Effect of C-terminal truncation on enzyme properties of recombinant amylopullulanase from <Emphasis Type="Italic">Thermoanaerobacter pseudoethanolicus</Emphasis> |
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Authors: | Fu-Pang Lin Yi-Hsuan Ho Hsu-Yang Lin Hui-Ju Lin |
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Institution: | (1) Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan;(2) Department of Life Science, National Taiwan Ocean University, Keelung, Taiwan;(3) Center of Excellence for Marine Bioenvironment and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan;(4) Department of Health, Food and Drug Administration, Executive Yuan, Taipei, Taiwan |
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Abstract: | The smallest and enzymatically active molecule, TetApuQ818, was localized within the C-terminal Q818 amino acid residue after
serial C-terminal truncation analysis of the recombinant amylopullulanase molecule (TetApuM955) from Thermoanaerobacter pseudoethanolicus. Kinetic analyses indicated that the overall catalytic efficiency, k
cat/K
m, of TetApuQ818 was 8–32% decreased for the pullulan and the soluble starch substrate, respectively. Changes to the substrate
affinity, K
m, and the turnover rate, k
cat, were decreased significantly in both enzymatic activities of TetApuQ818. TetApuQ818 exhibited less thermostability than
TetApuM955 when the temperature was raised above 85°C, but it had similar substrate-binding ability and hydrolysis products
toward various substrates as TetApuM955 did. Both enzymes showed similar spectroscopies of fluorescence and circular dichroism,
suggesting the active folding conformation was maintained after this C-terminal Q818 deletion. This study suggested that the
binding ability of insoluble starch by TetApuM955 did not rely on the putative C-terminal carbohydrate binding module family
20 (CBM20) and two FnIII regions of TetApu, though the integrity of the AamyC module of TetApuQ818 was required for the enzyme
activity. |
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Keywords: | |
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