Characterization of recombinant Phanerochaete chrysosporium cellobiose dehydrogenase mutants with increased oxidative stability from Pichia pastoris KM71H strain
Authors
Balaz, Ana MarijaPopov, Neda
Prodanović, Olivera
Ostafe, Raluca
Fischer, Rainer
Prodanović, Radivoje
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Phanerochaete chrysosporium is a white rot fungi and it has been known to secrete
flavocytochrome enzyme cellobiose dehydrogenase (CDH, EC 1.1.99.18) which contains
two domains, a flavine domain and cytochrome domain. Flavine domain contains FAD as
prostetic group and its catalytically active domain, whereas cytochrome domain serves as
electrone acceptor. Cellobiose and lactose, as well as other β – 1,4 – linked disaccharides
and oligosaccharides, have been oxidized by the cellobiose dehydrogenase to their
corresponding lactones. CDH can be used for constructing biosensors and therefore
directed evolution has been used to produce more active and stable variants of the enzyme.
Wild type CDH enzyme was expressed in S.cerevisiae INVSc1 cells and used for creation
of saturation mutagenesis libraries at M65, M685 and M738 and screening for increased
oxidative stability. More stable mutants that were found were recloned into Pichia pastoris
KM71H strain for higher expression yield.... They were afterwards, expressed in Pichia,
purified and kineticaly characterized.
Keywords:
Phanerochaete chrysosporium / cellobiose dehydrogenaseSource:
The 9th conference of the Serbian Biochemical Society: Diversity in Biochemistry, 2019, 74-75Publisher:
- Faculty of Chemistry, Serbian Biochemical Society
Funding / projects:
- Allergens, antibodies, enzymes and small physiologically important molecules: design, structure, function and relevance (RS-MESTD-Basic Research (BR or ON)-172049)
- Novel encapsulation and enzyme technologies for designing of new biocatalysts and biologically active compounds targeting enhancement of food quality, safety and competitiveness (RS-MESTD-Integrated and Interdisciplinary Research (IIR or III)-46010)
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Institut za multidisciplinarna istraživanjaTY - CONF AU - Balaz, Ana Marija AU - Popov, Neda AU - Prodanović, Olivera AU - Ostafe, Raluca AU - Fischer, Rainer AU - Prodanović, Radivoje PY - 2019 UR - http://rimsi.imsi.bg.ac.rs/handle/123456789/3043 AB - Phanerochaete chrysosporium is a white rot fungi and it has been known to secrete flavocytochrome enzyme cellobiose dehydrogenase (CDH, EC 1.1.99.18) which contains two domains, a flavine domain and cytochrome domain. Flavine domain contains FAD as prostetic group and its catalytically active domain, whereas cytochrome domain serves as electrone acceptor. Cellobiose and lactose, as well as other β – 1,4 – linked disaccharides and oligosaccharides, have been oxidized by the cellobiose dehydrogenase to their corresponding lactones. CDH can be used for constructing biosensors and therefore directed evolution has been used to produce more active and stable variants of the enzyme. Wild type CDH enzyme was expressed in S.cerevisiae INVSc1 cells and used for creation of saturation mutagenesis libraries at M65, M685 and M738 and screening for increased oxidative stability. More stable mutants that were found were recloned into Pichia pastoris KM71H strain for higher expression yield. They were afterwards, expressed in Pichia, purified and kineticaly characterized. PB - Faculty of Chemistry, Serbian Biochemical Society C3 - The 9th conference of the Serbian Biochemical Society: Diversity in Biochemistry T1 - Characterization of recombinant Phanerochaete chrysosporium cellobiose dehydrogenase mutants with increased oxidative stability from Pichia pastoris KM71H strain EP - 75 SP - 74 UR - https://hdl.handle.net/21.15107/rcub_rimsi_3043 ER -
@conference{ author = "Balaz, Ana Marija and Popov, Neda and Prodanović, Olivera and Ostafe, Raluca and Fischer, Rainer and Prodanović, Radivoje", year = "2019", abstract = "Phanerochaete chrysosporium is a white rot fungi and it has been known to secrete flavocytochrome enzyme cellobiose dehydrogenase (CDH, EC 1.1.99.18) which contains two domains, a flavine domain and cytochrome domain. Flavine domain contains FAD as prostetic group and its catalytically active domain, whereas cytochrome domain serves as electrone acceptor. Cellobiose and lactose, as well as other β – 1,4 – linked disaccharides and oligosaccharides, have been oxidized by the cellobiose dehydrogenase to their corresponding lactones. CDH can be used for constructing biosensors and therefore directed evolution has been used to produce more active and stable variants of the enzyme. Wild type CDH enzyme was expressed in S.cerevisiae INVSc1 cells and used for creation of saturation mutagenesis libraries at M65, M685 and M738 and screening for increased oxidative stability. More stable mutants that were found were recloned into Pichia pastoris KM71H strain for higher expression yield. They were afterwards, expressed in Pichia, purified and kineticaly characterized.", publisher = "Faculty of Chemistry, Serbian Biochemical Society", journal = "The 9th conference of the Serbian Biochemical Society: Diversity in Biochemistry", title = "Characterization of recombinant Phanerochaete chrysosporium cellobiose dehydrogenase mutants with increased oxidative stability from Pichia pastoris KM71H strain", pages = "75-74", url = "https://hdl.handle.net/21.15107/rcub_rimsi_3043" }
Balaz, A. M., Popov, N., Prodanović, O., Ostafe, R., Fischer, R.,& Prodanović, R.. (2019). Characterization of recombinant Phanerochaete chrysosporium cellobiose dehydrogenase mutants with increased oxidative stability from Pichia pastoris KM71H strain. in The 9th conference of the Serbian Biochemical Society: Diversity in Biochemistry Faculty of Chemistry, Serbian Biochemical Society., 74-75. https://hdl.handle.net/21.15107/rcub_rimsi_3043
Balaz AM, Popov N, Prodanović O, Ostafe R, Fischer R, Prodanović R. Characterization of recombinant Phanerochaete chrysosporium cellobiose dehydrogenase mutants with increased oxidative stability from Pichia pastoris KM71H strain. in The 9th conference of the Serbian Biochemical Society: Diversity in Biochemistry. 2019;:74-75. https://hdl.handle.net/21.15107/rcub_rimsi_3043 .
Balaz, Ana Marija, Popov, Neda, Prodanović, Olivera, Ostafe, Raluca, Fischer, Rainer, Prodanović, Radivoje, "Characterization of recombinant Phanerochaete chrysosporium cellobiose dehydrogenase mutants with increased oxidative stability from Pichia pastoris KM71H strain" in The 9th conference of the Serbian Biochemical Society: Diversity in Biochemistry (2019):74-75, https://hdl.handle.net/21.15107/rcub_rimsi_3043 .