Diseño de la variante mutante N233K de la enzima PETasa de Ideonella sakaiensis

Abstract

The exponential increase in polyethylene terephthalate waste represents a severe global environmental crisis, demanding efficient biotechnological alternatives. This work evaluates the impact of the N233K point mutation on the M5 variant of the Ideonella sakaiensis PETase enzyme. This modification aims to rigidify a flexible surface loop through an intramolecular salt bridge to increase the thermal stability of the biocatalyst, offering a green and sustainable pathway aligned with the circular economy. Methodologically, the mutant gene was expressed in Escherichia coli Rosetta (DE3), yielding 2.09 grams of wet biomass in 550 milliliters of LB medium. After cell lysis by sonication, the recombinant protein was isolated using immobilized metal affinity chromatography (IMAC-FPLC) with a gross elution volume of 15 milliliters. Subsequently, the pool was purified and desalinated by gravity size-exclusion chromatography into a final volumen of 2 milliliters. Analytical quantification was performed on a spectrophotometer applying a corrected mass-specific extinction coefficient of 1.239 liters per gram centimeter. In conclusion, the process consolidated a real net mass of 5.745 milligrams of pure enzyme, which is equivalent to an overall yield of 7.74 percent and a final volumetric productivity of 11.490 milligrams per liter of culture. Gravimetric assays at 50 degrees Celsius for 72 hours confirmed an average mass loss of 10.87 percent in the plastic films, validating that the substitution autonomously confers the necessary thermostability to catalyze the hydrolysis of real PET.