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Ultraviolet difference spectroscopic study on the interactions of cellulase fromTrichoderma reesei with cellodextrins

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Abstract

The formation of cellodextrin-cellobiohydrolase complex was studied by ultraviolet difference spectroscopy. Upon the binding of cellodextrins (G7‐G3), cellobiohydrolase (EC 3.2.1.91) purified fromTrichoderma reesei produced difference spectra having maxima at 289‐293 nm and 283‐286 nm. These spectra are consistent with prior observations reported for lysozyme and amylase. In this case, water soluble cellulose oligomers (i.e., cellodextrins) are shown to interact with tryptophan residue(s) on cellobiohydrolase. The difference spectral maxima observed at acidic or alkaline pH were shifted. This was accompanied by a marked decrease of binding ability of cellobiohydrolase for cellodextrins. The standard free energy change for the association of cellodextrins to the cellobiohydrolase was an order of 4 kcal/gmol. The association constant of enzyme for substrate decreases by 15‐20% as temperature increases from 20 to 48°C. At 25°C, the dissociation constants for the enzyme with respect to cellohexose and cellotriose were estimated to be 1.19 and 1.37 mM, respectively. A decrease in dissociation constants was observed with an increase in the number of glucosyl units from 3 to 6. This suggests that there may be six or more subsites in the active center of cellobiohydrolase.

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Authors and Affiliations

  1. Laboratory of Renewable Resources Engineering and Department of Agricultural Engineering, Purdue University, West Lafayette

    Wen-Juin Shieh, G. J. Tsai, M. R. Ladisch & G. T. Tsao

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  1. Wen-Juin Shieh
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  2. G. J. Tsai
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  3. M. R. Ladisch
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  4. G. T. Tsao
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Shieh, WJ., Tsai, G.J., Ladisch, M.R. et al. Ultraviolet difference spectroscopic study on the interactions of cellulase fromTrichoderma reesei with cellodextrins. Appl Biochem Biotechnol 22, 13–29 (1989). https://doi.org/10.1007/BF02922694

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  • DOI: https://doi.org/10.1007/BF02922694

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