No description available.

In the work presented here, it is claimed that bacteria have been isolated which are capable of growth at high temperatures utilizing molecular nitrogen as their sole nitrogen source. Soil bacteria were isolated which grew at 55 C in nitrogen-free media. They were found to be obligatory thermophiles in nitrogen-free media and facultative thermophiles in media containing organically bound nitrogen.

No description available.

L'objectif principal de mon projet de doctorat est d'étudier l'évolution génomique de l'ordre des Archaea Thermococcales. Je me suis intéressé à comprendre les mécanismes des éléments mobiles génétiques (MGE) pouvant influencer l'évolution des génomes. En utilisant une approche multidisciplinaire, nous avons pu explorer les différents aspects de ce phénomène in silico, in vitro et in vivo. Grâce à des analyses in silico de tous les génomes de Thermococcales complètement séquencés disponibles, nous avons montré que cet ordre affiche un niveau élevé de réarrangements pouvant perturber les modèles d'expression génique. Dans une première approche, nous avons étudié l'existence de l'organisation chromosomique. L'inefficacité dans la prédiction de l'origine et de la terminaison de la réplication sur la seule base de la composition de l'ADN chromosomique ou skew, nous a motivé à utiliser une approche différente basée sur des séquences biologiquement pertinentes. Nous avons donc déterminé la position de l'origine de la réplication (oriC) dans tous les 21 génomes séquencés Thermococcales. La position potentielle de la terminaison a été prédite dans 19 génomes à ou près du site dif, où les dimères chromosomiques sont résolus avant la ségrégation de l'ADN. Le calcul du génome central a révélé un certain nombre de grappes de gènes essentiels avec une position chromosomique remarquablement stable à travers les espèces, en utilisant oriC comme référence. D'autre part, les régions core-free semblent correspondre à des éléments mobiles intégrés putatifs. Ces observations indiquent qu'un degré remarquable d ' «ordre» a été maintenu à travers les Thermococcales, même s'ils présentent des chromosomes fortement brouillés, les inversions étant particulièrement fréquentes. La découverte et la caractérisation d'un nouvel organisme, Thermococcus nautili nous ont permis de mieux comprendre le mécanisme sous-jacent causant ces inversions. En effet, le séquençage et l'analyse in silico de son génome ont fortement suggéré l'implication d'une nouvelle classe de tyrosine recombinases dans la plasticité génomique. Le plasmide pTN3 de T. nautili, qui est intégré dans le chromosome et auto-réplicable, code une intégrase appartenant à la classe des tyrosine recombinases. Des plasmides similaires ont également été trouvés intégrés dans le chromosome d'autres séquences de Thermococcales (par exemple TKV4 dans T. kodakarensis). Afin de tester son activité enzymatique, l’integrase codée par le plasmide pTN3 a été surproduite et purifiée. Les expériences in vitro ont d'abord permis de déterminer le segment de séquence minimal requis pour l'activité de l'intégrase et optimisé la réaction enzymatique in vitro. Ces résultats nous ont permis, en suite, de démontrer la réaction d'excision / d'intégration observée avec d'autres recombinases de tyrosine. De plus, l'excision in vivo d'un élément intégré apparenté (TKV4 de T. kodakarensis) par l'intégrase pTN3 a été réalisée au cours de cette étude. Pour cela, le gène IntpTN3 a été clone dans un vecteur de la bactérie E. coli / Thermococcus pour la transformation et l'expression dans T. kodakarensis. Après incubation, les cellules ont montré la présence de l'élément TKV4-intégré dans la forme circulaire libre. Enfin, nous avons pu imiter l'inversion chromosomique in vitro en utilisant des substrats synthétiques contenant des séquences cibles d'intégration. Nous avons également pu montrer que l'intégrase pTN3 possède une activité qui peut intervenir sur des inversions génomiques à grande échelle en utilisant différents sites et donc expliquer les réarrangements observés dans Thermococcales (Cossu et al, in prep). / The main goal of my PhD project is to investigate the genomic evolution of the Archaea Thermococcales order. I am interested in understanding how mobile genetic elements (MGE) can influence the evolution of genomes. Using a multidisciplinary approach, we were able to explore the different aspects of this phenomenon in silico, in vitro and in vivo. Through in silico analyses of all available completely sequenced Thermococcales genomes, we showed that this order displays a characteristic