Single-pot conversion of cephalosporin C to 7-aminocephalosporanic acid in the absence of hydrogen peroxide
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In this study, d-amino acid oxidase (DAAO) and catalase (CAT) in the permeabilized recombinant Pichia pastori cells were well investigated. It appeared that their thermal stability was negatively correlated with the apparent enzymatic activities. The frozen-melted cells presented the best stability and the lowest apparent activities of DAAO and CAT, whereas the cetyltrimethylammonium bromide (CTAB) permeabilized cells displayed the weakest stability and the highest apparent activities of the two enzymes. Simultaneous action of DAAO and CAT in the CTAB-permeabilized cells and glutaryl-7-aminocephalosporanic acid acylase (GA) immobilized on carrier contributed to the conversion of cephalosporin C (CPC) to 7-aminocephalosporanic acid (7-ACA) with a yield of 76.2%. During such a reaction cycle, no visible activity loss occurred at the immobilized GA, whereas the loss rates of DAAO and CAT activities were about 0.029 and 1.13 U min−1, respectively. Nevertheless, this problem could be easily solved by continuous feeding of the new permeabilized cell suspension at the rate of 6 ml h−1 to the reactor. Following such a fed-batch strategy, these permeabilized cells and the immobilized GA could be efficiently reused for 6 and 15 reaction cycles, respectively, yielding around 76% 7-ACA at each reaction cycle.