Effect of maleate counterion on malease activity: Production of d-malate in a crystal-liquid two-phase system

Abstract

Malease activity of permeabilized cells of Pseudomonas pseudoalcaligenes was strongly affected by the maleate salt used as the substrate. With Na 2-maleate as the substrate, malease activity decreased strongly with increasing substrate concentrations. By using counterions other than sodium, the concentration of available dissociated maleate, the “real” substrate for the enzyme, could be reduced significantly, owing to metal-substrate complex formation. Use of metal ions that resulted in the formation of an insoluble metal-substrate complex, such as Ca 2+ and Ba 2+, further reduced the maleate 2− concentration. The use of these metal ions resulted in the formation of a crystal-liquid two-phase system, owing to the low solubility of both the metal-substrate and the metal-product complex. Ca 2+ was found to be the best counterion for the conversion of high concentrations (up to 160 g l −1) of either maleate or citraconate by malease into d-malate and d-citramalate, respectively, with yields >99%. The use of this metal ion gave the highest malease activities and no substrate inhibition could be observed using this counterion.