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 suchapplications need to satisfy certain criteria, such as exceptional sensitivity, substrate selectivity, stability and activity..

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..

Supplementary data for the article: Kovačević, G.; Ostafe, R.; Balaž, A. M.; Fischer, R.; Prodanović, R. Development of GFP-Based High-Throughput Screening System for Directed Evolution of Glucose Oxidase. Journal of Bioscience and Bioengineering 2019, 127 (1), 30–37. https://doi.org/10.1016/j.jbiosc.2018.07.002

Supplementary material for: [https://doi.org/10.1016/j.jbiosc.2018.07.002]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/336

Horseradish peroxidase C1A wild type gene and its variants expressed in Pichia pastoris KM71H strain

Enzyme immobilization enables maintenance of enzyme activity and structural stability even in adverse conditions 1. Structural changes in enzymes that can occur due to the action of organic solvents, inhibitors or increased temperature can be prevented by immobilization of the enzymes in metal–organic frameworks (MOFs). It is reported that several enzymes, such as cytochrome c and horseradish peroxidase (HRP) have been successfully incorporated into MOFs 2. The aim of this work is to produce wild type horseradish peroxidase, isoform C1A, and several mutants specially designed to increase the activity and stability of HRP while immobilized within selected MOFs. Wild type and its variants were produced in metalotrophic yeast, Pichia pastoris KM71H strain, their activity and basic kinetic parameters were determined and compared prior imobilization

Horseradish peroxidase C1A wild type gene and its variants expressed in Pichia pastoris KM71H strain

Enzyme immobilization enables maintenance of enzyme activity and structural stability even in adverse conditions 1. Structural changes in enzymes that can occur due to the action of organic solvents, inhibitors or increased temperature can be prevented by immobilization of the enzymes in metal–organic frameworks (MOFs). It is reported that several enzymes, such as cytochrome c and horseradish peroxidase (HRP) have been successfully incorporated into MOFs 2. The aim of this work is to produce wild type horseradish peroxidase, isoform C1A, and several mutants specially designed to increase the activity and stability of HRP while immobilized within selected MOFs. Wild type and its variants were produced in metalotrophic yeast, Pichia pastoris KM71H strain, their activity and basic kinetic parameters were determined and compared prior imobilization.Book of Abstract

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

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

Production of a novel opine dehydrogenase

Opine dehydrogenases are a family of NAD(P)H dependent oxidoreductases, whichcatalyze the reductive condensation of an α amino group from an amino acid with an αketo acid during anaerobic glycolysis by regenerating NAD. They are widespread incephalopods and mollusks. Opines are associated with crown gall tumor pathogenesiscaused by A. tumefaciens providing nutrients to the pathogen, and novel opine compoundsacting as metallophores have been identified. Besides, opine-type secondary aminedicarboxylic acids are chiral intermediates of angiotensin-converting enzyme inhibitors. Anovel enzyme originating from an extremophile bacterium, with assumed opinedehydrogenase function was successfully expressed in Escherichia coli STAR cells andpurified by affinity chromatography. Molecular mass determined by SDS-PAGE wasapproximately 40 kDa. The activity was measured by using pyruvate and alanine assubstrates, by which proved that it has opine dehydrogenase activity

Modelling of catalytic activity and enzyme-MOF interactions using combined in silico approach

Enzymes as industrial biocatalysts offer numerous advantages over traditional chemical processes concerning sustainability and process efficiency. Immobilization of enzymes on solid supporters is one of the key strategies for improving the practical performances of enzymes. Metal-organic frameworks (MOFs) are promising candidates for enzyme immobilization. MOFs are porous coordination polymers consisting of metal-containing nodes and organic ligands linked through coordination bonds. It has been demonstrated that proteins can be successfully immobilized even in MOF pores whose apertures are smaller than the molecular dimension of the protein due to its conformational flexibility. For our study, we selected horseradish peroxidase (HRP) encapsulated in MOF PCN-888(Al). We report the modelling of PCN-888(Al) MOF and the design of novel HRP mutants, which determine their enzymatic activity and magnitude of intermolecular interactions with MOF. Using a combined in silico approach, consisting of Informational Spectrum Method (ISM) bioinformatics method, molecular docking and molecular dynamics simulations, we propose new HRP mutants, which show higher/lower specific catalytic activity and higher/lower MOF-HRP dissociation constant, compared to the wild type of enzyme.Book of Abstract