Producción heteróloga y determinación de la actividad hidrolasa de p-nitrofenil-palmitato (p-npp), de la lipasa lipb de amycolatopsis sp. atcc 39116

Ramírez Camacho, Alejandra

Publicación:
Producción heteróloga y determinación de la actividad hidrolasa de p-nitrofenil-palmitato (p-npp), de la lipasa lipb de amycolatopsis sp. atcc 39116

dc.contributor.advisor	Hernandez Torres, Jorge
dc.contributor.advisor	Serna Daza, Oriana Danuta
dc.contributor.author	Ramírez Camacho, Alejandra
dc.date.accessioned	2024-03-04T00:38:56Z
dc.date.available	2020
dc.date.available	2024-03-04T00:38:56Z
dc.date.created	2020
dc.date.issued	2020
dc.description.abstract	Las lipasas microbianas son muy apetecidas en la industria por su gran potencial biotecnológico. Su uso se ve limitado por los costos de producción e importación, por lo cual, se está a la búsqueda de una producción local de lipasas, especialmente en modelos de expresión bacterianos. Anteriormente, en el CINBIN se adelantó la caracterización bioinformática de una secuencia de lipasa potencial de Amycolatopsis sp., con características estructurales y funcionales similares a CalB de Candida antarctica, un referente en la industria. No obstante, no se sabe nada sobre su actividad enzimática in vitro. En este trabajo de grado, se expresó el gen LipB de Amycolatopsis sp. en Escherichia coli y se demostró la capacidad de la enzima Amyco_LipB para hidrolizar ésteres de cadena larga. Inicialmente, se diseñó un gen LipB compatible con el sistema de expresión de E. coli, basados en la secuencia proteica alojada en Genbank. Posteriormente, el gen LipB sintético se expresó en E. coli con el vector pET-22b. Se encontró que la enzima recombinante Amyco_LipB se expresa en cantidad suficiente para pruebas de actividad con extractos crudos, pero es insoluble y
dc.description.abstractenglish	Microbial lipases are highly appreciated in the industry because of their vast biotechnological potential. Their use is limited by production and import costs, in the case of Colombia. Currently, there is a search for the local production of lipases, especially in bacterial expression models. Previously, in the CINBIN, the bioinformatic characterization of a potential lipase sequence of Amycolatopsis sp. was performed, revealing structural and functional characteristics similar to that of CalB of Candida antarctica, a reference in the industry. However, nothing is known about its in vitro enzymatic activity. In this work, the LipB gene of Amycolatopsis sp. was expressed in Escherichia coli, and the ability of Amyco_LipB to hydrolyze long-chain esters was demonstrated. Initially, a version of the LipB gene compatible with the E. coli expression system was designed, based on the protein sequence registered in Genbank. Later, a synthetic LipB gene was expressed in E. coli with the pET-22b vector. It was found that the recombinant enzyme Amyco_LipB was sufficiently expressed for activity tests with crude extracts, but it is mostly insoluble and must be reconstituted for proper functioning. To test the hydrolytic activity of Amyco_LipB, enzyme activity tests were performed with crude extracts, using p-Nitrophenyl palmitate as a substrate. Results showed a specific activity of ~13.5 U/mg, similar to the value obtained by Barraza (2016) for the Serratia marcescens lipase. However, additional solubilization (0.8 M urea) and folding steps (16 mM CaCl2) increased the enzymatic activity to 40 U/mg of total protein, i.e., 300%. Compared to the specific activity of CalB, Amyco_LipB shows promising lipase activity levels. The results show that Amyco_LipB can hydrolyze long-chain esters, and the enzymatic activity values pledges for its potential use in transesterification reactions.
dc.description.degreelevel	Pregrado
dc.description.degreename	Biólogo
dc.format.mimetype	application/pdf
dc.identifier.instname	Universidad Industrial de Santander
dc.identifier.reponame	Universidad Industrial de Santander
dc.identifier.repourl	https://noesis.uis.edu.co
dc.identifier.uri	https://noesis.uis.edu.co/handle/20.500.14071/39642
dc.language.iso	spa
dc.publisher	Universidad Industrial de Santander
dc.publisher.faculty	Facultad de Ciencias
dc.publisher.program	Biología
dc.publisher.school	Escuela de Biología
dc.rights	http://creativecommons.org/licenses/by/4.0/
dc.rights.accessrights	info:eu-repo/semantics/openAccess
dc.rights.creativecommons	Atribución-NoComercial-SinDerivadas 4.0 Internacional (CC BY-NC-ND 4.0)
dc.rights.license	Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)
dc.rights.uri	http://creativecommons.org/licenses/by-nc/4.0
dc.subject	Lipasa
dc.subject	Amycolatopsis sp
dc.subject	Actividad enzimática
dc.subject	Hidrolisis
dc.subject	Solubilidad.
dc.subject.keyword	Lipase
dc.subject.keyword	Amycolatopsis sp
dc.subject.keyword	Enzymatiyc Activity
dc.subject.keyword	Hydrolysis
dc.subject.keyword	Solubility.
dc.title	Producción heteróloga y determinación de la actividad hidrolasa de p-nitrofenil-palmitato (p-npp), de la lipasa lipb de amycolatopsis sp. atcc 39116
dc.title.english	Heterologous production and determination of the hydrolase activity of pNitrophenylPalmitate (pNPP), of the lipase LipB of Amycolatopsis sp. ATCC 39116.
dc.type.coar	http://purl.org/coar/version/c_b1a7d7d4d402bcce
dc.type.hasversion	http://purl.org/coar/resource_type/c_7a1f
dc.type.local	Tesis/Trabajo de grado - Monografía - Pregrado
dspace.entity.type	Publication