Purification and Properties of a Cellobiose Phosphorylase (CepA) and a Cellodextrin Phosphorylase (CepB) from the Cellulolytic Thermophile Clostridium Stercorarium

Abstract

Two phosphorolytic enzymes displaying activity towards the soluble cellulose degradation products cellobiose and cellodextrins were purified from the crude extract of the cellulolytic thermophile Clostridium stercoruriurn. Both phosphorylases have monomeric structures with molecular masses of 93 and 91 kDa, respectively. Although the N-terminal amino acid sequences are highly similar, a clear distinction of the two enzymes could be made on the basis of their substrate specificities: the enzyme designated cellobiose phosphorylase cleaved exclusively the disaccharide substrate, whereas the enzyme designated cellodextrin phosphorylase accepted only oligosaccharides as substrates. Kinetic constants were determined for the cleavage of cellobiose and cellodextrins. Maximal activity was observed at 65°C in the pH range 6.0-7.0 for both enzymes. The sequences of the genes cepA and cepB encoding the cellobiose phosphorylase and the cellodextrin phosphorylase, respectively, have been submitted to the GenBank database.Keywords: phosphorylase ; cellobiose ; cellodextrin ; Clostridium; thermophilic.Enzymes capable of degrading cellobiose and cellodextrins are considered as essential components of microbial cellulolytic enzyme systems. They convert the cellobiose and cellodextrins formed during the enzymatic degradation of cellulose by the synergistic action of endoglucanases and exoglucanases to fermentable sugars. As cellulolytic enzymes are generally subject to product inhibition, an adequate level of cellobiose and cellodextrin removing enzymes is required for efficient breakdown of cellulose. The metabolism of soluble cellulose degradation products involves hydrolytic and phosphorolytic cleavage (Coughlan and Mayer, 1992). Hydrolysis is catalyzed by p-glucosidases releasing glucose from the disaccharide and the nonreducing ends of the oligosaccharides. Phosphorolysis is energetically advantageous and might constitute the primary route of cellobiose and cellodextrin utilization in particular in anaerobic environments. Cellobiose phosphorylase (cel1obiose:orthophosphate n-D-glUcoSyl-tranSferaSe) and cellodextrin phosphorylase

Engineering Escherichia coli Cells for Cellobiose Assimilation through a Phosphorolytic Mechanism

“…Because the phosphorylation uses inorganic phosphate as a donor, the phosphorolytic mechanism is an ATP-saving mechanism more often associated with some cellulolytic bacteria (5,12,15,20). All known cellobiose phosphorylases are cytoplasmic, as they lack signal peptides, and experimental data support this notion (1,19).…”

Lic16A of Clostridium thermocellum, a non-cellulosomal, highly complex endo-β-1,3-glucanase bound to the outer cell surface

“…C. thermocellum was described as a cellulose-hydrolysing bacterium, which grows on cellulose and cellodextrins, but not on pentoses and moreover, does not even readily grow on glucose. It was discussed that the uptake of oligocellodextrins is energetically favourable, because (1) energy is consumed for only one transportation event for more glucose residues, and (2) oligodextrins are degraded by intracellular cellobiose-and cellodextrin-phosphorylases producing glucose 1-phosphate (Tanaka et al, 1995;Reichenbecher et al, 1997;Lynd et al, 2002). It is not known if cellobiose or cellodextrin phosphorylases hydrolyse b-1,3-oligodextrins.…”

Cloning and Characterization of the Glucooligosaccharide Catabolic Pathway β-Glucan Glucohydrolase and Cellobiose Phosphorylase in the Marine HyperthermophileThermotoga neapolitana

“…Conversely, when a ␤-glucosidase hydrolyzes cellobiose, glycosidic bond energy is lost, and activation of the two glycosyl products requires two ATP molecules rather than one. To date, only cellobiose phosphorylases from C. gilvus (34), C. thermocellum (1,38), and C. stercorarium (30) have been purified and characterized. The CbpA from T. neapolitana is the first report of a cellobiose phosphorylase from any hyperthermophile.…”

“…The enzyme was incubated at 85°C for 15 min with 10 mM cellobiose in PC buffer (pH 5.5). The reaction was stopped by boiling for 10 min, and the amount of glucose 1-phosphate produced was determined by a coupled enzyme assay measuring the appearance of NADPH at 340 nm (30). The reaction mixture contained phosphoglucomutase (4 U ml Analytical methods and enzymatic characterization.…”

“…AB013109), Clostridium stercorarium (30), and Cellvibrio gilvus (26), respectively. It is also noted that the cellobiose phosphorylases, including T. neapolitana CbpA, have Ͼ50% similarity to the C-terminal domains of R. meliloti NdvB (21) and B. abortus Cgs proteins, which are responsible for ␤-(1,2) glucan synthetase activity (22).…”