dc.creator | Balaž, Ana Marija | |
dc.creator | Ostafe, Raluca | |
dc.creator | Fischer, Rainer | |
dc.creator | Prodanović, Radivoje | |
dc.date.accessioned | 2019-12-19T14:38:38Z | |
dc.date.available | 2019-12-19T14:38:38Z | |
dc.date.issued | 2017 | |
dc.identifier.uri | http://www.bds.org.rs/en/conferences.php | |
dc.identifier.uri | https://cer.ihtm.bg.ac.rs/handle/123456789/3279 | |
dc.description.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. | en |
dc.language.iso | en | sr |
dc.publisher | Faculty of Chemistry, Serbian Biochemical Society | sr |
dc.relation | info:eu-repo/grantAgreement/MESTD/Integrated and Interdisciplinary Research (IIR or III)/46010/RS// | sr |
dc.rights | openAccess | sr |
dc.rights.uri | https://creativecommons.org/licenses/by-nc-nd/4.0/ | |
dc.source | Serbian Biochemical Society Seventh Conference "Biochemistry of Control in Life and Technology" - Proceedings | sr |
dc.subject | Cellobiose dehydrogenase | sr |
dc.subject | oxidoreductases | sr |
dc.subject | electrophoresis | sr |
dc.subject | mutants | sr |
dc.title | Semi - rational design of cellobiose dehydrogenase from Phanerochaete chrysosporium for increased oxidative stability and high-throughput screening of library mutants | en |
dc.type | conferenceObject | sr |
dc.rights.license | BY-NC-ND | sr |
dcterms.abstract | Остафе, Ралуца; Балаж, Aна Марија; Фисцхер, Раинер; Продановић, Радивоје; | |
dc.identifier.rcub | https://hdl.handle.net/21.15107/rcub_cer_3279 | |
dc.identifier.fulltext | https://cer.ihtm.bg.ac.rs/bitstream/id/15651/01_biochem2017.pdf | |
dc.type.version | publishedVersion | sr |