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.
Keywords: D-Lactonohydrolase; D-Pantoic acid; Enzymatic resolution; Heterologous expression; Pichia pastoris.
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