high level of rearrangements potentially disrupting gene expression patterns. In a first approach, we investigated the existence of chromosomal organization. The inefficiency in predicting origin and termination of replication on the sole basis of chromosomal DNA composition or skew, motivated us to use a different approach based on biologically relevant sequences. We determined the position of the origin of replication (oriC) in all 21 sequenced Thermococcales genomes. The potential position of the termination was predicted in 19 genomes at or near the dif site, where chromosome dimers are resolved before DNA segregation. Computation of the core genome uncovered a number of essential gene clusters with a remarkably stable chromosomal position across species, using oriC as reference. On the other hand, core-free regions appear to correspond to putative integrated mobile elements. These observations indicate that a remarkable degree of “order” has been maintained across Thermococcales even if they display highly scrambled chromosomes, with inversions being especially frequent. The discovery and characterization of a new organism, Thermococcus nautili allowed us to better understand the underlying mechanism causing these inversions. The sequencing and in silico analysis of its genome strongly suggested the involvement of a new class of tyrosine recombinases in genomic plasticity. T. nautili pTN3 plasmid, which is found integrated into the chromosome and also self-replicating encodes an integrase belonging to this class. Similar plasmids have also been found integrated in the chromosome of other sequenced Thermococcales (e.g. TKV4 in T. kodakarensis). In order to test its enzymatic activity, we overproduced and purified the integrase encoded by pTN3. In vitro experiments first determined the minimal sequence segment required for integrase activity and optimized the enzymatic reaction in vitro. Due to this early results, we were able to demonstrate the excision/integration reaction observed with other tyrosine recombinases. Additionally, the in vivo excision of a related integrated element (TKV4 from T. kodakarensis) by the pTN3 integrase was performed during this study. The IntpTN3 gene has been cloned into an E. coli/Thermococcus shuttle vector for transformation and expression in T. kodakarensis. After incubation, cells showed the presence of the TKV4-integrated element in free circular form. Finally, we were able to mimic in vitro chromosomal inversion using synthetic substrates containing integration target sequences. We were also able to show that pTN3 integrase possesses an activity which can mediate large scale genomic inversions using different sites and therefore explain the rearrangements observed in Thermococcales).

Reservatórios de petróleo são ambientes únicos por apresentarem uma combinação de condições extremas referentes à temperatura, pressão e salinidade, e que sustentam o desenvolvimento de procariotos. Várias espécies dos Domínios Bactéria e Archaea têm sido isoladas deste ambiente, com destaque aos microrganismos redutores de sulfato (BRS), metanogênicos e fermentadores. Estes últimos utilizam como fonte de energia uma grande variedade de compostos orgânicos e uma grande parte de seus representantes em reservatórios pertence ao Filo Thermotogae. Estas bactérias apresentam uma estrutura característica envolvendo a célula, semelhante a uma bainha, chamada toga. Alguns gêneros deste Filo são habitantes exclusivos de reservatórios, como por exemplo, o gênero Petrotoga. O presente trabalho teve como objetivo isolar e caracterizar bactérias da classe Thermotogae a partir de fluidos de água/óleo de poços de um reservatório de petróleo localizado na região Nordeste, com vistas a contribuir para o conhecimento da diversidade e metabolismo microbianos deste tipo de ambiente. O reservatório tem salinidade média de 7%, temperatura média de 60ºC, o pH dos fluidos situa-se entre 6,6-6,8, e a profundidade média dos poços é de 1.100 metros. Dois meios propostos na literatura para o cultivo de Petrotoga foram testados para o isolamento deste gênero: meio P-mexicana e meio P. olearia. Os meios foram preparados em anaerobiose, sob atmosfera de N2 e salinidade de 70g/L de NaCl. Inóculos de cultivos pré-existentes em meios enriquecidos foram feitos nestes meios e incubados a 60ºC. Ambos os meios se mostraram seletivos para linhagens de Petrotoga. Os isolados foram identificados pelo sequenciamento do gene RNAr 16S, e um deles, o isolado MG414-03, foi submetido a testes de crescimento sob as variáveis temperatura, salinidade e fonte de carbono. MG414-03 