterial communities was also negligible, while species richness at different sampling times was the same when comparing the control treatment and the treatment containing "pure" glyphosate. The beta-diversity analysis found no significant difference between the communities present in the control and glyphosate treatments, while a few bacteria were differentially abundant between the two treatments, the vast majority being less abundant in the glyphosate treatment. This thesis has thus highlighted the importance of the initial nature of the organic matter on microbial activity as well as on the recruitment and dynamics of bacterial communities during composting, while the presence of glyphosate was shown to be a weak determinant of decomposition processes and species abundance. This information should help to optimize the treatment of organic matter by composting and to better assess the potential risks associated with composting contaminated biomass.

Molecular diagnostic assays have been developed and utilized to diagnose and to confirm the diagnoses of many plant-parasitic nematodes. We screened several gene sequences of soybean cyst nematode (SCN) [Heterodera glycines, Ichinohen] for their use as molecular markers. A methodology then was developed to use them to detect and quantify the number of H. glycines directly from Mississippi soil. A novel procedure utilizing multiple databases containing nematode DNA and EST sequences was developed to assist in the selection of SCN primers used in the PCR and qPCR assays. In vitro testing demonstrated that the DNA primers and probes developed from the novel procedure for the qPCR assays could accurately detect the presence of SCN. Subsequent testing resulted in a trend of increasing observed numbers of SCN contributing to increasing estimates by qPCR.

M. Tech. (Department of Biotechnology, Faculty of Applied and Computer Sciences), Vaal University of Technology / Lipases have been one of the important biocatalysts that catalyse the transformation of lipids to yield very important products that can be of beneficial in food, agriculture, pharmaceutical medicine and for the biodiesel production. In the search for novel biocatalysts, notably lipases, the conventional culture-based techniques were used but it can only address sourcing the biomolecule from 1-10% of the microbial population leaving the wealth of the biomolecules packed in 90-99% of the microbial community unaccounted for. Metagenomic technique, which is culture-independent, was developed as a comprehensive approach to address literally 100% of the microbial population thereby maximizing the chances of obtaining novel biocatalysts with superior physico-chemical and catalytic traits. In principle, any biomolecule including lipase could be sourced from any biologically-active environment, of which animal rumen is one. However, among the rumenant animals goat has diverse feeding habit, thereby laying ground for increased microbial diversity in its gastro-intestinal tract. It was thus, postulated that goat rumen could be potential source of novel lipase isoforms. Therefore, the aim of the study was to extract metagenomic DNA from goat rumen and construct a metagenomic fosmid library and screen the library for lipase isoforms. The fosmid clones were functionally screened using 1% tributyrin as a substrate and five positive clones were selected. From the five clones, two fosmids were selected for further study. Following nucleotide sequencing and in-silico analysis of the insert of the two selected clones, one lipase encoding open reading frame (Lip-VUT3 and Lip-VUT5) from each fosmid clones of approximately 212 and 248 amino acids, respectively, was identified. The amino acid sequences of the Lip-VUT3 ORF contained a classical conserved lipase GSDL sequence motif while the amino acid sequences of the Lip-VUT5 ORF contained a classical G-L-S-L-G conserved lipase/esterase motif sequence. The two genes (Lip-VUT3 and Lip-VUT5) were successfully expressed in Escherichia coli BL21 (DE3) and the purified enzymes exhibited respective temperature optima of 60 °C and 70 °C, and respective pH optima of 6.0 and 10.0. Further biochemical characterisation indicated that Lip-VUT3 and Lip-VUT5 lipases showed tolerance towards a wide concentration (50%-100%) of methanol. Lip-VUT3 had a Km value of 0.287 mM while Lip-VUT5 had a Km value of 0.556 Mm. This shows that Lip-VUT3 lipase has a higher affinity for olive oil than Lip-VUT5. Lip-VUT3 and Lip-VUT5 were characterised to be true lipases that have been recovered from the rumen environment through metagenomic approach. Therefore, the study proved that metagenomic approach helps in recovering novel lipase isoforms with potential down stream industrial and therapeautic applications from goat rumen metagenome, a rich but untapped source.

No description available.

