Physico-chemical Approach of the Amylolytic Action Pattern of a Thermostable Amylopullulanase |
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Authors: | Houda Boussarsar Barbara Rogé Mohamed Mathlouthi |
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Institution: | (1) Laboratoire de Chimie Physique Industrielle, UMR 614 Fractionnement des Agroressources et Emballage, Université de Reims Champagne Ardenne, Centre Europol’Agro, B.P. 1039, F-51687 Reims Cedex 2, France |
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Abstract: | This paper describes the amylolytic action pattern of Thermococcus hydrothermalis recombinant amylopullulanase (Th-ApuΔ2) E.C 3.2.1.41]. A comparison was made between amylose hydrolysis catalyzed by this
enzyme and by two other amylolytic enzymes: α-amylase E.C 3.2.1.1] (from Aspergillus oryzae) and glucoamylase E.C. 3.2.1.3] (from Aspergillus niger), respectively taken as models for “endo” and “exo” catalytic patterns. Different independent physico-chemical methods were
used to characterize the hydrolysis products obtained with the studied enzymes. Viscosity results were correlated to reducing
sugars analysis to show a similarity between glucoamylase E.C. 3.2.1.3] and Th-ApuΔ2 E.C 3.2.1.41] behavior. On the other
hand, whereas α-amylase E.C 3.2.1.1] action rapidly decreased the viscosity of medium, glucoamylase and Th-ApuΔ2 hydrolysates
have only shown a negligible reduction in viscosity. Glass transition temperatures of glucoamylase and Th-ApuΔ2 hydrolysates
were found comparable (225–226°C) and significantly different from that of α-amylase (197°C). Thin-layer chromatographic analysis
of hydrolysates mainly revealed the presence of glucose in the case of glucoamylase and Th-ApuΔ2 activities and in addition
to glucose the Th-ApuΔ2 chromatograms have shown oligosaccharides with polymerization degree ranging from 2 to 7. These results
incite us to conclude that Th-ApuΔ2 has a dual “endo” and “exo” catalytic action pattern. Analysis of the Fourier transform
infrared (FTIR) results shows a comparable general aspect for all spectra. The presence of more numerous differentiated and
intense peaks in the spectrum of Th-ApuΔ2 hydrolysate reveals the presence of short-chain oligosaccharides. These results
confirm thin-layer chromatography results and support a dual action pattern. |
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Keywords: | Amylose Enzymatic action pattern Amylopullulanase Glucoamylase Alpha-amylase |
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