pertence ao gênero Petrotoga, com similaridade de 99,2% com a espécie Petrotoga miotherma. Suas células têm forma de bastonete e são envolvidas por toga; são Gram-negativas; têm comprimento médio de 1,8±0,9 μm e largura média de 0,7±0,1 μm, podendo ser individuais ou formar filamentos. Motilidade foi observada em células individuais e em arranjos de dois. Endósporos não foram observados em nenhuma fase de crescimento. Forma colônias circulares lisas, com borda lisa, transparentes, com diâmetro máximo de 1,0 mm. O isolado apresentou temperatura e salinidade ótimas de 60ºC e 4%, respectivamente. Utiliza glicose, xilose, maltose e sacarose como fontes de carbono, mas não utiliza xilana e carboximetilcelulose (CMC). É inibido pelos antibióticos canamicina e cloranfenicol nas concentrações de 10 μg/mL e 100 μg/mL, respectivamente. As atividades enzimáticas sobre os substratos xilana e carboximetilcelulose (CMC) foram testadas para o isolado MG414-03 e para as linhagens Petrotoga mobilis-DSM10674 e Petrotoga mexicana-DSM14811. O método utilizado foi o ensaio colorimétrico com DNS (Ácido Dinitrossalicílico) e o isolado MG414-03 não apresentou atividade enzimática sobre os substratos testados. Petrotoga mobilis-DSM10674 e Petrotoga mexicana-DSM14811 foram utilizadas como padrões no ensaio pois ambas são descritas como produtoras de xilanases, fato confirmado pelos resultados apresentados no teste. Petrotoga mobilis-DSM10674 apresentou atividade enzimática sobre o CMC, na ordem de 73±18 U/L, fato não registrado na literatura para esta espécie. / Oil reservoirs are unique environments for having a combination of extreme conditions relating to temperature, pressure, salinity, and that support the growth of microorganisms. Several species of Domains Bacteria and Archaea have been recovered from oil reservoirs, especially the sulfate-reducing microorganisms (SRB), methanogens and fermenters. The latter use as a carbon source a wide variety of organic compounds and a large fraction of the strains are members of the Phylum Thermotogae. Thermotogae species share a common characteristic: a balloon-like sheath or “toga” present outside the cell membrane. Some genera of the phylum have been exclusively recovered from oil reservoirs, as the genus Petrotoga. This study aimed to isolate and characterize strains members of Thermotogae from water/oil fluids of an onshore oil reservoir located in the Northeast of Brazil, in order to contributes to the knowledge of diversity and microbial metabolism of this type of environment. The reservoir has an average salinity of 7%, an average temperature of 60 º C, the pH of the fluid is between 6.6-6.8 and the average depth of the wells is 1,100 meters. Two culture media proposed in the literature for Petrotoga cultivation were tested: P-mexicana Medium and P. olearia medium. The media was prepared anaerobically under N2 flush and salinity adjusted for 70g/L NaCl. Aliquots from pre-existing enriched cultures were inoculated in those media and incubated at 60 º C. Both media proved be selective for Petrotoga strains. The isolates were identified by 16S rRNA gene sequencing, and the isolate MG414-03 was tested for the growth under variables temperature, salinity and carbon source. MG414-03 belongs to the genus Petrotoga, with 99.2% similarity with Petrotoga miotherma species. Cells are rods, 1.8 (± 0.9) x 0.7 (± 0.1) μm in size, with an outer sheath-like structure (toga), occurring singly or in sheaths. It stains Gram-negative. Motility was observed in single or in double cells. No spore formation was detected. Colonies are circular, with a smooth edge, transparent, with a maximum diameter of 1.0 mm. Optimum salinity at 4% and optimum temperature at 60ºC. Ferments glucose, xylose, maltose and sucrose, but not xylan and carboxymethylcellulose (CMC). It is inhibited by the antibiotics kanamycin and chloramphenicol at concentrations of 10 μg/mL and 100 μg/mL, respectively. The enzymatic activity on xylan and carboxymethylcellulose (CMC) were tested for isolate MG414-03, Petrotoga mobilis DSM10674 and Petrotoga Mexicana DSM14811. The method used was the colorimetric assay with DNS (dinitrosalicylic acid) and isolate MG414-03 showed no enzymatic activity on the tested substrates. Petrotoga mobilis DSM10674 and Petrotoga Mexicana DSM14811 were used as standards in the DNS assay, as both strains are described as xylanase producers, and this ability was confirmed by the results presented in the test. Petrotoga mobilis-DSM10674 showed enzymatic activity on CMC, of 73 ± 18 U/L, which was not recorded in the literature for this species.

