LARGE-SCALE PRODUCTION OF MANDELATE RACEMASE BY PSEUDOMONAS-PUTIDA ATCC-12633 - OPTIMIZATION OF ENZYME-INDUCTION AND DEVELOPMENT OF A STABLE CRUDE ENZYME PREPARATION

The production of mandelate racemase [EC 5.1.2.2] from Pseudomonas put ida ATCC 12633 was optimized with respect to (i) biomass and (ii) enzy me induction. The dependence of biomass yield and enzyme content on th e 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-b atch cultivations with repeated glucose- and mandelate-feed but mandel ate racemase induction was low. On the other hand, excellent enzyme-in duction was obtained in batch-cultivation when both glucose and mandel ate were used right from the start as carbon source. The optimal time for cell-harvest was indicated by a characteristic decolorization of t he growth medium from grey/blue to yellow. This latter phenomenon (whi ch is caused by the degradation of the blue Fe(III)-complex of catecho l, a late intermediate of the mandelate pathway) greatly simplified th e monitoring of the enzyme activity and the determination of the optim al time for harvest. Based on these results, a lyophilized crude enzym e preparation was prepared from a 10-liter bioreactor showing a total specific activity of 23 10(6) U (mu mol g(-1) min(-1)). This represent s roughly a 300-fold enhancement in activity as compared to the previo us protocol. (C) 1997 Elsevier Science B.V.