Starch digestion in tropical fishes: isolation, structural studies and inhibition kinetics of alpha-amylases from two tilapias Oreochromis niloticus and Sarotherodon melanotheron.
alpha-Amylases from the intestinal cavity of two tilapia species, Oreochromis niloticus (ONI-AMY) and Sarotherodon melanotheron (SME-AMY), were purified using ammonium sulfate precipitation, affinity chromatography and chromatofocusing procedures. The purification was approximately 100-fold. The amylolytic activity, specific activity, product distribution, pH and temperature profile of ONI-AMY and SME-AMY are quite similar. The molecular mass differs slightly: 56600 (ONI-AMY) vs. 55500 (SME-AMY). As shown by isoelectric focusing analysis, both amylases contain two isoforms A and B with distinct pI: 7.2 (A) and 7.8 (B), vs. 8.3 (A) and 8.8 (B), respectively. It was not possible to isolate B, since B converts into A with time. The kinetics of the inhibition of ONI-AMY and SME-AMY activity by alpha-, beta- and gamma-cyclodextrin (alpha-, beta- and gamma-CD) were investigated using amylose as the substrate. Statistical analysis of the kinetic data expressed using a general velocity equation and assuming rapid equilibrium showed that the inhibition is of the mixed noncompetitive type. Similar results were obtained with ONI-AMY and SME-AMY. beta- and gamma-CD are stronger inhibitors than alpha-CD. ONI-AMY and SME-AMY are then closely related and show the general features common to the members of the alpha-amylase class (family 13). They enable ONI and SME tilapias to digest starch in food.
IRD, c/o Institut Méditerranéen de Recherche en Nutrition, Faculté de Sciences et Techniques de St Jérôme, Université d'Aix-Marseille, av. Escadrille Normandie-Niemen, F-13397, Marseille cedex 20, France. yann.moreau@mpl.ird.fr
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Y
Moreau
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Desseaux
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Koukiekolo
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Marchis-Mouren
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Santimone
D000596:Amino Acids / Q000032:analysis; D000681:Amylases / Q000037:antagonists & inhibitors; D000818:Animals; D002846:Chromatography, Affinity; D003505:Cyclodextrins / Q000494:pharmacology; D004063:Digestion / Q000502:physiology; D006801:Humans; D007408:Intestinal Absorption / Q000187:drug effects; D007700:Kinetics; D008956:Models, Chemical; D008970:Molecular Weight; D013213:Starch / Q000378:metabolism*; D017210:Tilapia / Q000378:metabolism*; D000516:alpha-Amylases / Q000302:isolation & purification* / Q000502:physiology*
D000596:Amino Acids; D003505:Cyclodextrins; D013213:Starch; D000681:Amylases; D000516:alpha-Amylases