Isolation of four major subunits from Clostridium thermocellum cellulosome and their synergism in the hydrolysis of crystalline cellulose.
The cellulosome of Clostridium thermocellum, purified by affinity chromatography, was dissociated under mild conditions and separated by SDS-PAGE. Two major p-nitrophenylcellobiosidases (PNPCases I and II) corresponding to the S5 (103 kDa) and S8 (78 kDa) subunits and one major carboxymethylcellulase (CMCase) coinciding with the S11 (60.5 kDa) subunit were isolated and characterized using carboxymethylcellulose (CMC), H3PO4-swollen cellulose and cello-oligosaccharides. Both PNPCases showed little effect on the viscosity of CMC and released twice as much total sugar as reducing sugar from H3PO4-swollen cellulose. The CMCase released ten times more total sugar than reducing sugar from H3PO4-swollen cellulose and reduced the viscosity of CMC rapidly. None of these enzymes was active on cellotriose. Both PNPCases released cellobiose from cellotetraose, and cellobiose and cellotriose from cellopentaose. In contrast, CMCase was active only on cellopentaose and released mainly glucose. Use of MeUmb(Glc)n revealed that both PNPCases cleaved preferentially either the second or fourth linkage from the non-reducing end while the CMCase was specific for the internal glycosidic bonds. Thus, the PNPCases and CMCase behaved as typical exo- and endoglucanases, respectively. When tested individually, all three enzymes degraded Avicel only to a small extent. A 1.5-2.0-fold increase in sugar release was observed when CMCase was combined with either PNPCase I, II or both. Combining S1 with either PNPCase II or CMCase resulted in fourfold synergism in the hydrolysis of Avicel. Synergism was sevenfold when all three enzymes were combined with S1.
Bhat
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Department of Protein Engineering, Reading Laboratory, Earley Gate, UK.
Goodenough
P W
PW
Bhat
M K
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Owen
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D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002240:Carbohydrate Sequence; D002480:Cellulase; D002482:Cellulose / Q000737:chemistry / Q000378:metabolism*; D002846:Chromatography, Affinity; D003013:Clostridium / Q000737:chemistry* / Q000648:ultrastructure; D003460:Crystallization; D004357:Drug Synergism; D006026:Glycoside Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D006868:Hydrolysis; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry*; D009844:Oligosaccharides / Q000378:metabolism; D017434:Protein Structure, Tertiary
D001426:Bacterial Proteins; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D009844:Oligosaccharides; D002482:Cellulose; C093314:4-nitrophenyl cellobiosidase I; C093315:4-nitrophenyl cellobiosidase II; D006026:Glycoside Hydrolases; D002480:Cellulase; C019826:carboxymethylcellulase