Ciencia e Ingeniería en Alimentos y Biotecnología

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

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Diseño de la variante mutante N233K de la enzima PETasa de Ideonella sakaiensis
(Universidad Técnica de Ambato. Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología. Carrera de Biotecnología, 2026-07)
Orellana Cordova, Julio Jaker
;
García Solís, Mario Daniel
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.
Caracterización cinética y estructural de la enzima ancestral N1 PETasa
(Universidad Técnica de Ambato. Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología. Carrera de Biotecnología, 2026-01)
Vallejo Espín, Diana Carolina
;
Cerda Mejía, Liliana Alexandra
The contamination caused by polyethylene terephthalate (PET) represents one of the most critical environmental challenges today, due to its high global production, persistence, and the limited efficiency of conventional recycling methods. In this context, enzyme-based degradation strategies have emerged as sustainable and highly specific alternatives. The methodology included structural modeling using AlphaFold 3.0, multiple sequence alignments, comparative analysis with modern PETases, and the experimental evaluation of enzymatic activity on pNP-acetate, pNP-butyrate, and post-consumer PET. This research aligns with the development of biotechnologies applied to the enzymatic recycling of polymers. Structural analyses revealed that N1 PETase retains the α/β-hydrolase fold, the Ser–Asp–His catalytic triad, and functional motifs associated with polyester hydrolysis. The ancestral N1 PETase enzyme emerges as a promising candidate for designing optimized biocatalysts. It also provides evidence on the relationship between structural architecture, loop dynamics, and catalytic efficiency, contributing to the advancement of biotechnological solutions for the circular economy. In this context, the present study carried out the structural, functional, and kinetic characterization of the ancestral N1 PETase enzyme, reconstructed through phylogenetic methods, to determine its potential as a biocatalyst for PET depolymerization.
Diseño de la variante mutante Q119Y-L117F de la enzima PETasa de Ideonella sakaiensis
(Universidad Técnica de Ambato. Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología. Carrera de Biotecnología, 2026-01)
Molina Molina, Michael Ulices
;
García Solís, Mario Daniel
The massive accumulation of plastic waste poses a critical challenge to environmental sustainability. In 2022, global plastic production reached 400.3 million tons, with polyethylene terephthalate (PET) accounting for 6.2 percent of the total; however, only a limited fraction (35.5 million tons) was recycled, highlighting the need for efficient biocatalytic alternatives. PETase from Ideonella sakaiensis has emerged as a biotechnological alternative. Various studies have shown that modifications to key residues can improve its thermal stability and catalytic efficiency. In this study, the mutant variant IsPETaseQ119Y-L117F was constructed, whose plasmid was obtained through a specialized external organism. Subsequently, the recombinant enzyme was expressed and purified by affinity chromatography, ensuring its purity for functional analysis. Enzymatic activity was analyzed using commercially available PET pellets (post-consumer beverage bottles), with a reduction in mass recorded after incubation at 50 degrees Celsius for 9 days. The results obtained show that rational engineering of PETase can significantly improve its catalytic behavior and provides a scientific basis for continuing the design of more stable and efficient biocatalysts. This study thus contributes to the development of sustainable solutions aimed at the biodegradation of PET and the mitigation of the environmental impact associated with this polymer.
Diseño de la variante mutante W159H de la enzima PETasa de Ideonella sakaiensis
(Universidad Técnica de Ambato. Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología. Carrera de Biotecnología, 2026-01)
Dutan Atancuri, John Alexander
;
García Solís, Mario Daniel
The sustained increase in the production and consumption of polyethylene terephthalate (PET) has led to a significant accumulation of plastic waste that is difficult to manage, for which conventional recycling routes have technical and energy limitations. In this context, biodegradation mediated by PET hydrolase enzymes is emerging as a complementary alternative for the depolymerization of post-consumer PET. Ideonella sakaiensis PETase (IsPETase) stands out for its ability to act on semi-crystalline PET under moderate conditions; however, its activity and thermal stability are still insufficient for pre-industrial scale applications. The objective of this work was to design and evaluate the W159H mutant variant of the PETase enzyme from I. sakaiensis in order to improve its catalytic performance against post-consumer PET bottles. The point mutation W159H was introduced into the M3 variant of IsPETase, cloned into the pET-30a(+) vector, expressed in Escherichia coli Rosetta, and characterized by SDS-PAGE. Activity was evaluated by incubating micronized PET pellets at 50 degrees Celsius and pH 9.4, quantifying the loss of polymer mass. The W159H variant achieved a 44.9 percent degradation of the initial 125.2 milligrams of PET, demonstrating competitive catalytic performance. The results demonstrated that the W159H substitution represents a relevant optimization point of the IsPETase aromatic clamp and provides experimental evidence serving as a basis for the rational design of multiple variants aimed at the biological recycling of PET waste.
Diseño de la variante mutante S214H de la enzima PETasa de Ideonella sakaiensis
