Carrera de Biotecnología

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    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.
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    Construcción de las variantes mutantes I208V/N212A, I208V/S238Y y N212A/S238Y de la enzima PETasa de Ideonella sakaiensis
    (Universidad Técnica de Ambato. Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología. Carrera de Biotecnología, 2023-09) Gavilanes Flores, Vivian Samantha; García Solís, Mario Daniel
    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.
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    Expresión y purificación de una enzima PETasa obtenida mediante reconstrucción ancestral
    (Universidad Técnica de Ambato. Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología. Carrera de Biotecnología, 2023-03) Quispe Gudiño, Melanie Dayana; Cerda Mejía, Liliana Alexandra
    In the world, there are a wide variety of organic polymers that have come to represent a significant problem of environmental contamination. Currently, the scientific field began to design appropriate and efficient alternatives to facilitate the recycling of plastic materials ecologically. Biodegradation by enzymes has become highly relevant in recent years due to its good capacity for plastic degradation. The present investigation aimed to express and purify an PETase enzyme obtained from an ancestral reconstruction (ASR); the ancestral enzyme was expected to have superior characteristics to I. sakaiensis PETase respect to its activity against PET and other organic polymers. The ancestral PETase N1 enzyme was expressed in E.coli Ta cells under stable conditions of 1,0 mM IPTG at room temperature, over 16 hours. The enzyme purification was carried out through affinity chromatography (FPLC) complemented with an SDS-PAGE electrophoresis to verify the presence of the ancestral PETase N1 enzyme according to its molecular weight. Over more, the purification used an extra serine protease inhibitor (PMSF) together with EDTA metalloprotease inhibitor. The activity of the ancestral PETase N1 enzyme was evaluated through degradation assays in six high-value polymers by SEM. The images obtained showed degradation features in the HDPE polymer, while in the LDPE, PS, and PVC polymers there was low activity. On the other hand, PP and PET polymers experienced a null activity of the enzyme that could be influenced by various factors.
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    Construcción de variantes mutantes de la enzima PETasa de Ideonella sakaiensis
    (Universidad Técnica de Ambato. Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología. Carrera de Biotecnología, 2022-09) Sevilla Cevallos, María Eduarda; Cerda Mejía, Liliana Alexandra
    Indiscriminate plastic production has become a serious environmental problem due to the lack of an efficient treatment. Polyethylene terephthalate (PET) is one of the most used polymers, however chemical and physical options for its degradation are not efficient, neither environmentally friendly. During the last decade, PET biologic treatment has aroused great interest, since it allows the complete degradation of this polymer, and its degradation products work as raw material to produce new PET. Consequently, biological degradation makes it possible to establish a circular system for the use of PET. This investigation built mutant variants of the Ideonella sakaiensis PETase, based on a structural and computational analysis of the enzyme to rationally design variants that potentially increase the enzymatic activity on PET. I208V, N212A and S238Y mutations presented a greater difference in binding energies, suggesting high affinity for the substrate and a low one for the reaction products. Mutant variants were built by site-directed mutagenesis using QuickChange II kit, and through Sanger sequencing it was confirmed that at least two clones of each mutant variant got the desired sequence. Obtained mutations were expressed in E. coli Rosetta (DE3) under expression assay established conditions, 0.1 mM IPTG and 22°C overnight (approx. 15 h). Finally, expressed mutants were purified through affinity chromatography (FPLC) using a Ni-NTA column coupled to an FPLC system. The presence of the enzyme of interest was verified by SDS-PAGE electrophoresis.