The invention is drawn to a new enzyme that selectively hydrolyzes D-pantolactone in D,L-pantolactone and has the following characteristics:(a) action: acts on pantolactone to produce the corresponding acid;(b) specificity for substrate: acts specifically on D-pantolactone but not on L-pantolactone;(c) pH stability: stable at pH5-9;(d) optimal pH: 7.0-7.5;(e) optimal temperature: ca. 50.degree. C.; and(f) effect of various metal ions or inhibitors: inhibited by Cd.sup.2+, Hg.sup.2+ or Cu.sup.2+ EDTA, as well as a process for the preparation thereof. The enzyme is preferably produced by cultivating the microorganisms of the genera Fusarium, Cylindrocarpon, and Gibberella, and most preferably the microorganism Fusarium oxysporum IFO 5942.