Efficient depolymerization of polyethylene terephthalate (PET) and polyethylene furanoate by engineered PET hydrolase Cut190

AMB Express

Table 4 Comparison of PET hydrolyzing activities

Mutant	Total products (mM)	Degradation (%)	T_m Ca²⁺
Mutant	Total products (mM)	Degradation (%)	0 mM	2.5 mM
Cut190*	61.5	59.1	56.3	67.7
Cut190**	71.7	68.9	60.7	71.7
Cut190*SS	69.9 (75.1)	67.2 (72.3)	82.1	83.3
Cut190**SS	76.6	73.7	n.d	86.6
Cut190**SS/L136F	93.8	90.2	84.2	86.2
Cut**SS/L136F/Q138G	89.1	85.8	83.7	84.7*
Cut**SS/L136F/Q138G/F255I	− (94.9)	− (91.3)	82.7	83.5

The reaction mixture contained HEPES–NaOH buffer (pH 9.0), 2.5 mM CaCl₂, 24% glycerol, 50 ppm dodecyltrimethylamine chloride and approximately 2 μM Cut190* or its derivative. The concentrations of the buffer were 100 mM for Cut190* and Cut190**, and 150 mM for others. Preincubation was performed at room temperature for 10–20 min with PET powder 2 (20 mg/ml) in the reaction mixture except enzyme and CaCl₂. Then each enzyme and CaCl₂ were added, and the incubation was started at 63 and 65 °C with shaking for 3 days. Values at 65 °C are shown in parentheses. All the experiments were performed in duplicate
^*With 25 mM Ca²⁺. No difference was observed in the absence and the presence of 2.5 mM Ca²⁺. Nano-differential fluorometry (Nano Tempa Tycho NT.6, M&S TechnoSystems, Inc., Osaka, Japan) showed the same behavior against Ca²⁺, although T_m values were 91.5 and 91.4 °C with 0 and 2.5 mM Ca²⁺, respectively