Characterization and comparison of Clostridium cellulovorans endoglucanases-xylanases EngB and EngD hyperexpressed in Escherichia coli.
By the use of a T7 expression system, endoglucanases-xylanases EngB and EngD from Clostridium cellulovorans were hyperexpressed and purified from Escherichia coli. The two enzymes demonstrated both endoglucanase and xylanase activities. The substrate specificities of both endoglucanases were similar except that EngD had four-times-greater p-nitrophenyl beta-1,4-cellobiosidase activity. The two proteins were very homologous (80%) up to the Pro-Thr-Thr region which divided the protein into -NH2- and -COOH-terminals. The -COOH- region of EngB has high homology to the endoglucanases and a xylanase from Clostridium thermocellum and to an endoglucanase from Clostridium cellulolyticum and did not show strong binding to cellulose (Avicel). However, the -COOH- region of EngD, which had homology to the cellulose-binding domains of Cellulomonas fimi exo- and endoglucanases and to Pseudomonas fluorescens endoglucanase, demonstrated binding ability to cellulose even when the domain was fused to the N-terminal domain of EngB. By probing the Avicel-purified cellulase complex (F8) with anti-EngB and anti-EngD antibodies, both EngB and EngD were shown to be present on the cellulase complex of C. cellulovorans. Many proteins homologous to EngB and EngD were also present on the complex.
Foong
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Department of Biochemistry and Biophysics, University of California, Davis 95616.
Doi
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D001665:Binding Sites / Q000502:physiology; D015153:Blotting, Western; D002480:Cellulase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D043325:Xylan Endo-1,3-beta-Xylosidase
D002482:Cellulose; D006026:Glycoside Hydrolases; D043325:Xylan Endo-1,3-beta-Xylosidase; D002480:Cellulase