Cellodextrin Phosphorylase from Clostridium thermocellum YM4 Strain Expressed in Escherichia coli.

Kinetics and Relative Importance of Phosphorolytic and Hydrolytic Cleavage of Cellodextrins and Cellobiose in Cell Extracts of Clostridium thermocellum

“…Thus, the greater affinity observed for phosphorylation of cellodextrins is consistent with, and may contribute to, preferred utilization of the more bioenergetically advantageous substrate. Our results are generally consistent with those of Alexander and Krishnareddy et al, who used enzymes purified from C. thermocellum assayed at 37°C to measure K m values of 7.3 mM for CbP (6) and 0.8 mM for CdP (7,24).…”

Synthesis of highly ordered cellulose II in vitro using cellodextrin phosphorylase

“…The phosphorolysis rate on cellobiose was very low, but clearly detectable, even though CDP has been believed not cleave the glucosidic bond of cellobiose. As reported previously, 27 the kinetic parameters for cellotriose and cellotetraose were similar in the present study, whereas those for cellobiose were quite different, that is, the K m value for cellobiose was 85-fold larger than the value for cellotriose, and the k cat value for cellobiose was 33-fold smaller than that for cellotriose, resulting in a kinetic efficiency for cellobiose of less than 1/1000th of those for the cello oligosaccharides. In the synthetic direction, moreover, inorganic phosphate was also produced in the reaction mixture using glucose as a glucosyl acceptor at approximately 1.9% of the rate recorded under similar conditions using cellobiose as the acceptor.…”

“…A series of soluble cello oligosaccharides was formed after the reaction with cellobiose, in good agreement with previous reports. 24,27 In contrast, no significant production of soluble cello oligosaccharides was observed with glucose, and most of the glucose remained in the reaction mixture even after accumulation of the precipitate was observed (Fig. 2e), clearly indicating that increase of DP occurs preferentially when glucose is used as the acceptor, whereas all acceptor molecules in the reaction mixture are equally oligomerized when cellobiose is used as a glucosyl acceptor.…”

“…27 The enzyme activity was determined by quantifying the amount of inorganic phosphate produced from 39 The phosphorolytic reaction of CDP was determined by measuring the increase in aG1P generated from cello oligosaccharides and inorganic phosphate in 50 mM MOPS buffer (pH 7.5) at 37°C. The amount of aG1P was quantified by using the phosphoglucomutase/glucose 6-phosphate dehydrogenase method 40 after inactivating CDP by boiling the reaction mixture for 10 min.…”

“…[21][22][23] Cellodextrin phosphorylase (CDP; EC 2.4.1.49), for example, phosphorolyzes cello oligosaccharides with the degree of polymerization greater than or equal to three, but does not react with cellobiose. CDP has been found only in Clostridia [24][25][26][27] and utilized for the preparation of cello oligosaccharide derivatives. [28][29][30][31][32] Samain et al, 33 reported the preparation of crystalline cellodextrins from cellobiose and a-glucose 1-phosphate (aG1P), and the products showed electron diffraction patterns resembling those of cellulose II.…”