Cargando…

Purification and characterization of trypsin from Luphiosilurus alexandri pyloric cecum

Trypsin from L. alexandri was purified using only two purification processes: ammonium sulfate precipitation and anion exchange liquid chromatography in DEAE-Sepharose. Trypsin mass was estimated as 24 kDa through SDS-PAGE, which showed only one band in silver staining. The purified enzyme showed an...

Descripción completa

Detalles Bibliográficos
Autores principales: dos Santos, Claudio Wilian Victor, da Costa Marques, Maria Elizabeth, de Araújo Tenório, Humberto, de Miranda, Edma Carvalho, Vieira Pereira, Hugo Juarez
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5613698/
https://www.ncbi.nlm.nih.gov/pubmed/28955938
http://dx.doi.org/10.1016/j.bbrep.2016.08.003
_version_ 1783266310215434240
author dos Santos, Claudio Wilian Victor
da Costa Marques, Maria Elizabeth
de Araújo Tenório, Humberto
de Miranda, Edma Carvalho
Vieira Pereira, Hugo Juarez
author_facet dos Santos, Claudio Wilian Victor
da Costa Marques, Maria Elizabeth
de Araújo Tenório, Humberto
de Miranda, Edma Carvalho
Vieira Pereira, Hugo Juarez
author_sort dos Santos, Claudio Wilian Victor
collection PubMed
description Trypsin from L. alexandri was purified using only two purification processes: ammonium sulfate precipitation and anion exchange liquid chromatography in DEAE-Sepharose. Trypsin mass was estimated as 24 kDa through SDS-PAGE, which showed only one band in silver staining. The purified enzyme showed an optimum temperature and pH of 50 °C and 9.0, respectively. Stability was well maintained, with high levels of activity at a pH of up to 11.0, including high stability at a temperature of up to 50 °C after 60 min of incubation. The inhibition test demonstrated strong inhibition by PMSF, a serine protease inhibitor, and Kinetic constants km and kcat for BAPNA were 0.517 mM and 5.0 S(−1), respectively. The purified enzyme was also as active as casein, as analyzed by zymography. Therefore, we consider trypsin a promising enzyme for industrial processes, owing to its stability in a wide range of pH and temperature and activity even under immobilization.
format Online
Article
Text
id pubmed-5613698
institution National Center for Biotechnology Information
language English
publishDate 2016
publisher Elsevier
record_format MEDLINE/PubMed
spelling pubmed-56136982017-09-27 Purification and characterization of trypsin from Luphiosilurus alexandri pyloric cecum dos Santos, Claudio Wilian Victor da Costa Marques, Maria Elizabeth de Araújo Tenório, Humberto de Miranda, Edma Carvalho Vieira Pereira, Hugo Juarez Biochem Biophys Rep Research Article Trypsin from L. alexandri was purified using only two purification processes: ammonium sulfate precipitation and anion exchange liquid chromatography in DEAE-Sepharose. Trypsin mass was estimated as 24 kDa through SDS-PAGE, which showed only one band in silver staining. The purified enzyme showed an optimum temperature and pH of 50 °C and 9.0, respectively. Stability was well maintained, with high levels of activity at a pH of up to 11.0, including high stability at a temperature of up to 50 °C after 60 min of incubation. The inhibition test demonstrated strong inhibition by PMSF, a serine protease inhibitor, and Kinetic constants km and kcat for BAPNA were 0.517 mM and 5.0 S(−1), respectively. The purified enzyme was also as active as casein, as analyzed by zymography. Therefore, we consider trypsin a promising enzyme for industrial processes, owing to its stability in a wide range of pH and temperature and activity even under immobilization. Elsevier 2016-08-11 /pmc/articles/PMC5613698/ /pubmed/28955938 http://dx.doi.org/10.1016/j.bbrep.2016.08.003 Text en © 2016 The Authors http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Research Article
dos Santos, Claudio Wilian Victor
da Costa Marques, Maria Elizabeth
de Araújo Tenório, Humberto
de Miranda, Edma Carvalho
Vieira Pereira, Hugo Juarez
Purification and characterization of trypsin from Luphiosilurus alexandri pyloric cecum
title Purification and characterization of trypsin from Luphiosilurus alexandri pyloric cecum
title_full Purification and characterization of trypsin from Luphiosilurus alexandri pyloric cecum
title_fullStr Purification and characterization of trypsin from Luphiosilurus alexandri pyloric cecum
title_full_unstemmed Purification and characterization of trypsin from Luphiosilurus alexandri pyloric cecum
title_short Purification and characterization of trypsin from Luphiosilurus alexandri pyloric cecum
title_sort purification and characterization of trypsin from luphiosilurus alexandri pyloric cecum
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5613698/
https://www.ncbi.nlm.nih.gov/pubmed/28955938
http://dx.doi.org/10.1016/j.bbrep.2016.08.003
work_keys_str_mv AT dossantosclaudiowilianvictor purificationandcharacterizationoftrypsinfromluphiosilurusalexandripyloriccecum
AT dacostamarquesmariaelizabeth purificationandcharacterizationoftrypsinfromluphiosilurusalexandripyloriccecum
AT dearaujotenoriohumberto purificationandcharacterizationoftrypsinfromluphiosilurusalexandripyloriccecum
AT demirandaedmacarvalho purificationandcharacterizationoftrypsinfromluphiosilurusalexandripyloriccecum
AT vieirapereirahugojuarez purificationandcharacterizationoftrypsinfromluphiosilurusalexandripyloriccecum