Purification and Characterization of a Novel Lactonohydrolase from Agrobacterium tumefaciens

Abstract

  A novel lactonohydrolase, catalyzing the stereospecific hydrolysis of L-pantoyl lactone to L-pantoic acid, was purified 2,400-fold to apparent homogeneity with a 1.96% overall recovery from Agrobacterium tumefaciens AKU 316 through a purification procedure including ammonium sulfate fractionation, and column chromatographies on DEAE-Sephacel, phenyl-Sepharose CL-4B, Sephacryl S-200, Mono-Q and alkyl-Superose. The relative molecular mass of the native enzyme estimated on high-pressure gel permeation chromatography was 62,000 Da, and the subunit molecular mass was estimated to 26,500 Da on SDS-polyacrylamide gel electrophoresis. The enzyme hydrolyzes several aromatic lactones, such as 3,4-dihydrocoumarin and homogentisic acid lactone, other than L-pantoyl lactone. The K_m and V_max for L-pantoyl lactone were 3.59 mM and 13.7 μmol/min/mg, respectively. The enzymatic activity was inhibited by several chelating reagents, Fe²⁺, Sn²⁺, Pb²⁺, and Fe³⁺.