D-(-)-Pantolactone

Immobilized whole-cells of Pichia pastoris harboring recombinant d-lactonase were entrapped in calcium alginate gels and used as an efficient biocatalyst for catalytic kinetic resolution of d,l-pantolactone. The immobilized whole-cell biocatalyst exhibited good catalytic stability, which was applied for stereospecific hydrolysis of d-pantolactone for up to 56 repeated batch reactions without obvious loss in the catalytic activity and enantioselectivity.

d-Pantolactone is a key chiral intermediate for the synthesis of d-pantothenic acid and its derivatives. Biocatalytic kinetic resolution of d,l-pantoyl lactone using d-lactonase is an efficient route to synthesize d-pantolactone. In this study, we report the expression of a novel d-lactonase TSDL in Escherichia coli host. The recombinant TSDL exhibited high hydrolysis activity and enantioselectivity toward d-pantolactone. The reaction conditions of the recombinant TSDL-catalyzed kinetic resolution of d,l-pantolactone was systematically investigated by whole cell biocatalysis. In addition, a preparative-scale reaction for bioproduction of d-pantoic acid was examined under optimized reaction conditions. This study presented an alternative enzymatic process for kinetic resolution of d,l-pantolactone.

d-Pantoic acid (D-PA) is an essential intermediate for the production of d-pantolactone. Here, three d-lactonohydrolases (D-Lacs), namely, Fm-Lac from Fusarium moniliforme SW-902, Fp-Lac from Fusarium proliferatum Nirenberg ECU2002, and Fo-Lac from Fusarium oxysporum AKU3702 were heterogeneously expressed in Pichia pastoris. The constructed recombinant strains produced D-Lacs of 1263 U/mL, 1025 U/mL, and 948 U/mL in a 3-L fermenter, respectively. Simultaneously, these three D-Lacs were used to resolve racemic pantolactone (DL-PL), the hydrolysis rate by Fo-Lac over 40% and the enantiomeric excesses was 99% after 4 h reaction, which outperformed Fm-Lac and Fp-Lac. Under the 800 mL scale reaction, the hydrolysis rate of DL-PL reached 39.2% with a D-PA concentration of 144.6 g/L and space-time yield of 36.2 g/L/h correspondingly. This is the highest catalytic efficiency reported so far, which shows that D-Lac heterologously expressed by P. pastoris has excellent industrial application prospects.