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Screening D'activités Hydrolytiques REPUBLIQUE ALGERIENNE DEMOCRATIQUE ET POPULAIRE MINISTERE DE L’ENSEIGNEMENT SUPERIEUR ET DE LA RECHERCHE SCIENTIFIQUE UNIVERSITE MENTOURI-CONSTANTINE Institut de la Nutrition, de l’Alimentation et des Technologies Agro-alimentaires (INATAA) Département de Biotechnologie alimentaire N° d’ordre : Série : MEMOIRE Présenté en vue de l’obtention du diplôme de Magister en Sciences Alimentaires Option : Biotechnologie Alimentaire Screening d’activités hydrolytiques extracellulaires chez des souches bactériennes aérobies thermophiles isolées à partir de sources thermales terrestres de l’Est algérien Présenté par GOMRI Mohamed Amine Devant le jury composé de : Président : Pr. AGLI A. Professeur INATAA, UMC Rapporteur : Dr. KHARROUB K. Docteur INATAA, UMC Examinateurs : Pr. KACEM CHAOUACHE N. Professeur Faculté des SNV, UMC Dr. BARKAT M. Docteur INATAA, UMC Année universitaire 2011-2012 Remerciements Je rends grâce à Dieu, le miséricordieux, le tout puissant, pour ce miracle appelé vie, que sa lumière nous guide vers lui, et que son nom soit l’élixir de nos peines et douleurs. Tout d’abord, je tiens à remercier Madame KHARROUB Karima pour m’avoir donné la chance de travailler sous sa direction, pour sa confiance en moi et ses encouragements mais surtout pour sa générosité dans le travail, qu’elle trouve en ces mots toute ma gratitude. Mes remerciements sont adressés aux membres du Jury qui ont pris sur leur temps et ont bien voulu accepter de juger ce modeste travail : Mr le professeur AGLI qui m’a fait l’honneur de présider ce Jury Mr le professeur KACEM-CHAOUCH E qui a eu l’amabilité de participer à ce Jury Mme BARKAT qui a bien voulu examiner ce travail Je tiens à remercier Mlle AYAD Ryma, Mesdames ZOUBIRI Lamia et DJABALI Saliha, Messieurs ZOUAOUI Nassim, BOUGUERRA Ali, SLIMANI Zakaria et FARHAT Chouaib pour leur aide inestimable, mais aussi pour leur amitié précieuse, qu’ils trouvent ici les plus sincères marques d’affection. Je remercie également Mlle HIMED Louiza, Messieurs CHEMACH Loucif, BENSALEM Adel, BOUASSLA Abdallah et BOUFEDECHE Amine pour leurs conseils et leur disponibilité. Un grand merci à toutes les personnes que j’ai côtoyé au cours de la réalisation de cette étude : personnel et enseignants de l’INATAA, personnel d’Objectif Santé, et à tous les autres. Enfin, Merci à Ma famille….et surtout à mon père, Mr GOMRI Achour, lui qui continue de croire en moi malgré toutes les difficultés et tous les obstacles. GMA TABLE DES MATIERES Page Résumés LISTE DES ABREVIATIONS LISTE DES FIGURES LISTE DES TABLEAUX INTRODUCTION…………………………………………………………………………. 1 REVUE BIBLIOGRAPHIQUE Chapitre 1. Place des thermophiles dans le monde extrêmophile…………………………. 3 1. Extrêmophiles…………………………………………………………………………… 3 1.1. Notions d’environnements extrêmes et d’extrêmophilie……………………………… 3 2. Thermophiles……………………………………………………………………………. 5 2.1. Classification………………………………………………………………………….. 5 2.2. Niches écologiques des thermophiles…………………………………………………. 5 2.2.1. Biotopes naturels……………………………………………………………………. 6 2.2.2. Biotopes artificiels…………………………………………………………………... 7 2.3. Diversité taxonomique et métabolique des thermophiles……………………………... 8 2.3.1. Eucarya……………………………………………………………………………… 10 2.3.2. Bacteria……………………………………………………………………………… 11 2.3.3. Archaea……………………………………………………………………………… 13 2.4. Grandes voies métaboliques des procaryotes thermophiles…………………………... 14 2.4.1. Autotrophie…………………………………………………………………………. 14 2.4.2. Hétérotrophie………………………………………………………………………... 15 2.4.3. Oxygène…………………………………………………………………………….. 15 2.5. Bases moléculaires de la thermophilie……………………………………………… 16 2.5.1. Acides nucléiques…………………………………………………………………… 16 2.5.2. Protéines…………………………………………………………………………….. 18 2.5.3. Membrane cellulaire………………………………………………………………… 19 2.5.4. Limite supérieure de la vie à haute température……………………………………. 20 2.5.5. Thermophiles et origine de la vie………………………………………………….. 21 Chapitre 2. Applications des thermophiles et de leurs biomolécules……………………… 23 1. Evolution des méthodes d’obtention des actifs biotechnologiques…………………….. 23 1.1. Applications basées sur les cellules entières………………………………………….. 23 1.1.1. Agents de minéralisation……………………………………………………………. 23 1.1.2. Piles à combustible microbiennes……………………………………………………. 24 1.1.3. Production d'hydrogène……………………………………………………………... 24 1.2. Applications basées sur les biomolécules…………………………………………….. 25 1.2.1. Enzymes…………………………………………………………………………….. 25 1.2.1.1. Enzymes de dégradation et de modification des polysaccharides………………… 25 1.2.1.2. Enzymes produisant des sucres rares……………………………………………... 31 2.2.1.3. Protéases………………………………………………………………………….. 32 1.2.1.4. Lipases et estérases………………………………………………………………... 33 1.2.1.5. Enzymes de manipulation des acides nucléiques…………………………………. 