Binding Sites of Cellulose-binding Module
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Accession number;05A1044276
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| Title;Binding Sites of Cellulose-binding Module |
| Author;
KARITA SHUICHI
(Mie Univ., Fac. Bioresources, JPN)
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Journal Title;Shokuniku ni kansuru Josei Kenkyu Chosa Seika Hokokusho
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Journal Code:X0296A
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ISSN:
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VOL.23;NO.;PAGE.127-131(2005)
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| Figure&Table&Reference;FIG.2, TBL.1, REF.4 |
| Pub. Country;Japan |
| Language;Japanese |
| Abstract;Noncatalytic carbohydrate-binding modules (CBMs) target the enzymes to specific insoluble polysaccharides, such as celluloses and hemicelluloses, and enhance catalytic efficiency by increasing the effective concentration of the enzyme on the surface of insoluble substrates. An endoglucanase Cel 5 A from an anaerobic cellulolytic bacterium Clostridium josui, contains a family 17 CBM and a family 28 CBM in tandem and three-repeat surface-layer homologous (SLH) modules. Gene fragments encoding CBMs (CBM17, 28, and 17/28) were amplified by PCR and ligated into the plasmid pQE30 (Qiagen), the resulting plasmid being introduced into Escherichia coli JM109. The His-tagged proteins were purified with Ni-NTA agarose. The thermodynamic parameters for binding of CBMs with cellopentaose were determined using isothermal titration calorimetry (ITC) and were evaluated using MicroCal Origin software. The CBM17/28 could bind to non-crystalline cellulose, soluble cellulose derivatives, oat-spelt xylan but not bind to birch wood xylan and starch. The binding parameters were obtained for cellopentaose. The CBM17 and CBM28 showed similar binding constants, Ka, 5.41*10'5', and 5.16*10'5', respectively. However, .DELTA.H values were different, -5.69 kcal/mol and -21.2 kcal/mol, respectively. This result suggests that CBM28 is more exothermal than CBM17 for binding reaction and the binding of CBM28 to cellopentaose is enthalpically favorable with large negative .DELTA.H, while CBM17 binding is entropically favorable with positive .DELTA.S. These two CBMs have different binding mechanisms. The CBM17 could bind to the cellulose saturated with the CBM28, suggesting the CBM17 can recognize a part on cellulose surface different from the CBM28 binding site. These results might answer the question why so different CBMs exist in plant cell wall degrading enzymes. These CBMs bind to non-crystalline cellulose and recognize more fine structure on cellulose surface. (author abst.) |
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