Application of Escherichia coli maltodextrin-phosphorylase for the continuous production of glucose-1-phosphate

Abstract Recombinant maltodextrin-phosphorylase from Escherichia coli was produced in under control of its native promotor in E. coli JM 109 and used for glucose-1-phosphate (G-1-P) production from different α-glucans such as maltodextrins or soluble starch. A phosphorylase preparation free of contaminating phosphatase activity was recovered from crude cell extracts in approximately 75% yield by hydrophobic interaction followed by affinity chromatography. The conditions partially optimized for phosphorolytic α-glucan degradation were established at a pH of 7.5 and an inorganic phosphate concentration of 300 to 500 m m . Two factors clearly limit the attainable maximum product yield: A) the equilibrium constant of G-1-P to inorganic phosphate at 30°C and pH 7.5 (K eq = 0.22–0.24), and B) the maximum degree of conversion of a particular α-glucan by phosphorylase, i.e., 45 and 38% for starch and maltodextrins. These latter values were shown to be increased to 70 and 65% for the cases of starch and maltodextrins, respectively, by the synergistic, simultaneous, or subsequent action of pullulanase (50–100 IUg −1 substrate) and phosphorylase. Continuous, enzymatic conversion of maltodextrins in an ultrafiltration membrane reactor has been performed using limiting concentrations of either phosphate or pretreated maltodextrin. On a laboratory scale, productivities of 2.6 g (10 mmol) G-1-P per h −1 with a phosphorylase loading of 1 IU ml −1 reactor volume were reached and could be maintained for reaction times up to 600 h.