Ciencia e Ingeniería en Alimentos y Biotecnología
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Item 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 RicardoHydrocarbon 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.Item 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 DanielPlastic 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.Item 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 DanielThe 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.Item 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 EstherPlastic 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.Item Evaluación de la capacidad de biodegradación de contenedores fabricados a partir de recursos agroindustriales infrautilizados de la industria bananera(Universidad Técnica de Ambato. Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología. Carrera de Biotecnología, 2024-08) Méndez López, Tomás Sebastián; Arancibia Soria, Mirari YosuneAccording to the United Nations, approximately 430 million tons of plastic are produced each year. Consequently, this waste represents 85 percent of the total contamination in bodies of water, 36 percent of which are disposable containers. To combat this problem, resources have been focused on the research of more environmentally friendly materials. In Ecuador, banana, due to its high production and waste generation, emerges as one of the best alternatives for the development of bio containers to replace single-use plastics. For this reason, this research evaluated the biodegradation capacity of containers made from underutilized agro-industrial resources of the banana industry. For this purpose, a buried test was carried out with samples of biodegradable banana containers (CBB) in agricultural soil for 15, 30, 45 and 60 days, where values of up to 52.53 percent biodegradation based on dry weight were obtained. On the other hand, in vitro biodegradation capacity was evaluated by inoculation of Aspergillus niger, where it was demonstrated that this strain prefers CBB as a substrate, compared to filter paper and single-use plastics, due to the greater availability of nutrients for its growth. Finally, through microbiological analysis and bibliographic research, it was demonstrated that the shelf life of CBB is equal to or greater than 60 days, since their physicochemical and usability characteristics are not compromised during this period.Item Caracterización de una enzima celulasa obtenida mediante reconstrucción ancestral de secuencias(Universidad Técnica de Ambato. Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología. Carrera de Biotecnología, 2024-02) León Gaibor, Dania Sarahí; Cerda Mejía, Liliana AlexandraModern enzymes are biological alternatives to the degradation of cellulosic biomass derived from agro-industrial waste, facilitating the production of high value-added products such as biofuels. However, the properties of current enzymes are not always suitable for their application in industrial environments. Ancestral sequence reconstruction is an in-silico technique that allows the deduction of ancient protein sequences from existing ones, based on the premise that on the primitive earth enzymes were more stable. We proceeded to the reconstruction of an ancestral enzyme from representative cellulases of family 6 of glycosyl hydrolases, with the expectation of obtaining improved properties and proceeding to their characterization. For the prediction of optimal conditions, the statistical method known as response surface methodology was used, considering pH and reaction temperature as variables. The results revealed that ancestral cellulase acts on the crystalline structure of cellulose. In addition, the determination of optimum conditions showed that cellulase exhibits higher activity at pH 8.6, compared to its current counterparts, which exhibit higher catalysis under acidic conditions. Also, the predicted temperature exceeds 76 degrees Celsius, which when compared to its counterparts that present temperatures up to a maximum of 60 degrees Celsius, suggesting a higher thermostability.Item 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 DanielPlastic 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.Item 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 AlexandraIn 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.Item 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 AlexandraIndiscriminate 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.Item Prospección de la enzima PETasa de Ideonella sakaiensis(Universidad Técnica de Ambato. Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología. Carrera de Ingeniería Bioquímica, 2022-09) Manzano Rivera, Ruth Abigail; García Solís, Mario DanielPlastic is one of the most used materials worldwide, its use has been around for a long time because its composition is very resistant and malleable to be able to develop many necessary tools and products. One of the best-known plastics is PET, which tends to be very resistant when wanting to degrade it. Many types of degradation have been used such as chemical, physicochemical, and biological. However, it has great limitations by resorting to polluting reagents, exaggerated temperatures and poor handling of the waste that causes degradation. One of the enzymes that degrade PET IsPETase from the bacterium Ideonella sakaiensis. The action of different mutants and variants that increased from 1.4 to 120 times the activity of the enzyme was described, the crystal structures of their mutants were also shown with an image resolution ranging from 1.4 to 2.00 angstrom, the crystal structures were found in the PDB, each residue presented a suitable polarity to the degradation of the PET surface. The double mutant with the highest Tm value was W159H, F229Y with a degradation rate equivalent to 23.4 mg of PET per hour, per milligram of enzyme. The adjuvants found were adequate to measure the kinetic rate of product formation and the binding of the substrate to the active site, the binding of SDS and dodecyl trimethylammonium helped the substrate to enter more easily into the active site of the enzyme.