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

Abstract

Rates of phosphorolytic cleavage of ␤-glucan substrates were determined for cell extracts from Clostridium thermocellum ATCC 27405 and were compared to rates of hydrolytic cleavage. Reactions with cellopentaose and cellobiose were evaluated for both cellulose (Avicel)-and cellobiose-grown cultures, with more limited data also obtained for cellotetraose. To measure the reaction rate in the chain-shortening direction at elevated temperatures, an assay protocol was developed featuring discrete sampling at 60°C followed by subsequent analysis of reaction products (glucose and glucose-1-phosphate) at 35°C. Calculated rates of phosphorolytic cleavage for cell extract from Avicel-grown cells exceeded rates of hydrolytic cleavage by >20-fold for both cellobiose and cellopentaose over a 10-fold range of ␤-glucan concentrations (0.5 to 5 mM) and for cellotetraose at a single concentration (2 mM). Rates of phosphorolytic cleavage of ␤-glucosidic bonds measured in cell extracts were similar to rates observed in growing cultures. Comparisons of V max values indicated that cellobiose-and cellodextrin-phosphorylating activities are synthesized during growth on both cellobiose and Avicel but are subject to some degree of metabolic control. The apparent K m for phosphorolytic cleavage was lower for cellopentaose (mean value for Avicel-and cellobiose-grown cells, 0.61 mM) than for cellobiose (mean value, 3.3 mM).

Regulation of Cellulase Synthesis in Batch and Continuous Cultures of Clostridium thermocellum

“…Phosphorolytic cleavage of ␤-glucosidic bonds by cellobiose and cellodextrin phosphorylases may contribute to the increasing cell yields with increasing substrate chain length (34). Consistent with this, the rates of phosphorolytic cleavage of both cellobiose and cellopentaose are much higher (e.g., 20-fold higher) than the rates of hydrolytic cleavage in C. thermocellum cell extracts grown on either Avicel or cellobiose (57). Lower ATP expenditure for substrate transport has also been suggested as a factor that contributes to higher cell yields for substrates with greater chain lengths (34).…”

“…For determination of cellobiose concentrations below the high-performance liquid chromatography (HPLC) detection limit (ϳ0.15 g/liter), 0.8 ml of cell-free broth after centrifugation was mixed with 0.2 ml of 10% (wt/wt) sulfuric acid and then autoclaved for 40 min to convert the cellobiose to glucose. After neutralization to pH 4 to 6 with solid CaCO 3 and centrifugation, the concentration of glucose in the supernatant was measured using the Sigma Infinity glucose kit reagents at double the concentrations recommended (Sigma G2020), which can detect a concentration of glucose as low as 0.005 g/liter (57). Ethanol, lactate, acetate, cellobiose, and glucose in samples taken from steady-state continuous fermentors were acidified by adding 0.0565 ml of sulfuric acid (10%, wt/wt) to 1-ml samples, which were subsequently analyzed by HPLC by using a Bio-Rad HPX-87H column (Bio-Rad, Hercules, Calif.) operated at 55°C with a 0.01% (vol/vol) H 2 SO 4 running buffer and a refractive index detector.…”

“…C. thermocellum ATCC 27405 was a gift from Arnold Demain in 1983 and has been maintained in our lab since then as described elsewhere (34). Stock cultures were maintained in MTC medium (see below) containing 10 g of Avicel (PH105; FMC Corp., Philadelphia, Pa.) per liter as the carbon source (56,57).…”

Cellulase, Clostridia, and Ethanol

“…Two intracellular ␤-glucosidases have been purified, characterized, and cloned (3,103,148,153,294). Rates of phosphorolytic cleavage have been found to be 20-fold higher than those of hydrolytic cleavage (365). From a bioenergetic standpoint, phosphorolytic cleavage is more beneficial because it provides a route to ATP synthesis.…”

Structural and mechanistic analysis of a β-glycoside phosphorylase identified by screening a metagenomic library

“…GPs distinguish themselves from most CAZymes in that the hydrolytic free energy associated with the glycosidic ester linkage of the sugar-1-phosphate product is roughly equivalent to that of the glycosidic linkage in the glycan substrate. Accordingly, the equilibrium constants for the reactions catalyzed by most GPs is close to 1, thus the equilibrium position can be tipped in favour of glycoside synthesis by manipulation of reaction conditions [11][12][13] . Since the discovery of the first GP, glycogen phosphorylase, in 1938 14 only 29 distinct new GP activities have been identified; the majority in the past 15 years 15 .…”