Ciencia e Ingeniería en Alimentos y Biotecnología

URI permanente para esta comunidadhttp://repositorio.uta.edu.ec/handle/123456789/412

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2024 - 20254

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Tema: search.filters.subject.CINÉTICA ENZIMÁTICA

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  • Miniatura
    Item type:Ítem,
    Caracterización cinética de las variantes I208V/N212A, I208V/S238Y y N212A/S238Y de la enzima Petasa de Ideonella sakaiensis. Articulado al proyecto de investigación aprobado mediante Resolución Nro. UTA-CONIN-2024-0075-R y reformado mediante la Resolución Nro. UTACONIN-2025-0003-R
    (Universidad Técnica de Ambato. Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología. Carrera de Biotecnología, 2025-07)
    Mena Amaluiza Darwin Paúl
    ;
    García Solís Mario Daniel
    The accumulation of plastic waste, especially polyethylene terephthalate (PET), represents a serious global environmental problem that demands sustainable alternatives. One promising solution is enzymatic biodegradation, in which the PETase enzyme from Ideonella sakaiensis stands out for its ability to degrade PET under moderate conditions. However, its limited thermostability restricts its industrial use. In this context, the present study aimed to kinetically characterize three mutant variants of the PETase enzyme (I208V-N212A, I208V-S238Y, and N212A-S238Y), previously designed to improve catalytic efficiency and thermal stability. The methodology involved the production of the variants through expression in E. coli Rosetta (DE3), purification by affinity chromatography using a Ni-NTA column, and subsequent evaluation of kinetic parameters using p(NP)-acetate as a model substrate. Additionally, the effect of temperature on enzymatic activity was analyzed. The results indicated that all variants adequately followed the Michaelis-Menten model, but the I208V-S238Y variant showed the best overall performance, with the lowest Km value (2.77 mM), the highest Vmax (41.86 U), and the greatest specificity constant (CE = 10.08 1 sachet mM per s), indicating higher substrate affinity and superior catalytic efficiency. Moreover, this variant exhibited good protein expression and thermal stability, meeting the three established objectives. The I208V/S238Y variant therefore emerges as the most promising candidate for biotechnological applications focused on efficient PET degradation, offering a viable alternative for sustainably mitigating plastic pollution.
  • Miniatura
    Item type:Ítem,
    Caracterización cinética de la variante quíntuple mutante de la enzima Petasa de Ideonella sakaiensis
    (Universidad Técnica de Ambato. Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología. Carrera de Biotecnología, 2025-07)
    Guachi Ibarra Madeleine Jeannette
    ;
    Cerda Mejía Liliana Alexandra
    Plastic pollution is a global environmental problem, as its production has increased exponentially since its discovery, reaching 470 million tonnes in 2024. Polyethylene terephthalate (PET) is one of the most common plastics, as its thermal and mechanical resistance makes it widely used in the packaging industry. However, these same properties make it difficult to degrade naturally and turn it into a persistent pollutant. Currently, there are several techniques for recovering PET components, although in many cases their quality is lost. Following the discovery of Ideonella sakaiensis and the isolation of PET hydrolases (PETases), biodegradation strategies have focused on developing highly efficient and thermostable PETases for industrial applications. This thesis aims to characterise a quintuple mutant variant capable of efficiently degrading PET. The methodology included esterase activity assays using p-nitrophenyl acetate (pNPA) as an analogue substrate to determine the kinetic constants of the wild-type and mutant variants. Subsequently, their activity was evaluated directly on PET. The results demonstrated that the mutant variant has greater catalytic efficiency compared to the wild-type enzyme, both with the analogue substrate and with PET. In conclusion, this variant represents a significant advance towards the development of sustainable biotechnological processes and their industrial application in the biological recycling of PET.
  • Miniatura
    Item type:Ítem,
    Degradación de polietileno tereftalato (PET) mediante el uso de la enzima IsPETasa N212A/S238Y a escala de biorreactor
    (Universidad Técnica de Ambato. Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología. Carrera de Biotecnología, 2024-08)
    Caguana Tenelema, Wilma Alexandra
    ;
    García Solís, Mario Daniel
    Plastic production in 2022 reached 400.3 metric tons, of which only 8.9 percent was recycled, while the remainder ended up in the environment. PET accounts for 6.2 percent of total plastic production, which represents a source of pollution of concern due to its low level of degradation and a half-life of more than 2,500 years. Conventional recycling methods (physical and chemical) cause secondary contamination, so biological recycling (biodegradation) through enzymes has been chosen. In this study, the degradation of polyethylene terephthalate (PET) using the enzyme IsPETase N212A S238Y was evaluated at bioreactor scale. After production of PETase enzyme at bioreactor scale using whey as autoinduction medium, 5 grams micronized commercial PET (PETc) was combined with 0.67 micromol of PETase prepared in 200 milliliters of 100 millimolar KPO4 buffer pH 8 for the degradation process. After 48 hours, biphasic degradation kinetics was observed. In addition, scanning electron microscopy (SEM) analyses revealed cleavages and cracked edges in the polymer. The N212A S238Y PETase enzyme PETase showed a 2.8 percent yield in the degradation of commercial PET (PETc) at bioreactor scale. This study suggests a new technology for sustainable PET recycling.
  • Miniatura
    Item type:Ítem,
    Caracterización cinética de las variantes silvestre y mutantes I208V, N212A y S238Y de la enzima PETasa de Ideonella sakaiensis
    (Universidad Técnica de Ambato. Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología. Carrera de Biotecnología, 2024-08)
    Sierra Mena, Joselyn Patricia
    ;
    García Solís, Mario Daniel
    The accumulation of plastic waste has reached critical levels, with 8 million tons polluting the oceans annually. The resulting microplastics enter ecosystems and organisms, posing a serious threat to environmental health. In response, bioremediation has emerged as a sustainable alternative, employing microorganisms or enzymes to degrade polymers. The enzyme IsPETase from Ideonella sakaiensis is particularly more effective than other PET-degrading enzymes. Thus, mutations close to the active site (S238Y, N212A and I208V) have been recently designed to improve its activity and thermostability, evaluated in qualitative analyses. Therefore, in this research the kinetics of these mutants was characterized by evaluating the kinetic parameters of the reactions catalyzed by these enzymes, using p(NP)-acetate as substrate and varying the temperature (25, 35 and 45 degrees Celsius). The data were analyzed with GraphPad Prism. A reduction in KM was observed in the mutants, indicating that the mutations increased the affinity for the substrate, although the decrease in kcat, reflected a lower catalytic capacity, especially for the I208V variant. Overall, all three mutations are less efficient than the WT variant in hydrolyzing phenyl esters under saturated conditions. Regarding the effect of temperature, the N212A variant showed the highest activity at 45 degrees Celsius, standing out as the most thermostable. This study aims to deepen the catalytic efficiency of IsPETase mutants, contributing to the development of biotechnological recycling for plastic wastes.