Structural and Functional Analysis of the Recombinant Aureobasidium pullulans Cutinase Variants for Efficient...

Polycaprolactone (PCL) is a biodegradable polyester widely used in industry, but it degrades slowly in the environment. Enzymatic hydrolysis catalyzed by cutinases represents a promising approach for PCL waste utilization. Through screening, we selected the Aureobasidium pullulans VKM 1116 strain capable of degrading PCL. We amplified the ApCUT1 cutinase gene from the A. pullulans VKM 1116 genomic DNA. Heterologous expression in Komagataella phaffii yielded ~60 mg/L of the target protein. The recombinant enzyme exhibited maximum activity at pH 6.0-6.5 and 30-40°C. Using site-directed mutagenesis, we generated single mutants (Y58W and L186F) and a double mutant (Y58W/L186F). The double mutant demonstrated the highest catalytic efficiency toward the model substrate 4-nitrophenyl 16-methylsulfonyl hexadecanoate (4-NP-(16-MS-C16)), exhibiting a two-fold increase in the hydrolysis rate compared to the wild-type enzyme. During PCL hydrolysis, the Y58W mutant showed the highest activity, providing a 1.8-fold increase in the suspension degradation rate and complete polymer granule degradation at a rate of 1 mg/h per 1 mg of protein. Our molecular modeling suggests that the hydrophobic bridge at the entrance to the active site functions as a "molecular trap." For the low-molecular-weight substrate (4-NP-(16-MS-C16)), enzymatic activity is limited by the kinetics of the substrate retention step, which is consistent with the maximum catalytic efficiency of the Y58W/L186F mutant with a closing trap. Conversely, activity toward PCL is limited by the initial adsorption stage, for which an open conformation of the free enzyme - as observed in the Y58W variant - is critically important. These results demonstrate the potential of the ApCUT1_Y58W cutinase for developing advanced PCL recycling technologies.