Reservatórios de petróleo são ambientes únicos por apresentarem uma combinação de condições extremas referentes à temperatura, pressão e salinidade, e que sustentam o desenvolvimento de procariotos. Várias espécies dos Domínios Bactéria e Archaea têm sido isoladas deste ambiente, com destaque aos microrganismos redutores de sulfato (BRS), metanogênicos e fermentadores. Estes últimos utilizam como fonte de energia uma grande variedade de compostos orgânicos e uma grande parte de seus representantes em reservatórios pertence ao Filo Thermotogae. Estas bactérias apresentam uma estrutura característica envolvendo a célula, semelhante a uma bainha, chamada toga. Alguns gêneros deste Filo são habitantes exclusivos de reservatórios, como por exemplo, o gênero Petrotoga. O presente trabalho teve como objetivo isolar e caracterizar bactérias da classe Thermotogae a partir de fluidos de água/óleo de poços de um reservatório de petróleo localizado na região Nordeste, com vistas a contribuir para o conhecimento da diversidade e metabolismo microbianos deste tipo de ambiente. O reservatório tem salinidade média de 7%, temperatura média de 60ºC, o pH dos fluidos situa-se entre 6,6-6,8, e a profundidade média dos poços é de 1.100 metros. Dois meios propostos na literatura para o cultivo de Petrotoga foram testados para o isolamento deste gênero: meio P-mexicana e meio P. olearia. Os meios foram preparados em anaerobiose, sob atmosfera de N2 e salinidade de 70g/L de NaCl. Inóculos de cultivos pré-existentes em meios enriquecidos foram feitos nestes meios e incubados a 60ºC. Ambos os meios se mostraram seletivos para linhagens de Petrotoga. Os isolados foram identificados pelo sequenciamento do gene RNAr 16S, e um deles, o isolado MG414-03, foi submetido a testes de crescimento sob as variáveis temperatura, salinidade e fonte de carbono. MG414-03 pertence ao gênero Petrotoga, com similaridade de 99,2% com a espécie Petrotoga miotherma. Suas células têm forma de bastonete e são envolvidas por toga; são Gram-negativas; têm comprimento médio de 1,8±0,9 μm e largura média de 0,7±0,1 μm, podendo ser individuais ou formar filamentos. Motilidade foi observada em células individuais e em arranjos de dois. Endósporos não foram observados em nenhuma fase de crescimento. Forma colônias circulares lisas, com borda lisa, transparentes, com diâmetro máximo de 1,0 mm. O isolado apresentou temperatura e salinidade ótimas de 60ºC e 4%, respectivamente. Utiliza glicose, xilose, maltose e sacarose como fontes de carbono, mas não utiliza xilana e carboximetilcelulose (CMC). É inibido pelos antibióticos canamicina e cloranfenicol nas concentrações de 10 μg/mL e 100 μg/mL, respectivamente. As atividades enzimáticas sobre os substratos xilana e carboximetilcelulose (CMC) foram testadas para o isolado MG414-03 e para as linhagens Petrotoga mobilis-DSM10674 e Petrotoga mexicana-DSM14811. O método utilizado foi o ensaio colorimétrico com DNS (Ácido Dinitrossalicílico) e o isolado MG414-03 não apresentou atividade enzimática sobre os substratos testados. Petrotoga mobilis-DSM10674 e Petrotoga mexicana-DSM14811 foram utilizadas como padrões no ensaio pois ambas são descritas como produtoras de xilanases, fato confirmado pelos resultados apresentados no teste. Petrotoga mobilis-DSM10674 apresentou atividade enzimática sobre o CMC, na ordem de 73±18 U/L, fato não registrado na literatura para esta espécie. / Oil reservoirs are unique environments for having a combination of extreme conditions relating to temperature, pressure, salinity, and that support the growth of microorganisms. Several species of Domains Bacteria and Archaea have been recovered from oil reservoirs, especially the sulfate-reducing microorganisms (SRB), methanogens and fermenters. The latter use as a carbon source a wide variety of organic compounds and a large fraction of the strains are members of the Phylum Thermotogae. Thermotogae species share a common characteristic: a balloon-like sheath or “toga” present outside the cell membrane. Some genera of the phylum have been exclusively recovered from oil reservoirs, as the genus Petrotoga. This study aimed to isolate and characterize strains members of Thermotogae from water/oil fluids of an onshore oil reservoir located in the Northeast of Brazil, in order to contributes to the knowledge of diversity and microbial metabolism of this type of environment. The reservoir has an average salinity of 7%, an average temperature of 60 º C, the pH of the fluid is between 6.6-6.8 and the average depth of the wells is 1,100 meters. Two culture media proposed in the literature for Petrotoga cultivation were tested: P-mexicana Medium and P. olearia medium. The media was prepared anaerobically under N2 flush and salinity adjusted for 70g/L NaCl. Aliquots from pre-existing enriched cultures were inoculated in those media and incubated at 60 º C. Both media proved be selective for Petrotoga strains. The isolates were identified by 16S rRNA gene sequencing, and the isolate MG414-03 was tested for the growth under variables temperature, salinity and carbon source. MG414-03 belongs to the genus Petrotoga, with 99.2% similarity with Petrotoga miotherma species. Cells are rods, 1.8 (± 0.9) x 0.7 (± 0.1) μm in size, with an outer sheath-like structure (toga), occurring singly or in sheaths. It stains Gram-negative. Motility was observed in single or in double cells. No spore formation