Cystic fibrosis (CF) is an autosomal recessive genetic disorder involving a mutation in the CF transmembrane conductance regulator protein (CFTR), which causes dysfunctional transport of chloride ions across cell membranes. CF affects multiple body systems and a few of its symptoms include chronic cough, difficulty breathing, obstructive airway disease, bacterial pulmonary infections, maldigestion, malabsorption, pancreatitis, and male infertility. Until recently, treatment options have been limited to alleviating symptoms, but a new classification of drugs, CFTR modulators, provide an opportunity to slow the progression of the disease and improve clinical outcomes. The effect of CFTR modulators may be attributed to the reduction of persistently colonizing bacteria in CF lungs. Though, the effects of modulators on microbial communities colonizing the CF lung remains unknown, specifically with common respiratory pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus. Particularly, previous CF studies have been limited in scope due to focusing on only one type of modulator and by using low-yield sequencing techniques. To address this gap, we seek to study the changes in CF respiratory pathogens of patients initiating CFTR modulator therapy at Nemours Hospital using long-read metagenomic sequencing (Oxford Nanopore) of longitudinally collected respiratory samples. We have optimized a protocol for host DNA depletion and microbial metagenomic sequencing to characterize the respiratory microbiome. This study focuses on utilizing these sequencing data to compare the microbiome among two healthy controls to pre-CFTR-treatment microbial communities of two recruited pediatric CF patients.

No description available.

O sequenciamento de alto desempenho, pela redução dos custos nos últimos anos, vem sendo cada vez mais utilizado para prospectar e identificar vírus. Estes métodos são extremamente mais sensíveis que outros métodos moleculares, e capazes de sequenciar genomas virais sem conhecimento prévio, clonagem ou isolamento. Neste estudo, utilizamos o sequenciamento de alto desempenho para conhecer, e caracterizar genomas completos de arbovírus isolados nas Américas, incluindo a prospecção de vírus em amostras de pequenos mamíferos do estado de São Paulo, Brasil. Assim, sequenciamos e caracterizamos 44 Bunyavirales, 35 no gênero Orthobunyavirus, família Peribunyaviridae, oito no gênero Phlebovirus, família Phenuiviridae, e um orthonairovírus, família Nairoviridae. Entre os Bunyavirales identificamos uma provável nova estratégia de codificação da proteína não estrutural do segmento pequeno, e ainda identificados sete vírus que são reassortants naturais. Caracterizamos o genoma completo do vesiculovírus Piry, determinando sua relação filogenética com arbovírus pertencentes ao gênero Vesiculovirus, família Rhabdoviridae. Prospectamos novos vírus, os quais incluímos em três famílias, Parvoviridae, Anelloviridae e Hepeviridae. Na família Parvoviridae, identificamos 20 chapparvovírus endógenos e exógenos, oriundos de grande diversidade de hospedeiros vertebrados e invertebrados, e que representam uma nova subfamília, a Chapparvovirinae. Também, descrevemos onze novas espécies de Anelloviridae em roedores silvestres e marsupiais, fornecendo importantes informações sobre a diversidade, a taxonomia, e ainda ampliamos a gama de hospedeiros de anellovírus conhecidos. Por fim, identificamos e caracterizamos uma nova espécie de Orthohepevirus de roedores Sigmodontinae, nomeada \"Orthohepevirus E\". Acreditamos que estamos a fornecer relevantes informações sobre genômica, epidemiologia molecular, evolução e taxonomia de 45 arbovírus americanos, bem como sobre 13 novas espécies virais encontradas em pequenos mamíferos. Tais informações deverão dar subsídios para múltiplos futuros estudos visando compreender a importância destes novos vírus e a desenvolver métodos diagnósticos. / In last years, high-throughput sequencing (HTS) has been cost-effective and increasingly used for prospection and identification of viruses. These methods are extremely more sensitive than other molecular methods and are capable of sequencing viral genomes without prior knowledge, cloning or isolation. In this study, we used HTS approach to identify and characterize complete genomes of arbovirus isolated in the Americas, as well as viral prospection in samples of small mammals from São Paulo State, Brazil. Thus, we sequenced and characterized 44 viruses from Bunyavirales order, including 35 in Orthobunyavirus genus, family Peribunyaviridae, eight in Phlebovirus genus, family Phenuiviridae, and one in Orthonairovirus genus, family Nairoviridae. Among the Bunyavirales we identified a novel putative strategy for encoding the non-structural protein of the small segment, as well as we identified seven viruses that are natural reassortants. Also, we characterized the complete genome of the Piry vesiculovirus, determining its phylogenetic relationship with arboviruses belonging to the Vesiculovirus genus, family Rhabdoviridae. On the other hand, we have prospected novel viruses, which included in three families, Parvoviridae, Anelloviridae, and Hepeviridae. In the Parvoviridae family, we identified 20 endogenous and exogenous chapparvoviruses from a broad diversity of vertebrate and invertebrate hosts, representing a new subfamily, the Chapparvovirinae. Also, we have described eleven new species of Anelloviridae in wild rodents and marsupials, providing important information on diversity, taxonomy and even broadening the range of known anelloviruses hosts. Finally, we identified and characterized a novel species of orthohepevirus in Sigmodontinae rodent, named \"Orthohepevirus E\". We believe that we are providing relevant relevant on genomics, molecular epidemiology, evolution and taxonomy of 45 American arboviruses, as well as on 13 new viral species found in small mammals. Thus, these informations should provide support for multiple future studies to understand the importance of these new viruses, as well as to develop diagnostic methods.

