tailieunhanh - Báo cáo khoa học: Cloning, expression and characterization of a family-74 xyloglucanase from Thermobifida fusca

Thermobifida fusca xyloglucan-specific endo-b-1,4-gluca-nase (Xeg)74 and the Xeg74 catalytic domain (CD) were cloned, expressed inEscherichia coli, purified and enzymehas aglycohydrolase family-74CDthat is a specific xyloglucanase followed by a family-2 carbo-hydrate binding module at the C Michaelis constant (Km) andmaximal rate (Vmax) values for hydrolysis of tamarind seed xyloglucan (tamXG) are 966lmol xyloglucan oligosaccharides (XGOs) min )1 Ælmol protein )1 | Eur. J. Biochem. 270 3083-3091 2003 FEBS 2003 doi Cloning expression and characterization of a family-74 xyloglucanase from Thermobifida fusca Diana C. Irwin Mark Cheng Bosong Xiangt Jocelyn K. C. Rose and David B. Wilson Department of Molecular Biology and Genetics Cornell University Ithaca New York USA Thermobifida fusca xyloglucan-specific endo-P-1 4-gluca-nase Xeg 74 and the Xeg74 catalytic domain CD were cloned expressed in Escherichia coli purified and characterized. This enzyme has a glycohyd rolase family-74 CD that is a specific xyloglucanase followed by a family-2 carbohydrate binding module at the C terminus. The Michaelis constant Km and maximal rate Vmax values for hydrolysis of tamarind seed xyloglucan tamXG are M and 966 imol xyloglucan oligosaccharides XGOs min-1-pmol protein-1. More than 75 of th e activity was retained after a 16-h incubation at temperatures up to 60 C. Tire nn ynre was most active at pH . NMR analysis showed that its catalytic mechanism is inverting. The ogoc sachhr i ide products from hydrolysis of tamXG were determined by MS analysis. Cel9B an active ca 1 boxymethylcenulose CMG ase from T. fusca was also found to have activity on xyloglucan XG at 49 pmol-min-1 mol protein-1 but it could not hydrolyze XG units containing galactose. An XXacelluOsve composite was prepared by growing Gluconacetobacter xylinus on glucose with tamXG in the medium. Although a mixture of purified cellulases was unable to degrade this material the composite material was fully hydrolyzed when Xeg74 was added. T. fusca was not able to grow on tamXG but Xeg74 was found in the culture supernatant at the same level as was found in cultures grown on Solka Floc. The function of this enzyme appears to be to break down the XG surrounding cellulose fibrils found in biomass so that T. fusca can utilize the cellulose as a carbon source. Keywords xyloglucanase cellulase inverting regulation plant cell walls. Converting .

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