Characterization of a double cellulose-binding domain

Synergistic high affinity binding to crystalline cellulose

Markus Linder (Corresponding Author), Irma Salovuori, Laura Ruohonen, Tuula Teeri

    Research output: Contribution to journalArticleScientificpeer-review

    122 Citations (Scopus)

    Abstract

    Most cellulose-degrading enzymes have a two-domain structure that consists of a catalytic and a cellulose-binding domain (CBD) connected by a linker region. The linkage and the interactions of the two domains represent one of the key questions for the understanding of the function of these enzymes. The CBDs of fungal cellulases are small peptides folding into a rigid, disulfide-stabilized structure that has a distinct cellulose binding face. Here we describe properties of a recombinant double CBD, constructed by fusing the CBDs of two Trichoderma reesei cellobiohydrolases via a linker peptide similar to the natural cellulase linkers. After expression in Escherichia coli, the protein was purified from the culture medium by reversed phase chromatography and the individual domains obtained by trypsin digestion. Binding of the double CBD and its single CBD components was investigated on different types of cellulose substrates as well as chitin. Under saturating conditions, nearly 20 micromol/g of the double CBD was bound onto microcrystalline cellulose. The double CBD exhibited much higher affinity on cellulose than either of the single CBDs, indicating an interplay between the two components. A two-step model is proposed to explain the binding behavior of the double CBD. A similar interplay between the domains in the native enzyme is suggested for its binding to cellulase.
    Original languageEnglish
    Pages (from-to)21268-21272
    Number of pages5
    JournalJournal of Biological Chemistry
    Volume271
    Issue number35
    DOIs
    Publication statusPublished - 1996
    MoE publication typeA1 Journal article-refereed

    Fingerprint

    Cellulose
    Crystalline materials
    Cellulase
    Enzymes
    Cellulose 1,4-beta-Cellobiosidase
    Cellulases
    Peptides
    Trichoderma
    Chitin
    Escherichia coli Proteins
    Reverse-Phase Chromatography
    Chromatography
    Disulfides
    Trypsin
    Culture Media
    Digestion
    Substrates

    Cite this

    Linder, Markus ; Salovuori, Irma ; Ruohonen, Laura ; Teeri, Tuula. / Characterization of a double cellulose-binding domain : Synergistic high affinity binding to crystalline cellulose. In: Journal of Biological Chemistry. 1996 ; Vol. 271, No. 35. pp. 21268-21272.
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    abstract = "Most cellulose-degrading enzymes have a two-domain structure that consists of a catalytic and a cellulose-binding domain (CBD) connected by a linker region. The linkage and the interactions of the two domains represent one of the key questions for the understanding of the function of these enzymes. The CBDs of fungal cellulases are small peptides folding into a rigid, disulfide-stabilized structure that has a distinct cellulose binding face. Here we describe properties of a recombinant double CBD, constructed by fusing the CBDs of two Trichoderma reesei cellobiohydrolases via a linker peptide similar to the natural cellulase linkers. After expression in Escherichia coli, the protein was purified from the culture medium by reversed phase chromatography and the individual domains obtained by trypsin digestion. Binding of the double CBD and its single CBD components was investigated on different types of cellulose substrates as well as chitin. Under saturating conditions, nearly 20 micromol/g of the double CBD was bound onto microcrystalline cellulose. The double CBD exhibited much higher affinity on cellulose than either of the single CBDs, indicating an interplay between the two components. A two-step model is proposed to explain the binding behavior of the double CBD. A similar interplay between the domains in the native enzyme is suggested for its binding to cellulase.",
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    Characterization of a double cellulose-binding domain : Synergistic high affinity binding to crystalline cellulose. / Linder, Markus (Corresponding Author); Salovuori, Irma; Ruohonen, Laura; Teeri, Tuula.

    In: Journal of Biological Chemistry, Vol. 271, No. 35, 1996, p. 21268-21272.

    Research output: Contribution to journalArticleScientificpeer-review

    TY - JOUR

    T1 - Characterization of a double cellulose-binding domain

    T2 - Synergistic high affinity binding to crystalline cellulose

    AU - Linder, Markus

    AU - Salovuori, Irma

    AU - Ruohonen, Laura

    AU - Teeri, Tuula

    N1 - Project code: B5SU00132

    PY - 1996

    Y1 - 1996

    N2 - Most cellulose-degrading enzymes have a two-domain structure that consists of a catalytic and a cellulose-binding domain (CBD) connected by a linker region. The linkage and the interactions of the two domains represent one of the key questions for the understanding of the function of these enzymes. The CBDs of fungal cellulases are small peptides folding into a rigid, disulfide-stabilized structure that has a distinct cellulose binding face. Here we describe properties of a recombinant double CBD, constructed by fusing the CBDs of two Trichoderma reesei cellobiohydrolases via a linker peptide similar to the natural cellulase linkers. After expression in Escherichia coli, the protein was purified from the culture medium by reversed phase chromatography and the individual domains obtained by trypsin digestion. Binding of the double CBD and its single CBD components was investigated on different types of cellulose substrates as well as chitin. Under saturating conditions, nearly 20 micromol/g of the double CBD was bound onto microcrystalline cellulose. The double CBD exhibited much higher affinity on cellulose than either of the single CBDs, indicating an interplay between the two components. A two-step model is proposed to explain the binding behavior of the double CBD. A similar interplay between the domains in the native enzyme is suggested for its binding to cellulase.

    AB - Most cellulose-degrading enzymes have a two-domain structure that consists of a catalytic and a cellulose-binding domain (CBD) connected by a linker region. The linkage and the interactions of the two domains represent one of the key questions for the understanding of the function of these enzymes. The CBDs of fungal cellulases are small peptides folding into a rigid, disulfide-stabilized structure that has a distinct cellulose binding face. Here we describe properties of a recombinant double CBD, constructed by fusing the CBDs of two Trichoderma reesei cellobiohydrolases via a linker peptide similar to the natural cellulase linkers. After expression in Escherichia coli, the protein was purified from the culture medium by reversed phase chromatography and the individual domains obtained by trypsin digestion. Binding of the double CBD and its single CBD components was investigated on different types of cellulose substrates as well as chitin. Under saturating conditions, nearly 20 micromol/g of the double CBD was bound onto microcrystalline cellulose. The double CBD exhibited much higher affinity on cellulose than either of the single CBDs, indicating an interplay between the two components. A two-step model is proposed to explain the binding behavior of the double CBD. A similar interplay between the domains in the native enzyme is suggested for its binding to cellulase.

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