Recombinant expression of an l‐amino acid oxidase from the fungus Hebeloma cylindrosporum in Pichia pastoris including fermentation
Abstract l‐amino acid oxidases (LAAOs) are flavoenzymes that catalyze the oxidative deamination of l‐amino acids to the corresponding α‐keto acids, ammonia, and hydrogen peroxide. Here, we show the overexpression, purification, and the characterization of LAAO4 from the fungus Hebeloma cylindrosporu...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Wiley
2020-10-01
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| Series: | MicrobiologyOpen |
| Online Access: | https://doi.org/10.1002/mbo3.1112 |
| _version_ | 1819198841909411840 |
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| author | Marc Christian Heß Svenja Bloess Joe Max Risse Karl Friehs Gabriele Fischer von Mollard |
| author_facet | Marc Christian Heß Svenja Bloess Joe Max Risse Karl Friehs Gabriele Fischer von Mollard |
| author_sort | Marc Christian Heß |
| collection | DOAJ |
| description | Abstract l‐amino acid oxidases (LAAOs) are flavoenzymes that catalyze the oxidative deamination of l‐amino acids to the corresponding α‐keto acids, ammonia, and hydrogen peroxide. Here, we show the overexpression, purification, and the characterization of LAAO4 from the fungus Hebeloma cylindrosporum in the yeast Pichia pastoris with a 9His‐tag and compare this with the recently characterized 6His‐hcLAAO4 expressed in E. coli. The expression of the enzyme with an ER‐signal sequence in P. pastoris resulted in a glycosylated, secreted protein. The enzymatic activity without activation was higher after expression in P. pastoris compared to E. coli. Due to treatment with acidic pH, a striking increase of activity could be detected for both expression systems resulting in similar specific activities after acid activation. Regarding the substrate spectrum, temperature stability, Km, and vmax values, hcLAAO4 showed very few differences when produced in these two expression systems. A higher yield of hcLAAO4 could be obtained by fermentation. |
| first_indexed | 2024-12-23T03:06:51Z |
| format | Article |
| id | doaj.art-f5ea4f73003e42ceafc821358957183d |
| institution | Directory Open Access Journal |
| issn | 2045-8827 |
| language | English |
| last_indexed | 2024-12-23T03:06:51Z |
| publishDate | 2020-10-01 |
| publisher | Wiley |
| record_format | Article |
| series | MicrobiologyOpen |
| spelling | doaj.art-f5ea4f73003e42ceafc821358957183d2022-12-21T18:02:18ZengWileyMicrobiologyOpen2045-88272020-10-01910n/an/a10.1002/mbo3.1112Recombinant expression of an l‐amino acid oxidase from the fungus Hebeloma cylindrosporum in Pichia pastoris including fermentationMarc Christian Heß0Svenja Bloess1Joe Max Risse2Karl Friehs3Gabriele Fischer von Mollard4Biochemistry III Department of Chemistry Bielefeld University Bielefeld GermanyBiochemistry III Department of Chemistry Bielefeld University Bielefeld GermanyFermentation Engineering Faculty of Technology Bielefeld University Bielefeld GermanyFermentation Engineering Faculty of Technology Bielefeld University Bielefeld GermanyBiochemistry III Department of Chemistry Bielefeld University Bielefeld GermanyAbstract l‐amino acid oxidases (LAAOs) are flavoenzymes that catalyze the oxidative deamination of l‐amino acids to the corresponding α‐keto acids, ammonia, and hydrogen peroxide. Here, we show the overexpression, purification, and the characterization of LAAO4 from the fungus Hebeloma cylindrosporum in the yeast Pichia pastoris with a 9His‐tag and compare this with the recently characterized 6His‐hcLAAO4 expressed in E. coli. The expression of the enzyme with an ER‐signal sequence in P. pastoris resulted in a glycosylated, secreted protein. The enzymatic activity without activation was higher after expression in P. pastoris compared to E. coli. Due to treatment with acidic pH, a striking increase of activity could be detected for both expression systems resulting in similar specific activities after acid activation. Regarding the substrate spectrum, temperature stability, Km, and vmax values, hcLAAO4 showed very few differences when produced in these two expression systems. A higher yield of hcLAAO4 could be obtained by fermentation.https://doi.org/10.1002/mbo3.1112 |
| spellingShingle | Marc Christian Heß Svenja Bloess Joe Max Risse Karl Friehs Gabriele Fischer von Mollard Recombinant expression of an l‐amino acid oxidase from the fungus Hebeloma cylindrosporum in Pichia pastoris including fermentation MicrobiologyOpen |
| title | Recombinant expression of an l‐amino acid oxidase from the fungus Hebeloma cylindrosporum in Pichia pastoris including fermentation |
| title_full | Recombinant expression of an l‐amino acid oxidase from the fungus Hebeloma cylindrosporum in Pichia pastoris including fermentation |
| title_fullStr | Recombinant expression of an l‐amino acid oxidase from the fungus Hebeloma cylindrosporum in Pichia pastoris including fermentation |
| title_full_unstemmed | Recombinant expression of an l‐amino acid oxidase from the fungus Hebeloma cylindrosporum in Pichia pastoris including fermentation |
| title_short | Recombinant expression of an l‐amino acid oxidase from the fungus Hebeloma cylindrosporum in Pichia pastoris including fermentation |
| title_sort | recombinant expression of an l amino acid oxidase from the fungus hebeloma cylindrosporum in pichia pastoris including fermentation |
| url | https://doi.org/10.1002/mbo3.1112 |
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