Functional production, characterization, and immobilization of a cold-adapted cutinase from Antarctic Rhodococcus sp.

Abstract

A lipolytic enzyme (Rcut) was discovered from the Rhodococcusstrain (RosL12) isolated from the Antarctic Ross Sea. The corresponding gene composed of 651 bases encoding 216 amino acids. It was found to be a cutinase gene through BLAST search. Rcut has a signal sequence consisting of 29 amino acids. An active Rcut was produced after the intact gene containing the signal sequence was transformed into Escherichia coli Rosetta-gami™ 2 (DE3) pLysS. Rcut was purified through a nickel-nitrilotriacetic acid purification system and a carboxymethyl Sepharose column chromatography. Its specific activity was 2190 U/mg. Rcut showed the highest activity at 40 °C and had a low activation energy of 3.16 kcal/mol. This means that it is a typical cold-adapted enzyme. Rcut showed high activity towards medium chain fatty acids (C4–C10). Rcut degraded polycaprolactone and polyethylene terephthalate, suggesting that it could be used for decomposition of synthetic plastics causing environmental pollution. Rcut was immobilized on methacrylate-divinyl benzene bead. This immobilized Rcut (immRcut) showed higher thermal stability than the free enzyme. ImmRcut performed transesterification of various esters and ethanol in a non-polar solvent, suggesting that it could be used for the synthesis of industrially useful ester compounds.