Purification and Characterization of Cutinase from Fusarium Verticillioides and Its Application in Butyl Valerate Ester Synthesis

Abstract

Fusarium verticillioides isolated from soil was found to produce extracellular cutinase under optimized culture conditions. The enzyme was purified using ion-exchange and size-exclusion chromatography, achieving a purification fold of 12.5 and a final yield of 62%. The molecular weight of the purified cutinase was determined to be 25 kDa using SDS-PAGE. The purified enzyme exhibited stability over a temperature range of 30–60 °C and a pH range of 6.0–9.0 retaining more than 85% of its activity after 6 h of incubation. It was stable in the presence of various organic solvents (up to 30% v/v) and surfactants, whereas its activity was completely inhibited by the serine protease inhibitor, that is, phenyl methane sulfonyl fluoride (PMSF), confirming the presence of an active serine residue in the catalytic site. Cutinase was highly efficient in catalyzing the synthesis of butyl valerate ester, achieving a yield of 92% under optimized conditions: 0.25 M butanol, 0.25 M valeric acid, 30 mg of enzyme, and n-hexane as the organic solvent at 50 °C for 8 h. The ester yield and purity were confirmed by GCMS analysis. This study highlights the potential of cutinase as a robust biocatalyst for ester synthesis with demonstrated efficiency and high yields. Its application in the food industry as a sustainable alternative to chemically synthesized esters is promising especially for artificial flavoring and aroma production. However, future studies are needed to address scalability, long term storage stability, and the economic feasibility of large-scale production to fully realize its industrial potential.