Análisis bioquímico de extractos enzimáticos de Puya dasylirioides (Bromeliaceae), una especie endémica de Costa Rica, como potencial fuente de nuevas enzimas proteolíticas
Fecha
2024
Autores
Redondo López, Sergei Gerardo
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Editor
Universidad Nacional (Costa Rica)
Resumen
Resumen. Costa Rica posee más de 190 especies de la familia Bromeliaceae, de las cuales 32 son endémicas. Estas especies poseen enzimas proteolíticas de gran importancia biotecnológica por sus diversas aplicaciones en la industria. A pesar de esto, ninguna de estas especies ha sido estudiada en Costa Rica. El objetivo de esta investigación fue evaluar la capacidad de la especie Puya dasylirioides para producir enzimas proteolíticas y establecerse en condiciones in vitro a partir de plantas en estado silvestre mediante técnicas enzimológicas, espectrometría de masas y herramientas de biotecnología vegetal para su potencial uso en la industria. Para el establecimiento in vitro se utilizó semillas y segmentos de hoja. Estos se expusieron a una batería de ensayos con el fin de determinar el método de desinfección, tipo y concentración de reguladores de crecimiento y tipo de recipiente de cultivo que brindara mejores resultados para el crecimiento de las plantas en condiciones in vitro. Con respecto a las enzimas proteolíticas, se realizaron extractos crudos a partir del tallo y hoja, se purificaron mediante fraccionamiento con etanol frío y cromatografía líquida de alta eficiencia (HPLC), y se realizaron ensayos enzimáticos para verificar la presencia de enzimas proteolíticas en los tejidos. Como parte de los resultados, se logró el establecimiento in vitro de la planta utilizando la semilla como explante, y se desarrolló un procedimiento de multiplicación de las plantas en condiciones in vitro. Además, se confirmó la presencia de enzimas proteolíticas en los tejidos del tallo y partes de la hoja, además de lograr la purificación de una enzima proteolítica extraída a partir del tallo de la planta con una masa aparente cercana a los 45 kDa. Esta investigación corresponde al primer informe sobre enzimas proteolíticas y establecimiento in vitro de una planta de la familia Bromeliaceae endémica de Costa Rica.
Abstract. Costa Rica has more than 190 species of the Bromeliaceae family, 32 of which are endemic. These species possess proteolytic enzymes of great biotechnological importance due to their diverse applications in industry. However, none of these species has been studied in Costa Rica. The objective of this research was to evaluate the ability of Puya dasylirioides species to produce proteolytic enzymes and establish in vitro from wild plants using enzymatic techniques, mass spectrometry and plant biotechnology tools for their potential use in industry. Seeds and leaf segments were used for in vitro establishment. These were exposed to a battery of tests in order to determine the method of disinfection, type and concentration of growth regulators and type of culture vessel that would provide the best results for plant growth under in vitro conditions. Regarding proteolytic enzymes, crude extracts were made from the stem and leaf, purified by fractionation with cold ethanol and high performance liquid chromatography (HPLC), and enzyme assays were performed to verify the presence of proteolytic enzymes in the tissues. As part of the results, in vitro establishment of the plant was achieved using the seed as explant, and a plant multiplication procedure was developed under in vitro conditions. In addition, the presence of proteolytic enzymes in the stem tissues and leaf parts was confirmed, and the purification of a proteolytic enzyme extracted from the plant stem with an apparent mass of about 45 kDa was achieved. This research corresponds to the first report on proteolytic enzymes and in vitro establishment of a plant of the Bromeliaceae family endemic to Costa Rica.
Abstract. Costa Rica has more than 190 species of the Bromeliaceae family, 32 of which are endemic. These species possess proteolytic enzymes of great biotechnological importance due to their diverse applications in industry. However, none of these species has been studied in Costa Rica. The objective of this research was to evaluate the ability of Puya dasylirioides species to produce proteolytic enzymes and establish in vitro from wild plants using enzymatic techniques, mass spectrometry and plant biotechnology tools for their potential use in industry. Seeds and leaf segments were used for in vitro establishment. These were exposed to a battery of tests in order to determine the method of disinfection, type and concentration of growth regulators and type of culture vessel that would provide the best results for plant growth under in vitro conditions. Regarding proteolytic enzymes, crude extracts were made from the stem and leaf, purified by fractionation with cold ethanol and high performance liquid chromatography (HPLC), and enzyme assays were performed to verify the presence of proteolytic enzymes in the tissues. As part of the results, in vitro establishment of the plant was achieved using the seed as explant, and a plant multiplication procedure was developed under in vitro conditions. In addition, the presence of proteolytic enzymes in the stem tissues and leaf parts was confirmed, and the purification of a proteolytic enzyme extracted from the plant stem with an apparent mass of about 45 kDa was achieved. This research corresponds to the first report on proteolytic enzymes and in vitro establishment of a plant of the Bromeliaceae family endemic to Costa Rica.
Descripción
Redondo López, S. G (2024). Análisis bioquímico de extractos enzimáticos de Puya dasylirioides (Bromeliaceae), una especie endémica de Costa Rica, como potencial fuente de nuevas enzimas proteolíticas. [ Tesis de Licenciatura]. Universidad Nacional, Heredia, Costa Rica.
Palabras clave
ENZIMAS, BROMELIACEAE, ENZIMOLOGÍA, CULTIVO IN VITRO, BIOTECNOLOGÍA, ENZYMES, ENZYMOLOGY, BIOTECHNOLOGY, COSTA RICA