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High-Level Expression of Pro-Form Lipase from Rhizopus oryzae in Pichia pastoris and Its Purification and Characterization
A gene encoding Rhizopus oryzae lipase containing prosequence (ProROL) was cloned into the pPICZαA and electrotransformed into the Pichia pastoris X-33 strain. The lipase was functionally expressed and secreted in Pichia pastoris with a molecular weight of 35 kDa. The maximum lipase activity of reco...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Molecular Diversity Preservation International (MDPI)
2013
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3907806/ https://www.ncbi.nlm.nih.gov/pubmed/24368519 http://dx.doi.org/10.3390/ijms15010203 |
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author | Wang, Jian-Rong Li, Yang-Yuan Xu, Shu-De Li, Peng Liu, Jing-Shan Liu, Dan-Ni |
author_facet | Wang, Jian-Rong Li, Yang-Yuan Xu, Shu-De Li, Peng Liu, Jing-Shan Liu, Dan-Ni |
author_sort | Wang, Jian-Rong |
collection | PubMed |
description | A gene encoding Rhizopus oryzae lipase containing prosequence (ProROL) was cloned into the pPICZαA and electrotransformed into the Pichia pastoris X-33 strain. The lipase was functionally expressed and secreted in Pichia pastoris with a molecular weight of 35 kDa. The maximum lipase activity of recombinant lipase (rProROL) was 21,000 U/mL, which was obtained in a fed-batch cultivation after 168 h induction with methanol in a 50-L bioreactor. After fermentation, the supernatant was concentrated by ultrafiltration with a 10 kDa cut off membrane and purified with ion exchange chromatography using SP Sepharose Fast Flow chromatography. The optimum pH and temperature of the rProROL were pH 9.0 and 40 °C, respectively. The lipase was stable from pH 4.0 to 9.0 and from 25 to 55 °C. The enzyme activity was enhanced by Ca(2+) and inhibited by Hg(2+) and Ag(+). The lipase showed high activity toward triglyceride-Tripalmitin (C16:0) and triglyceride-Trilaurin (C12:0). |
format | Online Article Text |
id | pubmed-3907806 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | Molecular Diversity Preservation International (MDPI) |
record_format | MEDLINE/PubMed |
spelling | pubmed-39078062014-01-31 High-Level Expression of Pro-Form Lipase from Rhizopus oryzae in Pichia pastoris and Its Purification and Characterization Wang, Jian-Rong Li, Yang-Yuan Xu, Shu-De Li, Peng Liu, Jing-Shan Liu, Dan-Ni Int J Mol Sci Article A gene encoding Rhizopus oryzae lipase containing prosequence (ProROL) was cloned into the pPICZαA and electrotransformed into the Pichia pastoris X-33 strain. The lipase was functionally expressed and secreted in Pichia pastoris with a molecular weight of 35 kDa. The maximum lipase activity of recombinant lipase (rProROL) was 21,000 U/mL, which was obtained in a fed-batch cultivation after 168 h induction with methanol in a 50-L bioreactor. After fermentation, the supernatant was concentrated by ultrafiltration with a 10 kDa cut off membrane and purified with ion exchange chromatography using SP Sepharose Fast Flow chromatography. The optimum pH and temperature of the rProROL were pH 9.0 and 40 °C, respectively. The lipase was stable from pH 4.0 to 9.0 and from 25 to 55 °C. The enzyme activity was enhanced by Ca(2+) and inhibited by Hg(2+) and Ag(+). The lipase showed high activity toward triglyceride-Tripalmitin (C16:0) and triglyceride-Trilaurin (C12:0). Molecular Diversity Preservation International (MDPI) 2013-12-24 /pmc/articles/PMC3907806/ /pubmed/24368519 http://dx.doi.org/10.3390/ijms15010203 Text en © 2014 by the authors; licensee MDPI, Basel, Switzerland http://creativecommons.org/licenses/by/3.0/ This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). |
spellingShingle | Article Wang, Jian-Rong Li, Yang-Yuan Xu, Shu-De Li, Peng Liu, Jing-Shan Liu, Dan-Ni High-Level Expression of Pro-Form Lipase from Rhizopus oryzae in Pichia pastoris and Its Purification and Characterization |
title | High-Level Expression of Pro-Form Lipase from Rhizopus oryzae in Pichia pastoris and Its Purification and Characterization |
title_full | High-Level Expression of Pro-Form Lipase from Rhizopus oryzae in Pichia pastoris and Its Purification and Characterization |
title_fullStr | High-Level Expression of Pro-Form Lipase from Rhizopus oryzae in Pichia pastoris and Its Purification and Characterization |
title_full_unstemmed | High-Level Expression of Pro-Form Lipase from Rhizopus oryzae in Pichia pastoris and Its Purification and Characterization |
title_short | High-Level Expression of Pro-Form Lipase from Rhizopus oryzae in Pichia pastoris and Its Purification and Characterization |
title_sort | high-level expression of pro-form lipase from rhizopus oryzae in pichia pastoris and its purification and characterization |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3907806/ https://www.ncbi.nlm.nih.gov/pubmed/24368519 http://dx.doi.org/10.3390/ijms15010203 |
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