Cooperativity and substrate specificity of an alkaline amylase and neopullulanase complex of Micrococcus halobius OR-1 |
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Authors: | Kamakshi P Rajdevi Ganesa Yogeeswaran |
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Institution: | (1) Division of Medical Biotechnology, Research and Development, Tamilnad Hospitals Academic Trust-Research Council, Cheran Nagar, 601 302 Perumbakkam, Chennai, India;(2) Present address: Division of Biotechnology R&D (III Floor), Sri Ramachandra Medical College and Deemed University, 600 116 Porur, Chennai, India |
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Abstract: | The saccharifying alkaline amylase and neopullulanase complex of Micrococcus halobius OR-1 hydrolyzes both α-(1,4)- and α-(1,6)-glycosidic linkages of different linear and branched polysaccharides. The following
observations were made concerning the analysis of the coexpressed amylase and neopullulanase enzymes. Even though the enzymes
were subjected to a rigorous purification protocol, the activities could not be separated, because both the enzymes were found
to migrate in a single peak. By contrast, two independent bands of amylolytic activity at 70 kDa and pullulanolytic activity
at 53 kDa were evident by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), reducing and nonreducing PAGE,
and zymographic analysis on different polysaccharides. Preferential chemical modification of the enzyme and concomitant high-performance
thin-layer chromatographic analyses of the saccharides liberated showed that amylase is sensitive to 1-(dimethylamino-propyl)-3-ethyl
carbodiimide-HCl and cleaved α-(1,4) linkages of starch, amylose, and amylopectin producing predominantly maltotriose. On
the other hand, formalin-sensitive neopullulanase acts on both α-(1,4) and α-(1,6) linkages of pullulan and starch with maltotriose
and panose as major products. It is understood that neopullulanase exhibits dual activity and acts in synergy with amylase
toward the hydrolysis of α-(1,4) linkages, thereby increasing the overall reaction rate; however, such a synergism is not
seen in zymograms, in which the enzymes are physically separated during electrophoresis. It is presumed that SDS-protein intercalation
dissociated the enzyme complex, without altering the individual active site architecture, with only the synergism lost. The
optimum temperature and pH of amylase and neopullulanase were 60°C and 8.0, respectively. The enzymes were found stable in
high alkaline pH for 24 h. Therefore, the saccharifying alkaline amylase and neopullulanase of M. halobius OR-1 evolved from tapioca cultivar shows a highly active and unique enzyme complex with several valuable biochemical features. |
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Keywords: | Micrococcus substrate specificity amylase neopullulanase |
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