Practical resolution system for DL-pantoyl lactone using the lactonase from Fusarium oxysporum.

We developed an enzymatic resolution system for DL-pantoyl lactone that uses immobilized mycelia of Fusarium oxysporum, which produce a lactone-hydrolyzing enzyme (lactonase). The lactonase catalyzes the stereospecific hydrolysis of D-pantoyl lactone. One hundred eighty repeated batch reactions (total reaction time, 3780 h) were made with mycelia entrapped in calcium alginate gels as the catalyst, in the presence of 90 mM CaCl2. With a 300 gl(-1)DL-pantoyl lactone solution as the substrate, the hydrolysis rate for DL-pantoyl lactone was > 40% and the optical purity of D-pantoic acid was 90% enantiomer excess. Immobilized mycelia retained 70% of their initial lactonase activity, even after 180 batch reactions. The estimated half-life of the lactonase activity of the immobilized mycelia was 6000 h, which is 35 times higher than that of the free mycelia. The process has been exploited commercially since 1999.

Expression of the Fusarium oxysporum lactonase gene in Aspergillus oryzae: molecular properties of the recombinant enzyme and its application.

The lactonase gene of Fusarium oxysporum was expressed in Aspergillus oryzae for optical resolution of DL-pantoyl lactone. When the chromosomal gene encoding the full-length form of the lactonase, which has its own NH2-terminal signal peptide, was introduced in the host cells, the resulting transformant produced an enzyme of 46,600 Da, which corresponded to the wild-type enzyme. In contrast, A. oryzae transformed with the cDNA coding the mature enzyme produced a protein of 41,300 Da. Deglycosylation analysis with an endoglycosidase revealed that the difference in molecular mass arose from the different sugar contents of the recombinant enzymes. The mycelia of the transformant were used as a catalyst for asymmetric hydrolysis of DL-pantoyl lactone. The initial velocity of the asymmetric hydrolysis reaction catalyzed by the transformant was estimated to be 30 times higher than that by F. oxysporum. When the mycelia of the transformant were incubated with a 20% DL-pantoyl lactone solution for 4 h, 49.9% of the racemic mixture was converted to D-pantoic acid (>95% ee).