eau 23 : Effe Sourc
5. Conclusion générale
Les enzymes actives sur l’amidon ont depuis longtemps suscité un intérêt pour leur utilisation potentielle en industrie (amidonnerie) pour l’hydrolyse de l’amidon ou la
production de dérivés de l’amidon. Par ailleurs, dans le domaine médical, l’-amylase,
enzyme recombinante, peut être utilisée comme aide digestive.
Dans ce contexte, cette thèse a pour principaux objectifs : la production et la
quantification de l’-amylase de P. camemberti PL21, sa purification à homogénéité , son
immobilisation et sa caractérisation .
Pour sa production, le plan statistique de Plackett et Burman a permis la sélection des
facteurs l’influençant ainsi que la production de biomasse. L’analyse statistique révèle, pour une production optimale de cette enzyme, le milieu de fermentation, à base de déchets d'oranges à 2 %, doit être supplémenté par : 10 g/l d'amidon, 5 g/l de l'extrait de levure, 20 ml/l de « Corn Seep Liquor », 5 ml/l de glycérol et 0.125g/l de CaCl2 à 20°C. Le pH doit être ajusté à pH 5 par un tampon phosphate à 0.1M et l’agitation fixée à 100 rpm.
La cinétique de production de l’enzyme et de la biomasse de la souche P. camemberti PL21, en fermenteur de 2 litres (conditions contrôlées de pH et de température, pH 5 et T° 20°C) montre un taux de croissance optimal (biomasse) de 28.6 g/l après 156 heures de
fermentation. Pour la même durée de fermentation, l’activité -amylasique maximale affiche
350.2 U
La purification partielle de l'α-amylase de P. camemberti PL21 , réalisée par les
méthodes classiques (Précipitation par le sulfate d’ammonium (NH4)2SO4 ; Dialyse ;
Chromatographie tamisage moléculaire sur Sephadex G -100 et Chromatographie échangeuse d’anions DEAE-Sepharose CL-6B) donne une purification de 23.1 fois avec un rendement de 38.5 .La masse moléculaire déterminée par la mobilité relative en électrophorèse PAGE révèle une masse moléculaire de 60,5 KDa., valeur comparable à l’enzyme fongique de A. oryzae ( 59.0 KDa)
Conclusion
125
Les constantes cinétiques Km et Vmax pour l’hydrolyse de l’amidon soluble
sont respectivement de 30.5 mg/ml et 92 U. La température optimale de l’enzyme
partiellement purifiée est de 30°C et un pH optimal de 5.5. Soumise à un traitement
thermique de 50°C pendant 2 heures, elle garde 60% de son activité ; à 70°C elle perd
complètement son activité au bout de 2 heures Les ions Ca2+ et Mg2+ sont des activateurs
pour cette enzyme, par contre les ions Cu2+, Fe2+, et Hg2+ sont des inhibiteurs.
Dans le but d’augmenter sa thermostabilité, l’α-amylase partiellement purifiée de P.camemberti PL21 est immobilisée par inclusion sur un support naturel de billes d’alginate de calcium (3%) avec un de rendement de 68 %. L’enzyme immobilisée résiste mieux aux traitements thermiques : à 90°C pendant 120 minutes, elle conserve 50% de son activité initiale ; à 80°C, sa demi-vie se prolonge à 180 minutes. Cette stabilité thermique a permis à l’enzyme immobilisée d’être exploitée industriellement. Son immobilisation fait augmenter son temps de réaction à 120 minutes au lieu de 10 à 15 minutes pour l’enzyme libre
L’activité enzymatique diminue lorsque la concentration d'alginate dépasse la concentration de 3% (gène par encombrement stérique). Sa vitesse maximale diminue de 32
% par rapport à celle de l’enzyme libre : 169.8 U vs 94 U. Par contre, l’affinité de
l’enzyme immobilisée pour son substrat, demeure inchangée par rapport à l’enzyme libre : les Km respectifs sont de 0.90 mg/ml et 0.93 mg/ml. L’α-amylase immobilisée conserve son activité plus longtemps et peut être réutilisée jusqu'à 5 fois. La stabilité au stockage à 4°C est de 10 jours, alors qu’à 30°C elle est de 3 jours.
Toutes ces qualifications permettent une éventuelle exploitation industrielle de l’α-amylase de P. camemberti PL21 lorsqu’elle est immobilisée.
Perspectives :
Pour une exploitation industrielle, il est nécessaire de réaliser des cinétiques en « Scall Up » (en fermenteur de 20 L, en fermenteur de 50 L : en fermenteur de 100 L) pour s’assurer de la stabilité de l’enzyme en conditions industrielles. Le gène de l’ α-amylase de P. camemberti PL21 peut être transféré sur un microorganisme qui pousse relativement vite pour une production industrielle d’une α-amylase recombinante utile en médecine (aide digestive), dans les IAA et autres industries.
