Characterization of cutinolytic esterase from Fusarium solani for application in polyester fiber modification

Abstract

Microfungi were selectively isolated for production of polyethylene terephthalate (PET) fiber-degrading enzymes potentially to be used to modify the surface of polyester fabric. Over one hundred fungi were isolated from plant surfaces and soil samples using a polycaprolactone (PCL) plate-clearing assay technique, and screened for cutinolytic esterase (cutinase) activity. Twenty-two isolates showed clearing indicating the production of cutinase. The ability of the fungi to produce cutinase in mineral medium (MM) using either potato suberin or PET fiber as substrates was assessed based on the hydrolysis of p-nitrophenyl butyrate (p-NPB). All isolates exhibited activity towards p-NPB, isolate PBURU-B5 giving the greatest activity with PET fiber as an inducer. PBURU-B5 was identified as Fusarium solani based on its conidial morphology and also comparative nucleotide sequencing from internal transcribed spacer region of the ribosomal RNA gene (rDNA-ITS), ergosterol biosynthesis gene (ERG) and translation elongation factor 1-α gene (TEF). The highest esterase yield was obtained in the supernatant from cultures grown at 25 ℃, initial pH 11.0 for 4 days. The enzyme was purified by sequential use of 50-80% ammonium sulfate precipitation, Hitrap Q FF, Hitrap Phenyl HP and HiPrep 16/60 Sephacryl S-200 High Resolution Chromatography. It was purified approximately 69 fold with overall 11% recovery to a specific activity of 137.5 U/mg. The enzyme was homogeneous by SDS-PAGE. The enzyme was a single polypeptide with a molecular weight of about 19 kDa as determined by gel filtration and SDS-PAGE. The optimum pH and temperature for activity of the purified enzyme were pH 9 and 45 ℃. The Km value for p-NPB was 0.53 M-1. Though enzyme activity was reduced to 50% by 10 mM CuSO₄.5H₂O, this was the exception as the enzyme was unaffected by a range of the other metals. Enzymatic modification of PET cloth material properties using crude enzyme from strain PBURU-B5 showed hydrolysis of ester bonds of the PET fiber. The modification of the PET fabric resulted in increase of water and moisture absorption, and general enhancement of hydrophilicity of the fabric. Analytical data on enzyme treated PET fabrics to characterize the surface modifications were obtained by dyeing with basic dye, and also ATR-FTIR and SEM studies. The overall changes resulted in improvements could facilitate processing of fabric ranging from easier dyeing while also yielding a softer feeling fabric for the user.