PERMEABILIZATION OF TRIGONOPSIS VARIABILIS FOR ENHANCED D-AMINO ACID OXIDASE ACTIVITY

The permeabilization of T. variabilis for use as a whole-cell biocatalyst has been little studied even though it is one of the most important microbial sources of D-Amino Acid Oxidase (DAO) and is useful for the separation of racemic amino acid mixtures, and the production of -keto acids and 7-amino cephalosporanic acid. In the present work, for the first time a comparative study has been carried out on the efficiency of the permeabilization of the yeast T. variabilis (CBS 4095) grown in a culture medium with D-methionine as an inducer of the synthesis of DAO using different detergents, organic solvents, and freeze-thaw cycles. The best results were obtained with treatment with cetyltrimethylammonium bromide (CTAB), 2% of wet weight of cells, at 45 degrees C for 30min. The efficiency of the permeabilization procedure was seen to depend on the detergent/cell ratio, being independent of the concentration of detergent in the reaction medium over a broad range: 0.016-0.8% (w/v). The main advantages of the CTAB procedure are the high activity and reproducibility obtained, together with the fact that it can be used with either fresh or frozen cells with almost identical results, unlike the other detergents studied (CHAPS, Triton X-100, Sarcosyl), and especially treatment with toluene-ethanol at 0 degrees C, where a previous sample-freezing step is determinant in the cell permeabilization process. The permeabilization procedure reported here can be used for both accurate in vivo quantification of DAO activity in T. variabilis and to prepare a whole-cell biocatalyst with a high level of activity and stability.