Identification, heterologous expression and characterization of a new unspecific peroxygenase from Marasmius fiardii PR-910
Abstract Unspecific peroxygenases (UPOs) are glycosylated enzymes that provide an efficient method for oxyfunctionalizing a variety of substrates using only hydrogen peroxide (H2O2) as the oxygen donor. However, their poor heterologous expression has hindered their practical application. Here, a nov...
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| Format: | Article |
| Language: | English |
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SpringerOpen
2024-03-01
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| Series: | Bioresources and Bioprocessing |
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| Online Access: | https://doi.org/10.1186/s40643-024-00751-x |
| _version_ | 1827301397336424448 |
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| author | Xin Fu Kexin Lin Xiaodong Zhang Zhiyong Guo Lixin Kang Aitao Li |
| author_facet | Xin Fu Kexin Lin Xiaodong Zhang Zhiyong Guo Lixin Kang Aitao Li |
| author_sort | Xin Fu |
| collection | DOAJ |
| description | Abstract Unspecific peroxygenases (UPOs) are glycosylated enzymes that provide an efficient method for oxyfunctionalizing a variety of substrates using only hydrogen peroxide (H2O2) as the oxygen donor. However, their poor heterologous expression has hindered their practical application. Here, a novel UPO from Marasmius fiardii PR910 (MfiUPO) was identified and heterologously expressed in Pichia pastoris. By employing a two-copy expression cassette, the protein titer reached 1.18 g L−1 in a 5 L bioreactor, marking the highest record. The glycoprotein rMfiUPO exhibited a smeared band in the 40 to 55 kDa range and demonstrated hydroxylation, epoxidation and alcohol oxidation. Moreover, the peroxidative activity was enhanced by 150% after exposure to 50% (v/v) acetone for 40 h. A semi-preparative production of 4-OH-β-ionone on a 100 mL scale resulted in a 54.2% isolated yield with 95% purity. With its high expression level, rMfiUPO is a promising candidate as an excellent parental template for enhancing desirable traits such as increased stability and selectivity through directed evolution, thereby meeting the necessary criteria for practical application. Graphical Abstract |
| first_indexed | 2024-04-24T16:22:27Z |
| format | Article |
| id | doaj.art-7d67ed5da8614faf8a187ead4e490332 |
| institution | Directory Open Access Journal |
| issn | 2197-4365 |
| language | English |
| last_indexed | 2024-04-24T16:22:27Z |
| publishDate | 2024-03-01 |
| publisher | SpringerOpen |
| record_format | Article |
| series | Bioresources and Bioprocessing |
| spelling | doaj.art-7d67ed5da8614faf8a187ead4e4903322024-03-31T11:09:24ZengSpringerOpenBioresources and Bioprocessing2197-43652024-03-0111111210.1186/s40643-024-00751-xIdentification, heterologous expression and characterization of a new unspecific peroxygenase from Marasmius fiardii PR-910Xin Fu0Kexin Lin1Xiaodong Zhang2Zhiyong Guo3Lixin Kang4Aitao Li5State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei UniversityState Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei UniversityState Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei UniversityState Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei UniversityState Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei UniversityState Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei UniversityAbstract Unspecific peroxygenases (UPOs) are glycosylated enzymes that provide an efficient method for oxyfunctionalizing a variety of substrates using only hydrogen peroxide (H2O2) as the oxygen donor. However, their poor heterologous expression has hindered their practical application. Here, a novel UPO from Marasmius fiardii PR910 (MfiUPO) was identified and heterologously expressed in Pichia pastoris. By employing a two-copy expression cassette, the protein titer reached 1.18 g L−1 in a 5 L bioreactor, marking the highest record. The glycoprotein rMfiUPO exhibited a smeared band in the 40 to 55 kDa range and demonstrated hydroxylation, epoxidation and alcohol oxidation. Moreover, the peroxidative activity was enhanced by 150% after exposure to 50% (v/v) acetone for 40 h. A semi-preparative production of 4-OH-β-ionone on a 100 mL scale resulted in a 54.2% isolated yield with 95% purity. With its high expression level, rMfiUPO is a promising candidate as an excellent parental template for enhancing desirable traits such as increased stability and selectivity through directed evolution, thereby meeting the necessary criteria for practical application. Graphical Abstracthttps://doi.org/10.1186/s40643-024-00751-xUnspecific peroxygenaseMarasmius fiardiiHeterologous expressionCharacterizationSemi-preparative |
| spellingShingle | Xin Fu Kexin Lin Xiaodong Zhang Zhiyong Guo Lixin Kang Aitao Li Identification, heterologous expression and characterization of a new unspecific peroxygenase from Marasmius fiardii PR-910 Bioresources and Bioprocessing Unspecific peroxygenase Marasmius fiardii Heterologous expression Characterization Semi-preparative |
| title | Identification, heterologous expression and characterization of a new unspecific peroxygenase from Marasmius fiardii PR-910 |
| title_full | Identification, heterologous expression and characterization of a new unspecific peroxygenase from Marasmius fiardii PR-910 |
| title_fullStr | Identification, heterologous expression and characterization of a new unspecific peroxygenase from Marasmius fiardii PR-910 |
| title_full_unstemmed | Identification, heterologous expression and characterization of a new unspecific peroxygenase from Marasmius fiardii PR-910 |
| title_short | Identification, heterologous expression and characterization of a new unspecific peroxygenase from Marasmius fiardii PR-910 |
| title_sort | identification heterologous expression and characterization of a new unspecific peroxygenase from marasmius fiardii pr 910 |
| topic | Unspecific peroxygenase Marasmius fiardii Heterologous expression Characterization Semi-preparative |
| url | https://doi.org/10.1186/s40643-024-00751-x |
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