O Alquilbenzeno Linear Sulfonado (LAS) é um surfactante aniônico de degradação complexa. Via ensaio cinético em batelada ajustou-se modelo de inibição por excesso de substrato na remoção de LAS e matéria orgânica de água residuária de lavanderia comercial (ARLC) em co-digestão com esgoto doméstico (ED). A adição de 50 mg L-1 de etanol (EOH) resultou em maiores valores para velocidade específica de utilização do substrato (robs) e concentração mais elevada de LAS que fornece o maior robs (18,98 mg LAS L-1 e 2,39 mg LAS L-1 na presença e ausência de etanol, respectivamente). Areia com 1,0 mm de diâmetro foi escolhida como material suporte). Objetivou-se otimizar a remoção do surfactante de ARLC + ED (1:3 volume; &#8764; 20 mg LAS L -1) em RLF em escala aumentada (19,8L) através de: (i) adição de etanol em diferentes dosagens; (ii) variação da velocidade ascensional (vasc) aplicada ao leito; e (iii) aumento do tempo de detenção hidráulica (TDH). Desta forma, para TDH de 18 h foram realizadas as seguintes fases operacionais: (I) ARLC + ED + 1,3 velocidade mínima de fluidificação (vmf); (II) ARLC + ED + 50 mg EOH L -1 + 1,3 vmf; (III) ARLC + ED+200 mg EOH L-1 + 1,3 vmf; (IV) ARLC + ED +200 mg L-1 + 1,0 vmf; (V) ARLC + ED + 200 mg L-1 + 0,7 vmf; (VI) ARLC + ED + 100 mg L-1 + 1,0 vmf; e para TDH de 30 h: (VII) ARLC + ED + 1,0 vmf. Não se observou diferença significativa na eficiência de remoção de DQO e LAS (&#8764; 50%; p < 0,5) nas fases I à IV. O decréscimo da vasc (0,7 vmf) resultou em 29% de eficiência de remoção de LAS (V) e o aumento do TDH em 86% de eficiência de remoção de LAS (VII). Nas fases VI e VII observou-se maior remoção de DQO (&#8805; 70%). As menores vasc e 200 mg EOH L-1 favoreceram acúmulo de ácidos no sistema (IV e V). No efluente do RLF foram identificados 17 compostos recalcitrantes. Para vasc = 0,7 vmf, foi observada maior diversidade de compostos recalcitrantes, em sua maioria, ftalatos. Caracterização taxonômica e funcional dos microrganismos para as fases III, IV e V (variação da vasc) e VII (maior eficiência de remoção de LAS e DQO) foi realizada por metagenômica. Foram identificados microrganismos dos domínios Archaea e Bacteria, sendo que a diminuição da vasc resultou em maior abundância relativa de arqueias metanogênicas, como Methanosarcina e genes relacionados a F420 reducing hydrogenase que é transportadora central de elétrons na metanogênese. Diversidade de gêneros foram identificados do domínio Bacteria (Geobacter, Thauera, Pseudomonas, Chryseobacterium, Sulfuricurvum e Sulfurospirillum, etc.) e genes codificadores de enzimas que atuam nas diferentes etapas de degradação do LAS: (a) adição de fumarato (fumarate redutase); (b) beta-oxidação (3-hidroxiacil-CoA desidrogenase); (c) clivagem do anel benzênico (benzoyl-CoA reductase); (d) dessulfonação (Adenylyl-sulfate reductase). Na amostra da fase VII, foram identificados genes relacionados à etapa de dessulfonação com maior abundância relativa, se comparada às demais fases. Para maior vasc observou-se maior abundância relativa de genes relacionados à fosforilação oxidativa com Chryseobacterium como principal representante. / The linear alkylbenzene sulfonate (LAS) is