Cloning, expression and characterization of a family-74 xyloglucanase from Thermobifida fusca.
Thermobifida fusca xyloglucan-specific endo-beta-1,4-glucanase (Xeg)74 and the Xeg74 catalytic domain (CD) were cloned, expressed in Escherichia coli, purified and characterized. This enzyme has a glycohydrolase 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 2.4 micro m and 966 micro mol xyloglucan oligosaccharides (XGOs) min-1. micro mol protein-1. More than 75% of the activity was retained after a 16-h incubation at temperatures up to 60 degrees C. The enzyme was most active at pH 6.0-9.4. NMR analysis showed that its catalytic mechanism is inverting. The oligosaccharide products from hydrolysis of tamXG were determined by MS analysis. Cel9B, an active carboxymethylcellulose (CMC)ase from T. fusca, was also found to have activity on xyloglucan (XG) at 49 micro mol.min-1. micro mol protein-1, but it could not hydrolyze XG units containing galactose. An XG/cellulose composite was prepared by growing Gluconacetobacterxylinus 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.
Irwin
Diana C
DC
Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA.
Cheng
Mark
M
Xiang
Bosong
B
Rose
Jocelyn K C
JK
Wilson
David B
DB
D000091:Acetobacter / Q000737:chemistry; D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002240:Carbohydrate Sequence; D020134:Catalytic Domain; D002473:Cell Wall / Q000378:metabolism; D002482:Cellulose / Q000737:chemistry / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D005936:Glucans; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D018551:Solanum lycopersicum / Q000737:chemistry; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012639:Seeds / Q000737:chemistry; D029861:Tamarindus / Q000737:chemistry; D013696:Temperature; D014990:Xylans
D005936:Glucans; D009844:Oligosaccharides; D011134:Polysaccharides; D011994:Recombinant Proteins; D014990:Xylans; C029353:xyloglucan; D002482:Cellulose; D006026:Glycoside Hydrolases; C072818:xyloglucan endo(1-4)-beta-D-glucanase