was detected. Colonies are circular, with a smooth edge, transparent, with a maximum diameter of 1.0 mm. Optimum salinity at 4% and optimum temperature at 60ºC. Ferments glucose, xylose, maltose and sucrose, but not xylan and carboxymethylcellulose (CMC). It is inhibited by the antibiotics kanamycin and chloramphenicol at concentrations of 10 μg/mL and 100 μg/mL, respectively. The enzymatic activity on xylan and carboxymethylcellulose (CMC) were tested for isolate MG414-03, Petrotoga mobilis DSM10674 and Petrotoga Mexicana DSM14811. The method used was the colorimetric assay with DNS (dinitrosalicylic acid) and isolate MG414-03 showed no enzymatic activity on the tested substrates. Petrotoga mobilis DSM10674 and Petrotoga Mexicana DSM14811 were used as standards in the DNS assay, as both strains are described as xylanase producers, and this ability was confirmed by the results presented in the test. Petrotoga mobilis-DSM10674 showed enzymatic activity on CMC, of 73 ± 18 U/L, which was not recorded in the literature for this species.

It is widely known that fossil fuels are limited; consequently, the generation of new sources of energy in a clean and environmentally friendly manner is a research priority. Bioethanol appears to be one potential solution, especially second-generation production from renewable biomass. In order to use lignocellulosic feedstock to produce bioethanol, its polysaccharide components, cellulose and hemicellulose, must be hydrolysed into soluble sugars, which can then be converted into ethanol by fermentative microorganisms such as Geobacillus thermoglucosidasius TM242 used by the company ReBio Technologies Ltd. To date, the cost of commercial enzymes used during the hydrolysis process remains a major economic consideration in the production of second-generation bioethanol as an alternative fuel. The research project presented in this thesis aims to improve this rate-limiting step of microbial bioethanol production through an investigation of the different enzymes associated with hemicellulose hydrolysis. Firstly, the TM242 genome sequence revealed a number of genes encoding glycoside-hydrolases. Six of these genes were cloned and expressed in E. coli and the recombinant enzymes characterised; three of them, two β-xylosidases and an α arabinofuranosidase, are relevant to xylan hydrolysis, and were found to be highly active and thermostable. Crystallisation of one of the β-xylosidases permitted the determination of a high-resolution (1.7 Å) structure of the apo-enzyme along with a lower resolution (2.6 Å) structure of the enzyme-substrate complex, resulting in the first reported structure of a GH52 family member (Espina et al., 2014). Secondly, as the TM242 microorganism lacks xylanase enzymes, four genes encoding xylanases from closely-related Geobacillus strains were cloned and expressed in E. coli, with one of them being also successfully cloned and expressed in G. thermoglucosidasius TM242. This heterologous xylanase was secreted in active form representing an enhanced biomass utilisation by TM242. In conclusion, it is felt that the findings presented here have the potential to make a valuable contribution towards second-generation bioethanol production.

A reverse transcription reaction allows the production of complementary DNA (cDNA) using an RNA template and relies on polymerases displaying reverse transcriptase (RT) activity. This process, with major applications in both research and in medical diagnostics, is often limited by the nature of the RTs available. RNA secondary structure can prove problematic where mesophilic retroviral RTs are used while the alternative approach, using thermophilic DNA polymerases with RT activity, often results in error-prone cDNA production. <br /> This project recognised the need to study other possible sources of thermophilic RTs and outlines the study of four previously uncharacterised Group II Intronencoded proteins (IEP), with RT domains, from thermophilic bacteria. While cloning of the IEP genes and their expression on a small scale proved successful, difficulties were encountered when attempting purification. Despite a lack of overall purity, samples containing IEPs from Thermosinus carboxydivorans and Petrotoga mobilis were shown to have RT activity but characterisation of these IEPs was not carried out. However, an IEP from Bacillus caldovelox proved to be an excellent candidate for characterisation as successful purification was achieved. Enzyme engineering was also performed, fusing a Sac7d domain onto the C-terminus of this protein. These enzymes were shown to have optimum RT activity at 54ºC with activity still being displayed at 76ºC. Other studies on these enzymes showed that, unlike the retroviral RTs, the IEPs displayed no DNA-dependent DNA polymerase activity. The Sac7d fusion protein was also studied in terms of possible enhancements to the RT activity of an IEP. However, preliminary studies showed that, although this domain did not prove to be detrimental to the enzyme, it had little effect on improving the processivity of the RTs. <br /> Although this class of RT looks promising in terms of use as an alternative thermophilic RT, the IEPs studied in this report did incur major limitations during cDNA synthesis, which included lower than expected optimum reaction temperatures, very low fidelity and an inability to synthesise cDNA using complex RNA templates.

