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A Unique Mono- and Diacylglycerol Lipase from Penicillium cyclopium: Heterologous Expression, Biochemical Characterization and Molecular Basis for Its Substrate Selectivity
A cDNA gene encoding a mature peptide of the mono- and diacylglycerol lipase (abbreviated to PcMdl) from Penicillium cyclopium PG37 was cloned and expressed in Pichia pastoris GS115. The recombinant PcMdl (rePcMdl) with an apparent molecular weight of 39 kDa showed the highest activity (40.5 U/mL of...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4106778/ https://www.ncbi.nlm.nih.gov/pubmed/25051359 http://dx.doi.org/10.1371/journal.pone.0102040 |
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author | Tan, Zhong-Biao Li, Jian-Fang Li, Xue-Ting Gu, Ying Wu, Min-Chen Wu, Jing Wang, Jun-Qing |
author_facet | Tan, Zhong-Biao Li, Jian-Fang Li, Xue-Ting Gu, Ying Wu, Min-Chen Wu, Jing Wang, Jun-Qing |
author_sort | Tan, Zhong-Biao |
collection | PubMed |
description | A cDNA gene encoding a mature peptide of the mono- and diacylglycerol lipase (abbreviated to PcMdl) from Penicillium cyclopium PG37 was cloned and expressed in Pichia pastoris GS115. The recombinant PcMdl (rePcMdl) with an apparent molecular weight of 39 kDa showed the highest activity (40.5 U/mL of culture supernatant) on 1,2-dibutyrin substrate at temperature 35°C and pH 7.5. The rePcMdl was stable at a pH range of 6.5–9.5 and temperatures below 35°C. The activity of rePcMdl was inhibited by Hg(2+) and Fe(3+), but not significantly affected by EDTA or the other metal ions such as Na(+), K(+), Li(+), Mg(2+), Zn(2+), Ca(2+), Mn(2+), Cu(2+), and Fe(2+). PcMdl was identified to be strictly specific to mono- and diacylglycerol, but not triacylglycerol. Stereographic view of PcMdl docked with substrate (tri- or diacylglycerol) analogue indicated that the residue Phe(256) plays an important role in conferring the substrate selectivity. Phe(256) projects its side chain towards the substrate binding groove and makes the sn-1 moiety difficult to insert in. Furthermore, sn-1 moiety prevents the phosphorus atom (substitution of carboxyl carbon) from getting to the O(γ) of Ser(145), which results in the failure of triacylglycerol hydrolysis. These results should provide a basis for molecular engineering of PcMdl and expand its applications in industries. |
format | Online Article Text |
id | pubmed-4106778 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-41067782014-07-23 A Unique Mono- and Diacylglycerol Lipase from Penicillium cyclopium: Heterologous Expression, Biochemical Characterization and Molecular Basis for Its Substrate Selectivity Tan, Zhong-Biao Li, Jian-Fang Li, Xue-Ting Gu, Ying Wu, Min-Chen Wu, Jing Wang, Jun-Qing PLoS One Research Article A cDNA gene encoding a mature peptide of the mono- and diacylglycerol lipase (abbreviated to PcMdl) from Penicillium cyclopium PG37 was cloned and expressed in Pichia pastoris GS115. The recombinant PcMdl (rePcMdl) with an apparent molecular weight of 39 kDa showed the highest activity (40.5 U/mL of culture supernatant) on 1,2-dibutyrin substrate at temperature 35°C and pH 7.5. The rePcMdl was stable at a pH range of 6.5–9.5 and temperatures below 35°C. The activity of rePcMdl was inhibited by Hg(2+) and Fe(3+), but not significantly affected by EDTA or the other metal ions such as Na(+), K(+), Li(+), Mg(2+), Zn(2+), Ca(2+), Mn(2+), Cu(2+), and Fe(2+). PcMdl was identified to be strictly specific to mono- and diacylglycerol, but not triacylglycerol. Stereographic view of PcMdl docked with substrate (tri- or diacylglycerol) analogue indicated that the residue Phe(256) plays an important role in conferring the substrate selectivity. Phe(256) projects its side chain towards the substrate binding groove and makes the sn-1 moiety difficult to insert in. Furthermore, sn-1 moiety prevents the phosphorus atom (substitution of carboxyl carbon) from getting to the O(γ) of Ser(145), which results in the failure of triacylglycerol hydrolysis. These results should provide a basis for molecular engineering of PcMdl and expand its applications in industries. Public Library of Science 2014-07-22 /pmc/articles/PMC4106778/ /pubmed/25051359 http://dx.doi.org/10.1371/journal.pone.0102040 Text en © 2014 Tan et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Tan, Zhong-Biao Li, Jian-Fang Li, Xue-Ting Gu, Ying Wu, Min-Chen Wu, Jing Wang, Jun-Qing A Unique Mono- and Diacylglycerol Lipase from Penicillium cyclopium: Heterologous Expression, Biochemical Characterization and Molecular Basis for Its Substrate Selectivity |
title | A Unique Mono- and Diacylglycerol Lipase from Penicillium cyclopium: Heterologous Expression, Biochemical Characterization and Molecular Basis for Its Substrate Selectivity |
title_full | A Unique Mono- and Diacylglycerol Lipase from Penicillium cyclopium: Heterologous Expression, Biochemical Characterization and Molecular Basis for Its Substrate Selectivity |
title_fullStr | A Unique Mono- and Diacylglycerol Lipase from Penicillium cyclopium: Heterologous Expression, Biochemical Characterization and Molecular Basis for Its Substrate Selectivity |
title_full_unstemmed | A Unique Mono- and Diacylglycerol Lipase from Penicillium cyclopium: Heterologous Expression, Biochemical Characterization and Molecular Basis for Its Substrate Selectivity |
title_short | A Unique Mono- and Diacylglycerol Lipase from Penicillium cyclopium: Heterologous Expression, Biochemical Characterization and Molecular Basis for Its Substrate Selectivity |
title_sort | unique mono- and diacylglycerol lipase from penicillium cyclopium: heterologous expression, biochemical characterization and molecular basis for its substrate selectivity |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4106778/ https://www.ncbi.nlm.nih.gov/pubmed/25051359 http://dx.doi.org/10.1371/journal.pone.0102040 |
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