Ciencia e Ingeniería en Alimentos y Biotecnología

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

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2020 - 20265

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Fecha de inicio: 2020Tema: search.filters.subject.ENZIMAS

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  • Miniatura
    Item type:Ítem,
    Diseño de la variante mutante S188D 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)
    Robalino Sánchez, Génesis Aurora
    ;
    García Solís, Mario Daniel
    Polyethylene terephthalate PET accumulates in ecosystems at rates that conventional recycling methods cannot compensate for. The PETase from Ideonella sakaiensis, IsPETase, degrades PET, but its thermostability below 40 degrees Celsius limits its use in industrial applications. The structural comparison with the thermophilic cutinase LCC, whose D193 residue participates in salt bridge networks that sustain activity up to 70 degrees Celsius, motivated the S188D variation, which replaces the TCC codons with GAC, based on the ancestral mutant M8 architecture of IsPETase. The variant was cloned into the pET-30a(+) vector and expressed in E. coli Rosetta (DE3) under induction with IPTG 0.1 mM at 20 degrees Celsius. Purification by IMAC Ni NTA affinity chromatography produced 5,662 g of soluble protein, with fractions four and five showing the highest active protein concentration, 2,49 mg per mL, and the presence of the protein between the 26 to 34 kDa band was confirmed by SDS PAGE. The degradation assay showed that the activity of 1.5 mg of enzyme per g of micronized post consumer PET, without prior amorphization, at 50 degrees Celsius for 72 hours, reduced PET by an unspecified percentage compared with the control sample. This performance is acceptable, but it does not show significance compared with ancestral mutant variants. Therefore, it is necessary to integrate S188D with other cumulative mutations that strengthen the functional stability of IsPETase and consolidate it as a viable strategy for plastic biodegradation at an industrial scale.
  • Miniatura
    Item type:Ítem,
    Análisis proteómico in silico en la ruta de flavonoides del cacao ecuatoriano (Theobroma Cacao)
    (Universidad Técnica de Ambato. Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología. Carrera de Biotecnología, 2025-07)
    Chamorro Sevilla Patricio Javier
    ;
    Galarza Galarza Cristian Fernando
    This study presents an in silico proteomic analysis of the flavonoid biosynthesis pathway in Theobroma cacao, focusing on the key enzymes involved in this process. Flavonoids are bioactive compounds in cacao that offer various health benefits, such as antioxidant, anti-inflammatory, and cardioprotective properties. The objective of this research is to understand the genetic and functional roles of enzymes such as Chalcone Synthase (CHS), Trans-cinnamate-4-monooxygenase (C4H), and Chalcone Isomerase (CHI) in the flavonoid biosynthetic pathway. Using bioinformatics tools like KEGG, UniProt, and STRING, enzyme sequences were identified, aligned, and analyzed for homology and protein-protein interactions. The research also utilized molecular modeling tools like AlphaFold to predict the 3D structures of these enzymes and AutoDock for docking simulations, aiming to identify potential enzyme-ligand interactions that could enhance flavonoid production. The study found that CHS, C4H, and CHI play critical roles in the conversion of precursor molecules into flavonoids and contribute to the antioxidant properties of cacao. Additionally, protein transmembrane prediction revealed specific regions involved in enzyme localization within cellular membranes. These findings provide insights into the biosynthesis of flavonoids in cacao, offering potential biotechnological applications for improving the production of bioactive compounds in cacao plants. The research contributes to the broader goal of optimizing cacao cultivation for higher nutritional value and health benefits, with implications for both agricultural practices and the food industry.
  • Miniatura
    Item type:Ítem,
    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.
  • Miniatura
    Item type:Ítem,
    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.
  • Miniatura
    Item type:Ítem,
    Producción de la enzima recombinante Xyn30D de Paenibacillus barcinonensis para la degradación de residuos agroindustriales
    (Universidad Técnica de Ambato. Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología. Carrera de Ingeniería en Alimentos, 2020-01)
    Arévalo López, Yessenia Abigail
    ;
    Cerda Mejía, Liliana Alexandra
    The purpose of this study was to obtain the recombinant enzyme Xyn30D from Paenibacillus Barcinonensis, which after its expression in E. coli BL21 (DE3) and (TF2) and extraction, its degrading activity on agroindustrial waste was analyzed based on its specific enzyme activity (U/mg protein). Enzymatic activity was measured through the quantification of reducing sugars produced from the digestion of agroindustrial waste after the enzymatic reaction at 50 Celsius degrees and at different time intervals. Cocoa husk was the substrate that showed the highest enzymatic activity (468.3 Units per mg protein) with an incubation time of 15 minutes, followed by cane bagasse, corn stubble and rice bran. Additionally, the enzymatic activity on its optimal substrate (beech wood xylan) was calculated with enzymatic reactions at 15 and 30 minutes, obtaining values of 88.67 and 57.46 Units per mg protein. Finally, the identification of the products generated by the enzymatic reaction of Xyn30D on agroindustrial waste by means of the HPTLC technique, which showed the degradation products obtaining xylobiose and xylooligosaccharides as mentioned in the literature. On the other hand, when analyzing the degradation products of the synergy between Xyn30D and Cel6D, xylobiose, cellobiose, celooligosaccharides and xylooligosaccharides were obtained.