Ciencia e Ingeniería en Alimentos y Biotecnología
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Item 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 AlexandraThe 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.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 Evaluación del efecto de la enzima Xyn30D 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, 2024-08) Burbano Palacios, David Fernando; Cerda Mejía, Liliana AlexandraThe extraction and processing of raw materials in the wood, paper, and agricultural industries are responsible for greenhouse gas emissions and the destruction of biodiversity. In response, the use of renewable and earth-abundant raw materials, such as lignocellulose—a xylan-rich component—has been sought. However, the complex lignocellulosic structure presents a challenge to physical and chemical methods of degradation, leading to the proposed use of enzymes for this purpose. The bacterium Paenibacillus barcinonensis is known for its ability to produce extracellular enzymes that can break down biomass, including xylanases of the GH30 family. Among these enzymes, xylanase Xyn30D has demonstrated efficiency in lignocellulose degradation. In this project, we analyzed the activity of xylanase Xyn30D, from plasmid construction to expression in E. coli BL21 (DE3) and purification by immobilized metal affinity chromatography (IMAC). Furthermore, we evaluated its activity in four different agro-industrial wastes with high xylan content, which have a significant impact on the agricultural industry in Ecuador and worldwide. The substrates sugarcane bagasse and corn stover showed positive enzyme activity, while cocoa husk and rice husk did not exhibit significant values in terms of xylose units per gram of enzyme. This lack of activity is attributed to the presence of inhibitors in the chemical composition of the latter substrates.Item Determinación del ph y temperatura óptimos de la enzima xilanasa de Trichoderma reesei (TrXynIV) empleando xilano de haya como sustrato(Universidad Técnica de Ambato. Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología. Carrera de Biotecnología, 2024-08) Morales Valencia, Karla Pamela; Cerda Mejía, Liliana AlexandraIn recent years, the global population has doubled, leading to an increased demand for resources and environmental pollution due to the indiscriminate extraction of raw materials. This has generated the need to use more sustainable alternatives, such as lignocellulose, which is one of the most abundant sources of biomass. However, its complex structure requires costly and polluting pretreatments, making it less profitable. Despite this, alternative pretreatments have been developed, such as the use of enzymes capable of degrading lignocellulose. The fungus Trichoderma reesei is known for producing enzymes effective in the decomposition of biomass, such as xylanases from the GH30 family. Among these enzymes, XynVI stands out for its efficiency in lignocellulose degradation. In this project, the activity of xylanase XynVI was analyzed, from plasmid construction to its expression in E. coli BL21 (DE3) and purification by affinity chromatography (IMAC). Additionally, the optimal pH and temperature of the enzyme were evaluated using beechwood xylan as a substrate. As a result, deficient expression of the enzyme and low concentration after purification were observed. Nonetheless, XynVI showed an enzymatic activity of approximately 5 units per milligram of protein at a temperature of 60 degrees Celsius and pH of 5.1, with a difference of 10 degrees above the optimal temperature reported in the literature.Item Caracterización de una enzima xilanasa obtenida mediante reconstrucción ancestral de secuencias(Universidad Técnica de Ambato. Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología. Carrera de Biotecnología, 2024-02) Sánchez Aguiar, Alex Vinicio; Cerda Mejía, Liliana AlexandraAccelerated economic and demographic growth has promoted the development of agricultural, timber and paper industries, as well as their respective residues generation. Such by-products are mainly composed of lignocellulose, a recalcitrant polymer considered the most bioavailable source of biomass. Despite this, its potential has not been exploited enough, reason of why, part of science focus on lignocellulosic enzymes characterization. Some of them are xylanases belonging to the GH30 family, a catalytically diverse group, products of which, can be useful in value-added compounds elaboration, such as biofuels, polymers, prebiotics, among others. Additionally, there is some interest in enzymatic engineering of these proteins to reveal their behavior or improve their characteristics through rational design. However, ancestral reconstructions of sequences have also been used, under the idea that ancestors of existing enzymes possess improved properties related with promiscuity and resistance to extreme conditions such as temperature. Thus, the present study describes ancestral reconstruction of a GH30 xylanase, expressed in Escherichia coli BL21 and subsequently purified by IMAC, whose enzyme activity was tested in beechwood xylan. The ancestor, called Xyn_AN4, displays optimal activity at 55 Celsius degrees and pH 4.7 and has structural features similar to GH30-7 subfamily. Furthermore, its enzymatic activity is comparable to that observed in specific glucuronoxylanases and bifunctional xylanases, suggesting a possible connection with the catalyzed reaction.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 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.