Stabilization of maleate-hydratase activity of permeabilized Pseudomonas pseudoalcaligenes

As part of development of a continuous process for D-malate production, the stability of maleate hydratase in permeabilized Pseudomonas pseudoalcalige nes was characterized as a function of relevant process conditions. In a sy stem where D-malate is produced from a Ca-maleate suspension, these conditi ons were temperature, D-malate(2-), Ca2+, and biocatalyst concentration. Th e decrease of maleate-hydratase activity with time was described by first-o rder irreversible inactivation. The first-order inactivation rate constant increased with temperature between 20 degrees C and 35 degrees C and decrea sed with D-malate2- concentration between 0 and 50 mM; the temperature depe ndency increased with D-malate(2-) concentration. Although seemingly even m ore attractive with respect to biocatalyst stability, the effects of temper atures below 20 degrees C and D-malate(2-) concentrations exceeding 50 mM w ere not determined, as biocatalyst activity at these temperatures is extrem ely low and the D-malate(2-) concentration will not exceed 50 mM due to the low solubility of Ca-D-malate. Ca2+ and biocatalyst concentrations hardly affected the inactivation rate constant. However, Ca2+ can be used to contr ol the stability of the biocatalyst, as it controls the D-malate(2-) concen tration by shifting the dissociation equilibrium of Ca-D-malate towards Ca- D-malate formation with increasing Ca2+ concentration.