Conclusion 126 صخلملا ميزنا جاتنإ مت α-amylase رطف نم P. camemberti PL21 اساسأ ةنوكمو ةتبثم هيرمخت ةيئيب يف زيكرتب لاقتربلا بل ةلاضف قوحسم نم 2 % : ةيلاتلا رصانعلاب ةدوزم 10 اشنلا ل/غ , 5 لوريسيلجلا ل/لم , ةريمخلا صلختسم 10 ل/غ , ةرذلا عيقن صلختسم Corn-steep-liquor 20 ل/لم , مويسلاكلا ديرولك 0.125 جذومن تافوفصمل يبيرجت ططخم قفو كلذو. ل/غ Plackett and Burman
.ميزنلإا جاتنإ تيبثت لجأ نم مت .ةيئاوھ طورش تحت رمختلا ميزنلإا جاتنلإ ةيكرحلا ةساردلا جئاتن تلجس دعب 156 يف نيضحتلا نم ةعاس ةعسب رمخم 2 دنع رتل T° 22°C ; pH 5 ; Agitation 100rpm يزلايما طاشن ىصقأ و ةيويح ةلتك ربكا ةميقب 28.6 g/L و 350.2 U .يلاوتلا ىلع يعيبطلا ديملايركلاا لج لامعتسا دنع ايداحأ اطيرش تطعأ ميزنلأل ةيئزجلا ةيقنتلا PAGE نزوو ب ردق يئيزج 60.5 KDa ةرارحو ةضومح يتجرد وذ 5.5 و °C 30 ظفتحي . ميزنا α-amylase رطف نم P.camemberti PL21 ةبسنب 60% دنع 50°C ةدمل 120 ةميقب ةيكرح تارشؤم ميزنلالل .د 0.92 غم / لم و U 30.5 طاشن ةدحو نم لكل Km و Vmax نم لك تانويا لمعت .يلاوتلا ىلع Ca2+ و Mg2+ طيشنت ىلع دوجو يف طبثي امنيب ميزنلإا Cu2+, Fe2+ et Hg2+ . ميزنإ تيبثت مت امك α-amylase رطف هزرفا يذلاو ايئزج ىقنملا P. camemberti PL21 ةقيرطب زيكرتب تانيجللاا لج تابيبح لخاد ءاوتحلاا (3%) ذإ .تبثملا و رحلا ميزنلإا نم لك صاوخ ةسارد مت دقو تيبثتلا ةبسن تغلب 68 % . ميزنلإا نم لكل يئادتبلاا زيكرتلا ريثأت ةسارد للاخ نم لصحتملا جئاتنلا تحضوأ دقو دنع ىلثملا طورشلا يف لعافتلا ةيكرح ىلع ءاشنلا لعافتلا ةدام و 30°C و pH 5.0 ىوتسم ىلع ارييغت لا ارشؤم رحلا ميزنلإاب ةنراقم تبثملا ميزنلأل ةيكرحلا ت ةميق ترھظ ثيح Km تضفخنا نيح يف ةتباث ابيرقت ىوصقلا ةعرسلا ةميق Vmax ةبسنب 32 % ةجردب تبثملا ميزنلأا زيمتي . 5.5 pH ىلثم ةرارح ةجرد و °C 50 ةدمل يرارح تابث عم 120 دنع ةقيقد °C 90 ظفتحي . ةداعإ نكميو لوطأ ةدمل هطاشنب تبثملا ميزنلأا هلامعتسا 5 دنع نيزختلل هتابث ةردق عم تارم 4°C ةدمل 10 للاخ ةيلك هطاشن دقفي امنيب مايأ 3 مايأ دنع °C 30 . :حاتفملا تاملكلا Penicillium Camemberti PL21 ميزنإ , α-amylase ,لاقتربلا روشق , Plackett and Burman , .ةيقنت , رمخت , تانيجللاا لج تابيبح
Conclusion
127 Abstract
Alpha-amylase from Penicillium camemberti PL21 was produced in a 2l fermenter at 22°C, agitation 100 rpm, pH 5 and an aeration of 0.50 l-1 min-1 .The culture medium was prepared to include orange waste peel (2% w/v), and enriched by: yeast extracts (5g/l), the “corn-steep-liquor” (5ml/l), CaCl2 (0.125 mg/l), starch (10gr/l) and glycerol (5ml/l). After 156 h of incubation, 200 ml of the culture medium containing α -amylase were collected and determined their amylolytic activity and protein.
The α-amylase was purified by ammonium sulphate precipitation, dialysis, Sephadex G-100 and DEAE-Sepharose CL-6B column chromatography. A purification factor of 38.5 with a 23.1% yield and final specific activity of 154.2 U proteins were recorded. Purification to homogeneity of α-amylase was confirmed by SDS PAGE. The molecular weight was estimated to be 60.5 KDa. The enzyme shows maximal activity in the soluble starch hydrolysis at pH 5, 5 and is stable in a range of pH from 5.0 to 6.0. Thermal stability was in the range of temperature from 50 to 60 °C, and its optimal temperature was 30 °C. Ions Cu2+, Fe2+, and Hg2+significantly increase the activity of the enzyme, with ion Ca2+ as the highest activator. Km and Vmax were respectively 0.92 mg/mL 30.5 U calculated using the linearization of Lineweaver-Burk.
P. camemberti PL21 α-amylase was immobilized in calcium alginate gel beads. Immobilization resulted in 68 % relative activity. The immobilized α-amylase showed no decrease in activity for 180 min. Optimum conditions were not affected by immobilization, and optimum pH and temperature for free and immobilized enzyme were 5.0-5.5 and 30-50°C, respectively. Immobilized enzyme was more stable at low and high pH and high temperatures 120 min at 90°C. The kinetic parameters for the immobilized α-amylase were also determined: Km = 0.93 mg/ml and Vmax = 94.0 U. The immobilized α-amylase could be used for 5 cycles and retains its activity for 10 days without any decrease at 4°C.
Keywords: α-amylase, Penicillium camemberti PL21, orange wastes peel, Fermentation, Purification; SDS-PAGE, Immobilization, calcium alginate beads.
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