33 1.2.1.6. Autres enzymes…………………………………………………………………… 34 2.2.2. Solutés compatibles…………………………………………………………………. 35 2.2.3. Lipides et peptides…………………………………………………………………... 35 Chapitre 3. Taxonomie polyphasique……………………………………………………… 38 1. Taxonomie phénotypique……………………………………………………………….. 38 2. Taxonomie numérique…………………………………………………………………... 38 3. Taxonomie génotypique………………………………………………………………… 39 MATERIEL ET METHODES 1. Sources thermales étudiées……………………………………………………………… 41 2. Echantillonnage…………………………………………………………………………. 42 3. Isolement, purification et conservation des isolats……………………………………… 43 4. Souches témoin………………………………………………………………………….. 43 5. Identification des isolats………………………………………………………………… 43 5. 1. Caractérisations culturale et cellulaire des isolats ………………………...………….. 44 5.2. Caractérisations physiologique et biochimique………………………………………. 44 5.2.1. Température, pH et NaCl…………………………………………………………… 44 5.2.2. Utilisation de substrats carbonés……………………………………………………. 44 5.2.3. Utilisation du citrate………………………………………………………………… 45 5.2.4. Utilisation des sucres sur milieu Triple Sugar Iron…………………………………. 45 5.2.5. Test Mannitol-Mobilité……………………………………………………………... 45 5.2.6. Mise en évidence des enzymes respiratoires………………………………………... 45 5.2.7. Caractérisation du type fermentaire………………………………………………… 45 5.2.8. Recherche de la ȕ-galactosidase…………………………………………………….. 46 5.2.9. Recherche de l’uréase……………………………………………………………….. 46 5.2.10. Dégradation des acides aminés…………………………………………………….. 46 6. Mise en évidence d’activités hydrolytiques extracellulaires……………………………. 47 6.1. Amylase……………………………………………………………………………… 47 6.2. Protéases………………………………………………………………………………. 47 6.3. Activités lipolytiques …………………………………………………………………. 47 6.4. Cellulase……………………………………………………………………………… 48 7. Taxonomie numérique…………………………………………………………………... 48 RESULTATS ET DISCUSSION 1. Analyse physico-chimique des échantillons……………………………………………. 50 2. Isolement et purification des isolats…………………………………………………….. 51 3. Caractérisation phénétique des isolats………………………………………………….. 51 3.1. Caractérisation culturale et morphologique…………………………………………... 51 3.1.1. Aspect macroscopique………………………………………………………………. 51 3.1.2. Aspect microscopique………………………………………………………………. 54 3.2. Caractérisation physiologique des isolats…………………………………………….. 56 3.2.1. Température de croissance………………………………………………………….. 56 3.2.2. Tolérance au NaCl et spectre de pH……………………………………………… 56 3.2.3. Utilisation des substrats carbonés…………………………………………………… 57 3.3. Caractérisation biochimique des isolats…..…………………………………………... 57 3.3.1. Mise en évidence des enzymes respiratoires………………………………………... 57 3.3.2. Utilisation des sucres sur milieu Triple Sugar Iron…………………………………. 57 3.3.3. Test Mannitol-Mobilité……………………………………………………………... 59 3.3.4. Caractérisation du type fermentaire………………………………………………… 59 3.3.5. Recherche de la ȕ-galactosidase…………………………………………………….. 59 3.3.6. Recherche de l’uréase……………………………………………………………….. 59 3.3.7. Utilisation du tryptophane…………………………………………………………... 59 3.3.8. Production de décarboxylases (LDC, ODC) et ariginine dihydrolase (ADH)……… 59 4. Profiles des activités hydrolytiques extracellulaires des souches isolées……………….. 64 5. Taxonomie numérique…………………………………………………………………... 69 6. Discussion………………………………………………………………………………. 74 CONCLUSION GENERALE ET PERSPECTIVES…………………………………… 79 REFERENCES BIBLIOGRAPHIQUES………………………………………………… 81 ANNEXES GLOSSAIRE COMMUNICATIONS SCIENTIFIQUES Nom et prénom : GOMRI Mohamed Amine Année universitaire : 2011-2012 Thème : Screening d’activités hydrolytiques extracellulaires chez des souches bactériennes aérobies thermophiles isolées à partir de sources thermales terrestres de l’Est algérien Nature du diplôme : Magister en Sciences Alimentaires Option : Biotechnologie Alimentaire Résumé Les microorganismes thermophiles et hyperthermophiles autochtones des environnements chauds tels que les sources thermales terrestres possèdent des capacités d’adaptations moléculaires intéressantes et constituent ainsi une importante source de molécules bioactives peu conventionnelles issues de mécanismes biochimiques et moléculaires uniques. Les enzymes hydrolytiques de ces organismes offrent des avantages majeurs et fournissent de nouvelles possibilités soit de l’amélioration ou de la création de nouveaux procédés biotechnologiques. Dans la perspective d’étudier une partie de leur potentiel enzymatique, 83 souches bactériennes thermophiles aérobies ont été isolées à partir d’échantillons d’eau et de sédiments prélevés au niveau de plusieurs sites géothermaux répartis entre les régions de Guelma et de Ouargla situés respectivement au Nord-Est et au Sud-Est de l’Algérie. Une caractérisation phénétique a été réalisée, et a permis d’avoir accès à certaines propriétés morphologiques, biochimiques et physiologiques de ces microorganismes et qui ont
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