in the laundry detergent composition and it presents complex degradation. Through kinetics assays in batch tests, an inhibition kinetic model by subtrate excess was adjusted to the data in the removal of LAS and organic matter from laundry wastewater (LW) in co-digestion with domestic sewage (DS). The addition of 50 mg L-1 of ethanol (EOH) to the influent resulted in higher values for the specific substrate rate (robs) as well as higher LAS concentration that provided the maximum LAS utilization rate by the biomass (Sbm) (18.98 mg LAS L-1 and 2. 39 mg LAS L-1 in the presence and absence of ethanol, respectively). Sand with 1.0 mm of diameter was chosen as supporting material for the fluidized bed reactor (FBR). The purpose of the present study was to optimize the removal of surfactant in LW + DS (1: 3 volume; &#8764; 20 mg LAS L-1) by using an upscale FBR (19.8 L) through: (i) adding ethanol in different dosages; (ii) varying the upflow velocity (vup) applied to bed; and (iii) increasing the hydraulic retention time (HRT). Thus, for 18h of HRT, the following stages were performed: (I) LW + DS + 1,3 fluidization minimum velocity (vfm); (II) LW + DS + 50 mg EOH L-1 + 1.3 vfm; (III) LW + DS + 200 mg EOH L-1 + 1.3 vfm; (IV) LW + DS + 200 mg L-1 + 1.0 vfm; (V) LW + DS + 200 mg L-1 + 0.7 vfm; (VI) LW + DW + 100 mg L-1 + 1.0 vfm; and for 30 h of HRT: (VII) LW + DS + 1.0 vfm. There was no significant difference in the efficiency of COD and LAS removal (&#8764; 50%, p < 0.5) in stages I to IV. The vup decrease (0.7 vfm) resulted in LAS removal efficiency of 29% (V) and the HRT increase resulted in LAS removal efficiency of 86% (VII). In stages VI and VII, COD removal &#8805; 70% was observed. Lower vup as well as ethanol dosage of 200 mg L-1 favored system acidification (IV and V). In the FBR effluent, 17 recalcitrant compounds were identified. For vup = 0.7 vfm, large diversity of recalcitrant compounds, mostly phthalates, was observed. A taxonomic and functional characterization of the microorganisms was performed by metagenomics analysis in stages III, IV and V (vup variation) and VII (higher efficiency of LAS and COD removal). Microorganisms of Archaea and Bacteria domains were identified, and the decrease of vup resulted in a higher relative abundance of methanogenic archaea, mainly Methanosarcina. Genes related to F420, which are the central electron carrier in the methanogenesis, were identified. Genera diversity was classified in Bacteria domain (Geobacter, Thauera, Pseudomonas, Pseudomonas, Chryseobacterium, Sulfuricurvum and Sulfurospirillum, etc.). Enzyme-encoding genes that act on different stages of LAS degradation were found: (a) addition of fumarate (fumarate reductase); (b) beta-oxidation (3-hydroxyacyl-CoA dehydrogenase); (c) benzene ring cleavage (benzoyl-CoA reductase) and (d) desulfonation (Adenylyl-sulfate reductase). In stage VII sample, genes related to the desulfonation step were identified with higher relative abundance, when compared to the other stages. For a higher vup, a higher relative abundance of genes related to oxidative phosphorylationwas observed and the genus main representative in that category was Chryseobacterium.

