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Semi - rational design of cellobiose dehydrogenase from Phanerochaete chrysosporium for increased oxidative stability and high-throughput screening of library mutants

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2017
01_biochem2017.pdf (403.1Kb)
Authors
Balaž, Ana Marija
Ostafe, Raluca
Fischer, Rainer
Prodanović, Radivoje
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Abstract
Cellobiose dehydrogenase (CDH, EC 1.1.99.18) from Phanerochaete chrysosporium belongs to a group of oxidoreductases and has the ability to degrade different components of woody plants. CDH is secreted by wood degrading, phytopathogenic and saprotrophic fungi and this widespread appearance implies hers important function and makes her an important enzyme for applications in industrial and biotechnological processes, as well as biosensors and biofuel cells. CDH is also used in industry for bleaching cotton and in food industry for lactose detection. CDH is monomeric enzyme consisting of two domains, flavin domain containing FAD as cofactor and smaller hem b containing cytochrome domain, connected via flexible linker. Physiological role of CDH is reflected in the degradation of cellulose and lignin in cooperation with other cellulolytic enzymes, because CDH catalyzes oxidation of celobiose (Glc - β - 1,4 Glc) and other β - 1,4 - linked disaccharides and oligosacchari...des to the corresponding lactons. Enzymes used in biosensors and for bleaching cotton should have high stability, especially toward reactive oxygen species. In order to improve oxidative stability of CDH, we have mutated CDH and tested its stability in presence of hydrogen peroxide. After successful cloning of the CDH gene in pYES2 vector, saturation mutagenesis was used to make library mutants where tree methionine residues were mutated. Residual activity of mutants was measured after the enzyme incubation in 0.3 M hydrogen peroxide for 0, 2 and 6h. After analysis of large number of mutants, it was observed that three mutants are showing higher oxidative stability compared to the wild - type enzyme. Residual activities of these mutants after 6 hour incubation in the hydrogen peroxide were over 50%, whereas wild-type has 30%. Selected mutants were expressed in S. cerevisiae and purified on DEAE column. Purity and activity of the enzymes were detected on the electrophoresis gel, oxidative stability of purified mutants was measured once again and characterization of these mutants was done. Mutants showing increased oxidative stability were sequenced and we have decided to combine these mutations with each other in order to make combined mutants that will be tested for oxidative stability. Screening library mutants for improved features in microtitatar plates is a long time process, in order to shorten the time necessary for screening libraries with 106 mutants we are developing fluorescent assay for flou cytometry.

