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.
Optimización de las variables nutricionales y ambientales para el crecimiento y producción del hongo Pycnoporus spp
(Universidad Técnica de Ambato. Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología. Carrera de Biotecnología, 2025-07)
Ponce Monta María Fernanda
;
Sánchez Garnica Manoella Alejandra
The fungus Pycnoporus spp. has gained increasing relevance for its applications in biotechnology, the food industry, and pharmacology due to its enzymatic properties and bioactive metabolites, such as laccases and antioxidant compounds. However, the lack of standardized protocols for its cultivation limits large-scale production. This study focused on the optimization of nutritional and environmental variables to enhance their growth and production in different culture media, with emphasis on the use of lignocellulosic residues such as sugarcane bagasse, pine sawdust, wheat straw, and rice straw. A completely randomized experimental design was used with four treatments differentiated by substrate formulation, replicated four times, and variables such as mycelial colonization, dry biomass production, and fructification were evaluated. Mycelium isolation and propagation were carried out in solid (PDA) and liquid-supplemented media under controlled conditions of temperature, humidity, and pH. Growth kinetics were evaluated by absorbance, and statistical analyses (ANOVA and Tukey’s HSD) were applied to determine significant differences between treatments. The results showed that the T1 and T3 formulations presented higher biomass yields and morphological stability. In conclusion, the combination of cellulose-rich substrates supplemented with nitrogen sources and controlled culture conditions significantly favors the development of Pycnoporus spp. This proposal can be replicated in industrial processes to produce enzymes and bioactive metabolites with high added value in areas such as food, environmental, and pharmaceutical.
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.
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.
Evaluación del efecto de la enzima Cel6D de Paenibacillus barcinonensis en la degradación de residuos agroindustriales
(Universidad Técnica de Ambato. Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología. Carrera de Alimentos, 2025-02)
Zurita Gancino, Joaquín Fernando
;
Cerda Mejía, Liliana Alexandra
The accumulation of agro-industrial residues generated by agricultural activity in Ecuador represents a significant environmental challenge because inadequate management can cause contamination and affect public health. The use of specific enzymes for cellulose degradation has been widely studied. Thus, this study focused on evaluating the effect of the enzyme Cel6D from Paenibacillus barcinonensis on the degradation of these residues, seeking a biotechnological alternative for their use and reduction of environmental impact. The methodology employed included the construction of a vector for the expression of the enzyme in Escherichia coli, followed by purification by affinity chromatography. The enzymatic activity of Cel6D was evaluated on various swollen and non-swollen agro-industrial substrates. The results showed that the enzyme presented enhanced enzymatic activity on swollen substrates, highlighting a yield of 1.84 49 milliunits of cellulose activity per milligram on cocoa husk and 1.49 49 milliunits of cellulose activity per milligram on rice bran, suggesting that swelling facilitates the access of the enzyme to cellulose. It was concluded that Cel6D is effective in the degradation of agro-industrial wastes, especially when the substrates are treated by acid swelling to increase its accessibility. This research highlights the importance of optimizing the swelling and purification processes to maximize enzyme activity and its potential application in biorefineries, thus contributing to a circular and sustainable economy in waste management in Ecuador.
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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