Large-scale production of Mandelate racemase by Pseudomonas putida ATCC 12633: optimization of enzyme induction and development of a stable crude enzyme preparation

Abstract

The production of mandelate racemase [EC 5.1.2.2] from Pseudomonas putida ATCC 12633 was optimized with respect to (i) biomass and (ii) enzyme induction. The dependence of biomass yield and enzyme content on the concentration of carbon-sources (such as glucose and mandelate) was determined by applying batch- and fed-batch cultivation on a ten-liter scale. On the one hand, highest cell-densities were obtained in fed-batch cultivations with repeated glucose- and mandelate-feed but mandelate racemase induction was low. On the other hand, excellent enzyme-induction was obtained in batch-cultivation when both glucose and mandelate were used right from the start as carbon source. The optimal time for cell-harvest was indicated by a characteristic decolorization of the growth medium from grey/blue to yellow. This latter phenomenon (which is caused by the degradation of the blue Fe(III)-complex of catechol, a late intermediate of the mandelate pathway) greatly simplified the monitoring of the enzyme activity and the determination of the optimal time for harvest. Based on these results, a lyophilized crude enzyme preparation was prepared from a 10-liter bioreactor showing a total specific activity of 23 10 6 U ( μmol g −1 min −1). This represents roughly a 300-fold enhancement in activity as compared to the previous protocol.