Made available in DSpace on 2015-04-22T22:12:36Z (GMT). No. of bitstreams: 1 carolinie.pdf: 1105289 bytes, checksum: fb4a9af7cf4012e7784dfbcf92246455 (MD5) Previous issue date: 2010-03-09 / FAPEAM - Fundação de Amparo à Pesquisa do Estado do Amazonas / In the Amazon agricultural soils are found in soil called Anthropogenic Dark Earths (ADE), a soil rich in organic matter and minerals. The biodiversity is a source of very high value of non-cultivable microorganisms that can be isolated through construction of metagenomic libraries, using vectors of large fragments, with the goal of finding new biocatalytic enzymes. Enzymes widely used in the industrial market are amylase and protease, are applied in industry: chemical, pharmaceutical, textile, detergent and food industries. Amylases degrade starch into smaller units of saccharides, as proteases catalyze the hydrolysis of peptide bonds. The aim of this study was to isolate and partially characterize enzymes amylase and protease of clones isolated from a selection of functional metagenomic library of TPI in the Amazon region. In this study were isolated 1.344 clones, only 4 producing halo of starch hydrolysis and 3 proteolytic clones belonging to metagenomic library constructed with total DNA extracted from microorganisms present in soil samples of the TPI, were partially characterized the enzymes produced by 4 clones amilolytic and 1 proteolytic clone. After isolation, the clones were inoculated in culture medium Luria Bertani (LB) at 37ºC under agitation of 150 rpm and subjected to assays at pHs of 3 to 10 and temperatures ranging from 25 to 100ºC for up to 90 of incubation. Amylases had its highest production in 24 h (4.3 U/mL) and its activity was optimal at neutral pH to slightly alkaline, the clone P1C4 showed the highest production at 70°C (6.93 U/mL), since the clones P5C4 and P6C12 reported higher thermostability at 80°C, these results indicate that the gene for alpha-amylase to be cloned encoding a thermostable enzyme, such as the enzyme produced by Bacillus liqueniformis. Since the clone P3A3 produced 26.3 U/mL protease in 10 hours of production, its activity was optimal at neutral pH, and its optimum temperature at 30ºC (56.0 U/mL) and its thermostability was demonstrated in 50°C (22,0 U/mL). These features contribute to the implementation of these enzymes in industrial sectors as in food production, chemical industry and production of detergents. The enzymes isolated in this study demonstrated new features and performance when compared to enzymes described today, which proves the efficiency of the construction of metagenomic libraries for isolation of new bioproducts. / Na floresta Amazônica são encontrados solos agricultáveis denominado de solo de Terra Preta de Índio (TPI), um solo rico em matéria orgânica e em minerais. A biodiversidade da Amazônia constitui uma fonte de valor altíssimo de micro-organismos não-cultiváveis que podem ser isolados através das construções das bibliotecas metagenômicas, utilizando vetores de grandes fragmentos, com o objetivo de encontrar novas enzimas biocatalíticas. As enzimas de maior aplicação no mercado industrial são as amilases e proteases, aplicadas nas indústrias: química, farmacêutica, têxtil, de detergentes e alimentícia. As amilases são capazes de degradar o amido em unidades de sacarídeos menores, já as proteases catalisam a hidrólise de ligações peptídicas. O objetivo deste trabalho foi isolar e caracterizar parcialmente enzimas amilolíticas e proteolíticas de clones isolados a partir de uma seleção funcional da biblioteca metagenômica de TPI da região Amazônica construída com DNA total extraído dos micro-organismos presentes nas amostras de solos de TPI, inseridos no vetor fosmidial (pCC1Fos ) e clonado na célula hospedeira Escherichia coli (EPI300). De 1.344 clones pertencente a biblioteca metagenômica, apenas 4 clones foram produtores de halo de hidrólise de amido e 3 clones foram proteolíticos, além disso, todas as enzimas produzidas pelos 4 clones amilolíticos e 1 clone proteolítico foram caracterizadas parcialmente. Após o isolamento, os clones foram inoculados em meio de cultura Luria Bertani (LB) a 37oC, sob agitação de 150 rpm e submetidos aos ensaios enzimáticos nos pHs de 3 a 10 e temperatura variando de 25 a 100ºC por até 90 minutos de incubação. As amilases tiveram sua maior produção em 24 horas (4,3 U/mL) e sua atividade ótima foi em pH neutro a levemente alcalino, o clone P1C4 demonstrou a maior produção a 70ºC (6,93 U/mL), já os clones P5C4 e P6C12 demonstraram a maior termoestabilidade a 80°C, tais resultados indicam que o gene de alfa-amilase clonado deve ser codificador de uma enzima termoestável, como por exemplo, a enzima produzida por Bacillus liqueniformis. Já o clone P3A3 produziu 26,3 U/mL de proteases em 10 horas de produção, sua atividade ótima foi em pH neutro, sendo sua temperatura ótima em 30ºC (56,0 U/mL) e sua termoestabilidade foi demonstrada em 50ºC (22,0 U/mL). Estas características contribuem para a aplicação destas enzimas em setores industriais como na produção de alimentos, indústria química e produção de detergentes. As enzimas isoladas neste trabalho demonstraram características e atuações novas quando comparadas a enzimas descritas atualmente, a qual comprova a eficiência da construção de bibliotecas metagenômicas para o isolamento de novos bioprodutos.