Development of a Novel Biocatalyst for the Resolution of rac‐Pantolactone

A novel L ‐pantolactone hydrolase, Lph, from Agrobacterium tumefaciens Lu681 was characterized, which stereospecifically hydrolyses L ‐pantolactone to L ‐pantoic acid yielding D ‐pantolactone with > 95% enantiomeric excess. The enzyme was found to be a 30 kDa‐Zn²⁺‐hydrolase with a K_m for L ‐pantolactone of 7 mM and a V_max of 30 U/mg. The corresponding lph gene was identified as an 807 bp ORF and cloned into E. coli. It was overexpressed under control of P_tac and P_rha yielding enzyme activities of up to 600 U/g dry weight. Resolution of d,l ‐pantolactone in repeated batches with isolated Lph and enzyme recovery by membrane filtration gave D ‐pantolactone with 50% yield and 90–95% ee over 6 days. Covalent immobilization to EupergitC led to a stable biocatalyst easy to handle in a repeated batch production of D ‐pantolactone. Further improvements in the activity of Lph were achieved by directed evolution of the enzyme. Activities of mutants F62S, K197D and F100L were increased 2.3, 1.7, and 1.5 fold, respectively.