Partial purification and characterization of an esterase from Fusarium sporotrichioides

Kinetics analysis of the growth of Fusarium sporotrichioides T-424 at 15 degrees C and 25 degrees C in liquid culture for 35 days revealed that production of deacetylated trichothecenes was associated with an increased activity in fungal esterases. High temperature (25 degrees C) favored enzyme production and enhanced esterase activity. Electrophoresis of crude extracts from the mycelia of F. sporotrichioides T-424 with carboxylesterase staining revealed that several esterases were produced by the fungus. Four carboxylesterase isoenzymes (I-IV) were separated on a DEAE-Sephadex anion exchange column. Type (III) esterase, having activities with the substrate 4-nitrophenylacetate and acetanilide, as well as hydrolytic activity for T-2 toxin and acetyl-T-2 toxin, was partially purified with ammonium sulfate precipitation, immunoaffinity column chromatography, and DEAE-Sephadex A-50 chromatography. The esterase (III) had a molecular weight around 68 kDa in SDS-PAGE. For the deacylation of T-2 toxin and acetyl-T-2 toxin, type (III) esterase had a high specificity for the acetyl group at the C-3 and C-4 positions. The Km values for acetyl-T-2 and T-2 toxin were found to be 41.35 microM and 0.38 microM, respectively. The Km value for the acetyl group at C-3 is 110 times greater than for that at C-4.