Keywords:
Cellobiose dehydrogenase / oxidoreductases / electrophoresis / mutants
Source:
Serbian Biochemical Society Seventh Conference "Biochemistry of Control in Life and Technology" - Proceedings, 2017
Publisher:
  • Faculty of Chemistry, Serbian Biochemical Society
Funding / projects:
  • Novel encapsulation and enzyme technologies for designing of new biocatalysts and biologically active compounds targeting enhancement of food quality, safety and competitiveness (RS-46010)
[ Google Scholar ]
Handle
https://hdl.handle.net/21.15107/rcub_cer_3279
URI
http://www.bds.org.rs/en/conferences.php
https://cer.ihtm.bg.ac.rs/handle/123456789/3279
Collections
  • Radovi istraživača / Researchers' publications
Institution/Community
IHTM
TY  - CONF
AU  - Balaž, Ana Marija
AU  - Ostafe, Raluca
AU  - Fischer, Rainer
AU  - Prodanović, Radivoje
PY  - 2017
UR  - http://www.bds.org.rs/en/conferences.php
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/3279
AB  - Cellobiose dehydrogenase (CDH, EC 1.1.99.18) from Phanerochaete chrysosporium
 belongs to a group of oxidoreductases and has the ability to degrade different components
 of woody plants. CDH is secreted by wood degrading, phytopathogenic and saprotrophic
 fungi and this widespread appearance implies hers important function and makes her an
 important enzyme for applications in industrial and biotechnological processes, as well as
 biosensors and biofuel cells. CDH is also used in industry for bleaching cotton and in
 food industry for lactose detection. CDH is monomeric enzyme consisting of two
 domains, flavin domain containing FAD as cofactor and smaller hem b containing
 cytochrome domain, connected via flexible linker. Physiological role of CDH is reflected
 in the degradation of cellulose and lignin in cooperation with other cellulolytic enzymes,
 because CDH catalyzes oxidation of celobiose (Glc - β - 1,4 Glc) and other β - 1,4 - linked
 disaccharides and oligosaccharides to the corresponding lactons. Enzymes used in
 biosensors and for bleaching cotton should have high stability, especially toward reactive
 oxygen species. In order to improve oxidative stability of CDH, we have mutated CDH
 and tested its stability in presence of hydrogen peroxide. After successful cloning of the
 CDH gene in pYES2 vector, saturation mutagenesis was used to make library mutants
 where tree methionine residues were mutated. Residual activity of mutants was measured
 after the enzyme incubation in 0.3 M hydrogen peroxide for 0, 2 and 6h. After analysis of
 large number of mutants, it was observed that three mutants are showing higher oxidative
 stability compared to the wild - type enzyme. Residual activities of these mutants after 6
 hour incubation in the hydrogen peroxide were over 50%, whereas wild-type has 30%.
 Selected mutants were expressed in S. cerevisiae and purified on DEAE column. Purity
 and activity of the enzymes were detected on the electrophoresis gel, oxidative stability of
 purified mutants was measured once again and characterization of these mutants was done. Mutants showing increased oxidative stability were sequenced and we have decided to
 combine these mutations with each other in order to make combined mutants that will be
 tested for oxidative stability. Screening library mutants for improved features in
 microtitatar plates is a long time process, in order to shorten the time necessary for
 screening libraries with 106 mutants we are developing fluorescent assay for flou
 cytometry.
PB  - Faculty of Chemistry, Serbian Biochemical Society
C3  - Serbian Biochemical Society Seventh Conference "Biochemistry of Control in Life and Technology" - Proceedings
T1  - Semi - rational design of cellobiose dehydrogenase from Phanerochaete chrysosporium for increased oxidative stability and high-throughput screening of library mutants
UR  - https://hdl.handle.net/21.15107/rcub_cer_3279
ER  - 
@conference{
author = "Balaž, Ana Marija and Ostafe, Raluca and Fischer, Rainer and Prodanović, Radivoje",
year = "2017",
abstract = "Cellobiose dehydrogenase (CDH, EC 1.1.99.18) from Phanerochaete chrysosporium
 belongs to a group of oxidoreductases and has the ability to degrade different components
 of woody plants. CDH is secreted by wood degrading, phytopathogenic and saprotrophic
 fungi and this widespread appearance implies hers important function and makes her an
 important enzyme for applications in industrial and biotechnological processes, as well as
 biosensors and biofuel cells. CDH is also used in industry for bleaching cotton and in
 food industry for lactose detection. CDH is monomeric enzyme consisting of two
 domains, flavin domain containing FAD as cofactor and smaller hem b containing
 cytochrome domain, connected via flexible linker. Physiological role of CDH is reflected
 in the degradation of cellulose and lignin in cooperation with other cellulolytic enzymes,
 because CDH catalyzes oxidation of celobiose (Glc - β - 1,4 Glc) and other β - 1,4 - linked
 disaccharides and oligosaccharides to the corresponding lactons. Enzymes used in
 biosensors and for bleaching cotton should have high stability, especially toward reactive
 oxygen species. In order to improve oxidative stability of CDH, we have mutated CDH
 and tested its stability in presence of hydrogen peroxide. After successful cloning of the
 CDH gene in pYES2 vector, saturation mutagenesis was used to make library mutants
 where tree methionine residues were mutated. Residual activity of mutants was measured
 after the enzyme incubation in 0.3 M hydrogen peroxide for 0, 2 and 6h. After analysis of
 large number of mutants, it was observed that three mutants are showing higher oxidative
 stability compared to the wild - type enzyme. Residual activities of these mutants after 6
 hour incubation in the hydrogen peroxide were over 50%, whereas wild-type has 30%.
 Selected mutants were expressed in S. cerevisiae and purified on DEAE column. Purity
 and activity of the enzymes were detected on the electrophoresis gel, oxidative stability of
 purified mutants was measured once again and characterization of these mutants was done. Mutants showing increased oxidative stability were sequenced and we have decided to
 combine these mutations with each other in order to make combined mutants that will be
 tested for oxidative stability. Screening library mutants for improved features in
 microtitatar plates is a long time process, in order to shorten the time necessary for
 screening libraries with 106 mutants we are developing fluorescent assay for flou
 cytometry.",
publisher = "Faculty of Chemistry, Serbian Biochemical Society",
journal = "Serbian Biochemical Society Seventh Conference "Biochemistry of Control in Life and Technology" - Proceedings",
title = "Semi - rational design of cellobiose dehydrogenase from Phanerochaete chrysosporium for increased oxidative stability and high-throughput screening of library mutants",
url = "https://hdl.handle.net/21.15107/rcub_cer_3279"
}
Balaž, A. M., Ostafe, R., Fischer, R.,& Prodanović, R.. (2017). Semi - rational design of cellobiose dehydrogenase from Phanerochaete chrysosporium for increased oxidative stability and high-throughput screening of library mutants. in Serbian Biochemical Society Seventh Conference "Biochemistry of Control in Life and Technology" - Proceedings
Faculty of Chemistry, Serbian Biochemical Society..
https://hdl.handle.net/21.15107/rcub_cer_3279
Balaž AM, Ostafe R, Fischer R, Prodanović R. Semi - rational design of cellobiose dehydrogenase from Phanerochaete chrysosporium for increased oxidative stability and high-throughput screening of library mutants. in Serbian Biochemical Society Seventh Conference "Biochemistry of Control in Life and Technology" - Proceedings. 2017;.
https://hdl.handle.net/21.15107/rcub_cer_3279 .
Balaž, Ana Marija, Ostafe, Raluca, Fischer, Rainer, Prodanović, Radivoje, "Semi - rational design of cellobiose dehydrogenase from Phanerochaete chrysosporium for increased oxidative stability and high-throughput screening of library mutants" in Serbian Biochemical Society Seventh Conference "Biochemistry of Control in Life and Technology" - Proceedings (2017),
https://hdl.handle.net/21.15107/rcub_cer_3279 .

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