Hemicellulases are key components in the degradation of plant biomass and carbon flow in nature. Thermotoga hypogea is a bacterium that can grow anaerobically at 90°C. It utilizes carbohydrates and peptides as energy and carbon sources. Three hemicellulytic enzymes: β-xylosidase, α-L-arabinofuranosidase and acetylesterase were investigated. Xylan and xylose were the best substrates for the growth as well as for yielding high activity for all three enzymes in the cells. Glucose grown cells possessed the least amount of enzyme activity for all three enzymes. More than 87% ± 3.0 of β-xylosidase and α-L-arabinofuranosidase activities and 34% ± 11 of acetylesterase activity were associated with the cells. Arabinofuranosidase and acetylesterase were partially purified but β-xylosidase was purified to homogeneity using the Fast Performance Liquid Chromatography system. The latter enzyme has an apparent molecular mass of 75 kDa demonstrated through sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a nondenatured weight of 130 kDa estimated by Gel-filtration. Its optimal temperature and pH-value for activity were 70°C and 6.0, respectively. The purified enzyme had a half life of 22 min at 70°C and pH 6.0. Among all tested substrates, the purified enzyme had specific activities of 44, 32, 4.5, 1.71 U/mg on p-nitrophenyl-β-xylopyranoside (pNβxp), 4-nitrophenyl-β-D-glucopyranoside (pNβgp), 4-nitrophenyl-α-L-arabinofuranoside (pNαLaf) and 4-nitrophenyl-α-D-xylopyranoside (pNαxp) respectively. The apparent Km of the xylosidase with pNβxp, was 2.6 mM and Vmax was 196 U/mg and for pNβgp the Km and Vmax values were 0.31 mM and 24 U/mg respectively. Based on N-terminal analysis, xylosidase showed high homology with Family 3 β-glucosidases.

Hemicellulases are key components in the degradation of plant biomass and carbon flow in nature. Thermotoga hypogea is a bacterium that can grow anaerobically at 90°C. It utilizes carbohydrates and peptides as energy and carbon sources. Three hemicellulytic enzymes: β-xylosidase, α-L-arabinofuranosidase and acetylesterase were investigated. Xylan and xylose were the best substrates for the growth as well as for yielding high activity for all three enzymes in the cells. Glucose grown cells possessed the least amount of enzyme activity for all three enzymes. More than 87% ± 3.0 of β-xylosidase and α-L-arabinofuranosidase activities and 34% ± 11 of acetylesterase activity were associated with the cells. Arabinofuranosidase and acetylesterase were partially purified but β-xylosidase was purified to homogeneity using the Fast Performance Liquid Chromatography system. The latter enzyme has an apparent molecular mass of 75 kDa demonstrated through sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a nondenatured weight of 130 kDa estimated by Gel-filtration. Its optimal temperature and pH-value for activity were 70°C and 6.0, respectively. The purified enzyme had a half life of 22 min at 70°C and pH 6.0. Among all tested substrates, the purified enzyme had specific activities of 44, 32, 4.5, 1.71 U/mg on p-nitrophenyl-β-xylopyranoside (pNβxp), 4-nitrophenyl-β-D-glucopyranoside (pNβgp), 4-nitrophenyl-α-L-arabinofuranoside (pNαLaf) and 4-nitrophenyl-α-D-xylopyranoside (pNαxp) respectively. The apparent Km of the xylosidase with pNβxp, was 2.6 mM and Vmax was 196 U/mg and for pNβgp the Km and Vmax values were 0.31 mM and 24 U/mg respectively. Based on N-terminal analysis, xylosidase showed high homology with Family 3 β-glucosidases.