Stabilization of D-amino acid oxidase and catalase in permeabilized Rhodotorula gracilis cells and its application for the preparation of alpha-ketoacids*

The cellular D-amino acid oxidase (DAAO) and catalase activities of Rhodotorula gracilis were greatly increased upon the treatment of the cells with cetyltrimethylammonium bromide (CTAB). However, these enzymes, slowly leaks out from the permeabilized cells. The released DAAO was rapidly inactivated in the absence of ethylenediaminotetraacetic acid (EDTA), beta-mercaptoethanol, and glycerol. DAAO within the permeabilized cells did not require these stabilizing agents. Treating the CTAB-permeabilized cells with 0.2% glutaraldehyde (GA) at 4 degrees C for 10 min prevented the leakage of both DAAO and catalase. Alternately, stabilized whole cell DAAO and catalase was prepared by treating the whole yeast cells with 1% GA at 4 degrees C for 60 min, followed by permeabilization with CTAB, a method which was equally efficient but easy to scale up. CTAB-permeabilized cells converted D-phenylalanine to 97% phenylpyruvate and 3% phenylacetate, and these cells were reused up to 3 cycles in a batchwise reaction. On the other hand, GA-treated CTAB-permeabilized cells produced more than 99% phenylpyruvate and the cells could be reused up to 20 cycles.