Monokaryotic Pleurotus sapidus Strains with Intraspecific Variability of an Alkene Cleaving DyP-Type Peroxidase Activity as a Result of Gene Mutation and Differential Gene Expression
Abstract
:1. Introduction
2. Results and Discussion
2.1. Analysis of Monokaryons
2.1.1. Pre-Selection of Monokaryons by Analysis of the Radial Growth Rate
2.1.2. Profiling of Alkene Cleavage and Peroxidase Activity in Monokaryons
2.1.3. Phenotypic Stability of Alkene Cleavage Activity in Sequential Cultivations
2.1.4. Comparison of PsaPOX from Selected P. sapidus Strains
2.2. Analysis of the Recombinant PsaPOX Variants
2.2.1. Activity of the Recombinant PsaPOX Variants
2.2.2. Structural Analysis of the PsaPOX Variants
2.2.3. Comparative Biochemical Characterization of the PsaPOX Variants
2.3. Expression Profile of the PsaPOX Gene from Different P. sapidus Strains
3. Materials and Methods
3.1. Chemicals and Materials
3.2. Fructification of P. sapidus, Isolation of Basidiospores, and Screening of Monokaryons
3.3. Submerged Cultivation of P. sapidus Strains
3.4. Phenotype Stability of Different P. sapidus Strains
3.5. cDNA Synthesis and Gene Amplification
3.6. Heterologous Expression of the PsaPOX Variants in Komagataella phaffii
3.7. His-Tag Purification of the Recombinant PsaPOX Variants
3.8. Biotransformation
3.9. Peroxidase Activity
3.10. Comparative Biochemical Characterization of the PsaPOX Variants
3.11. Quantitative Real-Time PCR
3.12. Sequence Accession Numbers
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
ABTS | 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) |
DK | Dikaryon |
DSMZ | Deutsche Sammlung von Mikroorganismen und Zellkulturen |
DyP | Dye-decolorizing peroxidase |
gpd3 | Glyceraldehyd-3-phosphate dehydrogenase |
MK | Monokaryon |
phos | Purine phosphorylase |
PsaPOX | DyP of P. sapidus |
PsaPOX_DK | Parental PsaPOX variant |
PsaPOX_high | PsaPOX variant with higher activity than PsaPOX_DK |
PsaPOX_low | PsaPOX variant with lower activity than PsaPOX_DK |
RT-qPCR | Quantitative real-time PCR |
SNL | Standard nutrient liquid |
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PsaPOX Variant | Km (µM) | kcat (s−1) | kcat/Km (s−1 mM−1) |
---|---|---|---|
DK | 33.0 ± 2.5 | 6.8 ± 0.1 | 206 ± 4 |
high | 25.8 ± 1.6 | 10.5 ± 0.1 | 408 ± 14 |
low | 31.9 ± 2.3 | 3.8 ± 0.1 | 119 ± 2 |
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Krahe, N.-K.; Berger, R.G.; Witt, M.; Zorn, H.; Omarini, A.B.; Ersoy, F. Monokaryotic Pleurotus sapidus Strains with Intraspecific Variability of an Alkene Cleaving DyP-Type Peroxidase Activity as a Result of Gene Mutation and Differential Gene Expression. Int. J. Mol. Sci. 2021, 22, 1363. https://doi.org/10.3390/ijms22031363
Krahe N-K, Berger RG, Witt M, Zorn H, Omarini AB, Ersoy F. Monokaryotic Pleurotus sapidus Strains with Intraspecific Variability of an Alkene Cleaving DyP-Type Peroxidase Activity as a Result of Gene Mutation and Differential Gene Expression. International Journal of Molecular Sciences. 2021; 22(3):1363. https://doi.org/10.3390/ijms22031363
Chicago/Turabian StyleKrahe, Nina-Katharina, Ralf G. Berger, Martin Witt, Holger Zorn, Alejandra B. Omarini, and Franziska Ersoy. 2021. "Monokaryotic Pleurotus sapidus Strains with Intraspecific Variability of an Alkene Cleaving DyP-Type Peroxidase Activity as a Result of Gene Mutation and Differential Gene Expression" International Journal of Molecular Sciences 22, no. 3: 1363. https://doi.org/10.3390/ijms22031363