Production and Purification of Cutinase from <i>Fusarium oxysporum</i> Using Modified Growth Media and a Specific Cutinase Substrate

Abstract

Cutinases are hydrolytic enzymes used by phytopathogenic fungi to gain entry into plants by breaking down the cuticular barrier of higher plants. Cutinase displayed hydrolytic activity not only towards cutin, the main component of the plant cuticle, but also towards a variety of soluble synthetic esters, insoluble triglycerides and polyesters. Therefore, cutinase was evaluated for use in the chemical, food and cotton bio-scouring industry and for synthetic fibers modification. This research examined the production and purification of extracellular cutinase from the phytopathogenic fungus Fusarium oxysporum . The addition of apple cutin or its hydrolysate to the fungus growth medium resulted in an enhanced secretion of cutinase into the extracellular fluid. Testing 1-hexadecanol as an alternative to natural cutin to induce cutinase production resulted in a high process yield under modified growth conditions. Cutinase enzyme production was followed by an optimized purification method for enzyme preparation using high-performance liquid chromatography and high-specificity 4-nitrophenyl (16-methyl sulfide ester) hexadecanoate (pNMSEH) cutinase substrate. Electrophoresis sodiumdodecyl sulfate-polyacrylamide and isoelectric focusing gels enabled the final separation and identification of the protein. The purified cutinase had an approximate molecular weight of 20 kDa and an isoelectric point of 4.7. The method presented here could be modified and used for cutinase production and purification in other microorganisms that exhibit cutinolytic activity.