Carrera de Biotecnología

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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 Alexandra
Accelerated 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.
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.
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 Alexandra
In 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.