Protein engineering of cellobiose dehydrogenase from Phanerochaete chrysosporium for higher oxidative stability in biocatalysis

Celobioza – dehidrogenaza poreklom iz Phanerochaete chrysosporium, gljive bele truleži, pripada ekstracelularnim oksidoredukcionim enzimima i katalizuje oksidaciju β – 1,4 – glikozidno vezanih oligosaharida poput celobioze i laktoze. Oksidacijom laktoze dolazi do formiranja laktobionske kiseline koja pronalazi veliku primenu u farmaceutskoj i kozmetičkoj industriji gde se koristi prilikom distribucije lekova i za hidrataciju kože kao sastavni deo različitih krema, gde zajedno sa hijaluronskom kiselinu ima ulogu u smanjenju bora. Celobioza – dehidrogenaza prilikom oksidacije laktoze ili celobioze, kao prirodnih supstrata, katalizuje redukciju jednog dvoelektronskog ili dva jednoelektronska akceptora elektrona. Jedan od najkorišćenijih dvoelektronskih akceptora elektrona je upravo dihlor fenol indofenol (DCIP), dok jednoelektronski akceptori elektrona mogu biti citohrom c, ABTS, ali i Fe3+ i Mn3+ joni. Redukcijom Fe3+ jona u prisustvu molekulskog kiseonika dolazi do formiranja vodonik peroksida i posredstvom Fentonove reakcije do generisanja hidroksil radikala. Polazeći od ove činjenice, iskoristili smo upravo Fentonovu reakciju za razvoj fluorescentnog eseja za visoko efikasnu pretragu biblioteka gena celobioza – dehidrogenaze, baziranog na detekciji proizvedenih hidroksil radikala fluorogenom probom aminofenil – fluoresceinom (APF). Primena celobioza – dehidrogenaze u konstruisanju biosenzora i biogorivnih ćelija leži upravo u njenoj sposobnosti da katalizuje oksidaciju laktoze, celobioze i β – 1,4 – vezanih oligosaharida do odgovarajućih laktona koji potom spontano hidrolizuju do aldonskih kiselina. Enzimi koji nalaze primenu u konstruisanju biosenzora i biogorivnih ćelija, moraju da zadovoljavaju nekoliko kriterijuma, odnosno moraju da imaju veliku osetljivost i supstratnu specifičnost, ali i da pokazuju povećanu stabilnost...Cellobiose dehydrogenase from Phanerochaete chrysosporium, a white rot fungus, belongs to the extracellular oxidoreductive group of enzymes and catalyzes the oxidation of the β – 1,4 – glycoside bond of oligosaccharides such as cellobiose and lactose. During oxidation of lactose, formation of lactobionic acid occurs which has many applications in pharmaceutical and cosmetic industry. Such applications include the distribution of medicine, a component responsible for skin hydration and when combined with hyaluronic acid as an agent against wrinkles. During oxidation of lactose or cellobiose by cellobiose dehydrogenase, reduction catalysis occurs of one two electron or two one electron acceptors. One of the most utilized two electron acceptors is DCIP, while one electron acceptors are usually cytochrome c, ABTS, Fe3+ and Mn3+ ions. During reduction of Fe3+ ions in the presence of molecular oxygen, H2O2 is formed and due to the Fenton reaction formation of hydroxyl radicals occurs. Due to this occurrence we wanted to use the Fenton reaction in order to develop a fluorescent assay based on the production of hydroxyl radicals and the fluorescence of aminophenyl fluorescein (APF). This would allow us to efficiently analyze cellobiose dehydrogenase gene libraries. With this fact in mind, the Fenton reaction was used to develop a fluorescent assay for the high throughput screening of cellobiose dehydrogenase genes, based on the detection of hydroxyl radicals with the fluorescent probe APF. The possible application of cellobiose dehydrogenase in the construction of various biosensors and biofuel cells is due the its ability to catalyze the oxidation of lactose, cellobiose and similar β – 1,4 – oligosaccharides do their corresponding lactones which then spontaneously hydrolyze to aldonic acids. Enzymes used in such applications need to satisfy certain criteria, such as exceptional sensitivity, substrate selectivity, stability and activity..