Instituto de Biotecnologia UNAM

Grupo de la Dra. Gloria Saab

I. Directed Evolution of alpha-amylase for the production of alkyl glucosides

This project aims to make use of directed evolution techniques to develop a biocatalyst for the production of alkyl glucosides using alpha-amylase from Thermotoga maritima from starch and fatty alcohols. This will require the development of a method to detect alcoholysis reactions on a background of hydrolysis reactions which are also catalyzed by this enzyme. Once this method developed and validated so combinatorial be explored through site-directed mutagenesis of the various positions which are located in protein binding site to the aglycone to achieve on the one hand, increase the yield of the alcoholysis reactions, decreasing turn the performance of hydrolysis reactions, and secondly, to obtain specific enzymes for various alcohols are used as acceptors. The clones isolated after this process variability input / selection alcoholítica show greater capacity is analyzed at the DNA sequence to be expressed and the corresponding enzymes and purify thus can perform its biochemical characterization to evaluate the performance alcoholysis, and the resulting enzyme stability, both at room and at different concentrations of different alcohols used for the alcoholysis reaction. presente proyecto pretende hacer uso de técnicas de evolución dirigida para desarrollar un biocatalizador para la producción de alquil glucósidos utilizando la alfa-amilasa de Thermotoga maritima a partir de almidón y alcoholes grasos. Para esto se requerirá el desarrollo de un método que permita detectar las reacciones de alcohólisis sobre un fondo de reacciones de hidrólisis que también son catalizadas por esta enzima. Una vez desarrollado y validado este método se explorarán de manera combinatoria a través de mutagénesis sitio-dirigida varias posiciones de la proteína que se ubican en el sitio de unión al aglicón para lograr por un lado, incrementar el rendimiento de las reacciones de alcohólisis, disminuyendo a su vez el rendimiento de las reacciones de hidrólisis, y por otro, la obtención de enzimas específicas para diferentes alcoholes que se utilicen como aceptores. Las clonas aisladas después de este proceso de introducción de variabilidad/selección que muestren una mayor capacidad alcoholítica, se analizarán a nivel de secuencia de DNA y se expresarán para purificar las enzimas correspondientes y así poder llevar a cabo su caracterización bioquímica para evaluar el rendimiento de la alcohólisis, así como la estabilidad de las enzimas resultantes, tanto a temperatura como a diferentes concentraciones de los diferentes alcoholes utilizados para la reacción de alcohólisis.

I. Study of the effect of the topology in the folding of TIM barrels by circular permutation

The present project aims to determine the importance of topology for the proper folding of proteins with TIM barrel folding type and identify the smallest units of folding in this type of structure. The use of circular permutations allow identification of substructures is not found in the context of the remainder of the protein does not have sufficient stability to be observed. Unlike other work on the subject, our exploration intended to cover all possible places where you can swap the protein, including regions of secondary structure that have not been explored. Kinetic characterization of folding permuted variants of two model proteins with this type of structure used to assess the effect of the topology in the formation and stabilization of enzymes which have a TIM barrel folding. The similarities and differences between members of this family contribute to decipher the evolutionary origin of these enzymes. Finally, the identification of independent modules folding form chimeras allow us to be an origin point for generation of libraries that can provide novel properties either binding activity or various compounds.as que pueden aportar propiedades novedosas ya sea de actividad o de unión a compuestos diversos.

Dra. Gloria Saab
Jefe de Departamento
Investigador
Tutor de Maestría y Doctorado
Dra. Wendy Xolalpa
Investigador
Dr. Ruben Paul Gaytan
Jefe Operativo de la U. de Síntesis y Secuenciación de ADN
Técnico Académico
Ing. Leticia Olvera
Técnico Académico
M.C. Rodrigo Arreola
Estudiante
Biol. Manuel Alejandro Arévalo
Estudiante
M.C. Emma Liliana Arévalo
Estudiante
Lic. Miguel Angel Davila
Estudiante
Lic. Ekaterina Jalomo
Estudiante
M.C. Jorge Luis Jimenez
Estudiante
M.C. Alexey Llopiz
Estudiante
Ing. Cesar Alejandro Martinez
Estudiante
Lic. Tomas Francisco Maya
Estudiante
Felipe Abraham López
Estancia Temporal
Liliana Maricela Onofre
Servicio Social
Antonio Dorantes
Laboratorista
Dr. Alfonso Miranda
Servicios profesionales
Corina Mondragón
Auxiliar de Laboratorio
C.P. Rubí Margarita Robledo
Asistente Ejecutivo


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