A comprehensive method for the sequential separation of extracellular xylanases and -xylosidases/arabinofuranosidases from a new Fusarium species

Abstract

Several fungal species produce diverse carbohydrate-active enzymes useful for the xylooligosaccharide biorefinery. These enzymes can be isolated by different purification methods, but fungi usually produce other several compounds which interfere in the purification process. So, the present work has three interconnected aims: (i) compare -xylosidase production by Fusarium pernambucanum MUM 18.62 with other crop pathogens; (ii) optimise F. pernambucanum xylanolytic enzymes expression focusing on the pre-inoculum media composition; and (iii) design a downstream strategy to eliminate interfering substances and sequentially isolate -xylosidases, arabinofuranosidases and endo-xylanases from the extracellular media. F. pernambucanum showed the highest -xylosidase activity among all the evaluated species. It also produced endo-xylanase and arabinofuranosidase. The growth and -xylosidase expression were not influenced by the pre-inoculum source, contrary to endo-xylanase activity, which was higher with xylan-enriched agar. Using a sequential strategy involving ammonium sulfate precipitation of the extracellular interferences, and several chromatographic steps of the supernatant (hydrophobic chromatography, size exclusion chromatography, and anion exchange chromatography), we were able to isolate different enzyme pools: four partially purified -xylosidase/arabinofuranoside; FpXylEAB trifunctional GH10 endo-xylanase/-xylosidase/arabinofuranoside enzyme (39.8kDa) and FpXynE GH11 endo-xylanase with molecular mass (18.0kDa). FpXylEAB and FpXynE enzymes were highly active at pH56 and 6050°C.