Ciencia e Ingeniería en Alimentos y Biotecnología
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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 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 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.Item Diseño y validación in silico de primers para la construcción de las variantes mutantes S121E, S121D, D186H y R280A de la enzima PETasa de Ideonella sakaiensis mediante tres diferentes métodos de mutagénesis dirigida al sitio(Universidad Técnica de Ambato. Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología. Carrera de Ingeniería Bioquímica, 2022-03) Herrera Aldaz, Bryan Alexander; Cerda Mejía, Liliana AlexandraThe PETase enzyme from Ideonella sakaiensis (IsPETase) can be used to degrade PET, however, although it has been reported to date to have the highest enzymatic activity under normal conditions of all PET degrading enzymes, its low thermal stability limits its analysis and application. Therefore, by targeted mutagenesis, specific mutations can be introduced into the DNA to significantly increase its enzymatic activity. The purpose of this study was the design and in silico validation of primers for the construction of S121E, S121D, D186H and R280A mutant variants of IsPETase using three site-directed mutagenesis techniques: QuikChange, Q5 Site-Directed Mutagenesis and Phusion Site-Directed Mutagenesis. For the design of the primers, the parameters contained in the design guidelines for targeted mutagenesis methods were considered. The results showed that the 24 primers obtained meet the general criteria such as length, melting temperature, percentage of GC content and the position of the mutation in the primer. For the validation of the proposed primers, hairpin, autodimer and heterodimer analysis was performed with bioinformatics tools, the calculated values are within acceptable ranges. Finally, the specificity of the primers design was carried out by means of alignments, whose results indicate a high specificity for their respective targets. Likewise, an in silico PCR assay confirmed the specificity of the primers, which represent the expected amplification product, thus demonstrating the correct selection of the designed primers and the execution of the mutagenic PCR process.Item Análisis estructural de la enzima PETasa de Ideonella sakaiensis y enzimas homólogas(Universidad Técnica de Ambato. Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología. Carrera de Ingeniería Bioquímica, 2022-03) Aguirre Muñoz, Adriana Alexandra; García Solís, Mario DanielPolyethylene terephthalate (PET), a low-cost thermoplastic produced industrially from fossil raw materials, is massively used in industry, and has become a high-impact pollutant. Among the latest discoveries related to the enzymatic treatment of PET is PETase from I. sakaiensis (IsPETase), which is a hydrolase capable of degrading PET. The present work was elaborated to establish possible mutations and modifications that improve the catalytic activity and enzymatic stability of IsPETase, for which a structural analysis of the same was carried out. Using the UNIPROT and PDBeFOLD databases, the enzymes that share a high degree of homology with IsPETase were identified. From these results, an analysis was carried out based on their percentage of sequence identity and secondary structure identity (SEQ and SSE respectively), E-value, Score, RMSD, and Q. The enzymes of greatest interest were selected for this analysis, the main ones being PbLipaseT, RgPETase, BbHydrolase, and SvCUT190, establishing that the N212S mutation can be used to improve the enzymatic activity and stability, in addition, possible substitutions to be studied were described to evaluate the effect that these would cause in the enzyme (S188E, S188A, and S114H). In the analysis of mutant variants, the presence of different amino acids in each case was identified; therefore, different possible substitutions to be made in the IsPETase enzyme were proposed, among the most relevant ones are W159F and S238T.Item Análisis Filogenético de Estructura y Genoma de enzima PETasa de Ideonella sakaiensis(Universidad Técnica de Ambato. Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología. Carrera de Ingeniería Bioquímica, 2021-01) Garcés Cifuentes, Andrés Benjamín; Terán Mera, David AndrésThe bioinformatic studies presented on this work consisted of phylogenetic analysis, structure modeling and molecular docking. On the phylogenetic analysis, more than a hundred identified sequences like PETase from Ideonella sakaiensis were aligned with it. Said information was used to identify high homology sequences that codify for enzymes with possible PET hydrolytic activity. Through this procedure three enzymes were found that meet similar features. The enzyme Pbs from Acidovorax delafieldii (PbsA), DHL from Rizhobacter gummiphilus and DHL from Polyangium brachysporum. Protein structure modelling was performed using SWISSPROT. This needed since the three enzymes did not possess a crystallographic model present on any database. The templates used for the modelling reported are solution higher than 1.8 Armstroms. The molecular docking procedure was performed through AutodockTools. A PET monomer was used as ligand. As result the fixation energies of each enzyme were determined through enzymatic kinetics calculations. This value is a good indicator of substrateenzyme affinity. Finally, plasmid extraction, cell transformation and qualitative analysis of enzymatic activity was carried out in the E. coli (rosetta) cell line. The importance of this study lies in the discovery of PETase activity in already known proteins, which offers more biotechnological options for the treatment of plastic waste, which is an environmental problem that has progressively become a priority worldwide.