Construcción de las variantes mutantes I208V/N212A, I208V/S238Y y N212A/S238Y de la enzima PETasa de Ideonella sakaiensis

Abstract

Plastic production in 2021 reached 390.7 million tons, of which 6.2 percent corresponded to PET. Unfortunately, only 10 percent of this material was recycled, 14 percent was incinerated, and the rest was released into the environment. PET has a low level of degradation and a useful life of 450 years, so its accumulation in the environment is worrying. For this reason, strategies have been developed for its degradation, such as biodegradation, based on the use of microorganisms or their enzymes. The PETase enzyme from Ideonella sakaiensis has demonstrated a superior ability to break down PET up to 120 times more than other homologous enzymes. Despite the high rate of degradation of PET by PETase, it cannot yet be used as a large-scale method, due to its sensitivity to slight changes in temperature and salinity. Therefore, in the present investigation, the double variants I208V N212A, I208V S238Y, and N212A S238Y have been introduced into the enzyme by mutagenic PCR. And the result of the enzymatic catalysis was evaluated through SEM, a method that allows visualizing the morphology of the surface of the PET treated with the mutants. It was determined by SEM that the double mutant N212A S238Y exhibited the highest rate of PET degradation. In addition, the enzymes with the I208V N212A and N212A S238Y variations reached their maximum catalytic activity at 35 degrees Celsius, and the I208V S238Y mutant lost its activity.