Partial purification and characterization of an esterase from fusarium sporotrichioides

Abstract

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.

Accumulation Kinetics of Three Scirpentriol-Based Toxins in Oats Inoculated in Vitro with Isolates of Fusarium sporotrichioides and Fusarium poae

“…Thus, the sequential accumulation of 4,15-DAS, 15-MAS and SCIRP may reflect the inductive, and therefore successive, formation of esterases catalyzing the two steps from the esters to the parent alcohol SCIRP, with the appearance of substrate as the inducing event. This hypothesis is consistent with the observation of Park and Chu [29] that the production of deacetylated trichothecenes in F. sporotrichioides cultures on a liquid medium was associated with an increased total activity of fungal esterases. The successive formation of esterases apparently did not exclude the simultaneous presence of precursor and product during certain phases of fungal development (Figure 1, Figure 2).…”

“…Different rates of formation due to genetic and environmental factors have been described. Thus, the production of DAS and MAS by strains of F. sporotrichioides in a liquid medium absorbed into vermiculate, investigated during an incubation of 20–30 days with five day intervals, varied considerably with the strain tested and also, in part, with the incubation temperature (25 or 15 °C) [29]. Overall, it can be concluded that, in grain samples collected at harvest, the analysis of SCIRP should be considered in addition to the analysis of 4,15-DAS and 15-MAS.…”

“…Carboxylesterases are described for Fusarium species as a heterogenous group consisting of several isoenzymes [29]. They are considered to be position specific by Udell and Dewick [33].…”

Simultaneous determination of aflatoxins, ochratoxin A andFusariumtoxins in maize by liquid chromatography/tandem mass spectrometry after multitoxin immunoaffinity cleanup

“…In the T‐2 and HT‐2 analysis, the disappearance of T‐2 and the extremely high HT‐2 recoveries (about 180%) led us to assume that T‐2 was converted into HT‐2 by enzymatic hydrolysis. Hydrolytic enzymes involved in selective deacetylation at the C3 and/or C4 positions of T‐2 toxin have been identified in animal organs,30, 31 as well as in crude extracts of Fusarium species 32, 33. Although trichothecene esterase activity has not so far been investigated in maize, carboxylesterase activities toward pesticide or xenobiotic esters have recently been identified in maize and sorghum 34.…”

Fusarium Tri8 Encodes a Trichothecene C-3 Esterase

“…sporotrichioides esterases were partially purified and characterized by Park and Chu (19,20). The esterase isozyme III, which had primarily C-3 esterase activity when assayed by conversion of 3-acetyl T-2 toxin to T-2 toxin, is a possible product of Tri8.…”