(Universidad Técnica de Ambato. Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología. Carrera de Biotecnología, 2026-01)
Chancusig Chicaiza, Zulema Nicolle
;
Cerda Mejía, Liliana Alexandra
The global accumulation of plastic waste, particularly polyethylene terephthalate (PET), constitutes a critical environmental problem due to its high resistance to natural degradation. Although mechanical and chemical recycling methods exist, they present limitations associated with the loss of material quality, the use of aggressive reagents, and high operational costs. In this context, enzymatic biodegradation emerges as a sustainable alternative to recover PET monomers under moderate conditions. The PETase enzyme from Ideonella sakaiensis has stood out for its ability to hydrolyze PET; however, its low thermostability and catalytic efficiency restrict its application on an industrial scale. This project focused on the design, purification, and evaluation of the S214H mutant variant, with the aim of improving its performance against PET. The mutation was incorporated through gene synthesis, cloned into the pET30a(+) vector, and expressed in Escherichia coli Rosetta (DE3). The recombinant protein was purified by affinity chromatography and analyzed by SDS-PAGE. Catalytic assays revealed that the S214H variant increased biodegradation efficiency, achieving 24.78 percent PET degradation under controlled conditions, exceeding the values obtained for the wild-type enzyme. This behavior suggests that the substitution at position 214 favors the stabilization of the catalytic microenvironment and improves interaction with the substrate. Taken together, these results position the S214H variant as an efficient alternative for the biological recycling of PET.
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.
Bioprospección de microorganismos autóctonos productores de biosurfactantes aislados de suelos contaminados con aceites de lubricadoras en Ambato. Articulado al Proyecto de Investigación aprobado mediante Resolución Nro. UTA-CONIN-2023-0294-R
(Universidad Técnica de Ambato. Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología. Carrera de Biotecnología, 2025-02)
Guerrero Carranza, Yadira Lizbeth
;
Calero Cáceres, William Ricardo
Hydrocarbon contamination represents a global issue due to its high toxicity and recalcitrance. This is mainly attributed to the indiscriminate use of oils of various types, ranging from edible oils to motor lubricants. In particular, motor oils have a significant impact on soil, as their heavy nature and low dispersion in water reduce the bioavailability of hydrocarbons for microorganisms. This phenomenon deteriorates soil structure over time. In this study, bacteria present in soils contaminated with oils from lubricant workshops in the city of Ambato were isolated, identified, and characterized. It is essential to highlight that biosurfactants, surfactant compounds of microbial origin, play a key role in this context. To identify biosurfactant-producing bacteria, various tests were performed, including the drop collapse test, oil spreading, hemolytic activity, emulsification, and the use of CTAB agar. Subsequently, biosurfactant-producing strains were identified using the MALDI-TOF technique to determine their genus and species. The results revealed that the bacteria belonged to Pseudomonas aeruginosa, known for producing rhamnolipids, a type of biosurfactant. Additionally, their antimicrobial activity was evaluated, and the phylogenetic similarity of these strains was investigated using the ERIC-PCR technique. Finally, an oil biodegradation study was conducted, comparing conditions with and without substrate enrichment to analyze process differences.
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.
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.
Evaluación de la biodegradación y propiedades mecánicas de diferentes formulaciones de bioplástico obtenidas a partir del aprovechamiento de colágeno residual y almidón de papa (Solanum tuberosum)
(Universidad Técnica de Ambato. Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología. Carrera de Biotecnología, 2024-08)
Miquinga Lasluisa, Cinthya Alejandra
;
Flores Tapia, Nelly Esther
Plastic pollution is one of the most critical problems we face, as the population continues to prefer this material for its versatility in daily life. However, the accumulation of plastics causes serious problems in terrestrial and marine ecosystems, affecting all beings that inhabit them. In addition, poor waste management has similar repercussions. This study focused on evaluating the biodegradation and mechanical properties of a bioplastic made from biopolymers recovered from organic waste, such as starch and collagen. For the production of bioplastics, collagen and starch were extracted by thermal hydrolysis and the wet method. Different formulations were used, varying the concentrations of starch and collagen, and two plasticizers were used: polyvinyl alcohol and glycerin. The aim was to develop a rapidly degrading material with optimal mechanical strength characteristics. It was determined that the most efficient collagen extraction was achieved at 120 minutes, with the highest yield. The presence of glycerin increased the flexibility of the sheets, while polyvinyl alcohol gave them more rigidity. The sample with a 1:1 ratio of flesh collagen showed higher tensile strength compared to the others. In biodegradation tests, faster decay was observed in water than on land; On land, the sheets with flesh collagen degraded more quickly. Therefore, it can be deduced that sheets made with flesh collagen and wet blue have optimal characteristics for the manufacture of bioplastics.

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