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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...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Elsevier
2016
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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 |
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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 |
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