Cellulase Ce15A from alkalophilic Bacillus sp. 1139 contains a family 17 carbohydrate-binding module (BspCBM17) and a family 28 CBM (BspCBM28) in tandem. The two modules have significantly similar amino acid sequences, but amino acid residues essential for binding are not conserved. BspCBM28 was obtained as a discrete polypeptide by engineering the cel5A gene. BspCBM17 could not be obtained as a discrete polypeptide, so a family 17 CBM from endoglucanase Cel5A of Clostridium cellulovorans, CcCBM17, was used to compare the binding characteristics of the two families of CBM. Both CcCBM17 and BspCBM28 recognized two classes of binding sites on amorphous cellulose: a high affinity site (K-a similar to1 x 10(6) m(-1)) and a low affinity site ( similar to2 X 10(4) m(-1)). They did not compete for binding to the high affinity sites, suggesting that they bound at different sites on the cellulose. A polypeptide, BspCBM17/ CBM28, comprising the tandem CBMs from Ce15A, bound to amorphous cellulose with a significantly higher affinity than the sum of the affinities of CcCBM17 and BspCBM28, indicating cooperativity between the linked CBMs. Ce15A mutants were constructed that were defective in one or both of the CBMs. The mutants differed from the wild-type enzyme in the amounts and sizes of the soluble products produced from amorphous cellulose. This suggests that either the CBMs can modify the action of the catalytic module of Ce15A or that they target the enzyme to areas of the cellulose that differ in susceptibility to hydrolysis.
机构:PENCE Inc, Prot Engn Network Ctr Excellence, Natl Business Ctr, Heritage Med Res Ctr 750, Edmonton, AB T6G 2S2, Canada
Boraston, AB
;
Tomme, P
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机构:PENCE Inc, Prot Engn Network Ctr Excellence, Natl Business Ctr, Heritage Med Res Ctr 750, Edmonton, AB T6G 2S2, Canada
Tomme, P
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Amandoron, EA
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机构:PENCE Inc, Prot Engn Network Ctr Excellence, Natl Business Ctr, Heritage Med Res Ctr 750, Edmonton, AB T6G 2S2, Canada
Amandoron, EA
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Kilburn, DG
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PENCE Inc, Prot Engn Network Ctr Excellence, Natl Business Ctr, Heritage Med Res Ctr 750, Edmonton, AB T6G 2S2, CanadaPENCE Inc, Prot Engn Network Ctr Excellence, Natl Business Ctr, Heritage Med Res Ctr 750, Edmonton, AB T6G 2S2, Canada
机构:PENCE Inc, Prot Engn Network Ctr Excellence, Natl Business Ctr, Heritage Med Res Ctr 750, Edmonton, AB T6G 2S2, Canada
Boraston, AB
;
Tomme, P
论文数: 0引用数: 0
h-index: 0
机构:PENCE Inc, Prot Engn Network Ctr Excellence, Natl Business Ctr, Heritage Med Res Ctr 750, Edmonton, AB T6G 2S2, Canada
Tomme, P
;
Amandoron, EA
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h-index: 0
机构:PENCE Inc, Prot Engn Network Ctr Excellence, Natl Business Ctr, Heritage Med Res Ctr 750, Edmonton, AB T6G 2S2, Canada
Amandoron, EA
;
Kilburn, DG
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h-index: 0
机构:
PENCE Inc, Prot Engn Network Ctr Excellence, Natl Business Ctr, Heritage Med Res Ctr 750, Edmonton, AB T6G 2S2, CanadaPENCE Inc, Prot Engn Network Ctr Excellence, Natl Business Ctr, Heritage Med Res Ctr 750, Edmonton, AB T6G 2S2, Canada