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Introduction
My research focus lies in the investigation and application of oxidoreductases and decarboxylases. If you want to contact me, please message me directly via daniel.kracher@tugraz.at!
Additional affiliations
June 2019 - December 2019
November 2017 - present
July 2016 - July 2016
Publications
Publications (49)
A new paradigm for cellulose depolymerization by fungi focuses on an oxidative mechanism involving cellobiose dehydrogenases (CDH) and copper-dependent lytic polysaccharide monooxygenases (LPMO); however, mechanistic studies have been hampered by the lack of structural information regarding CDH. CDH contains a haem-binding cytochrome (CYT) connecte...
The fuel for fungal enzymes
Many microorganisms have specialized enzymes to target and break down plant biomass. In fungi, these enzymes, called lytic polysaccharide monooxygenases (LPMOs), partner with electron transfer partners to oxidatively cleave the polysaccharide backbone of lignocellulosic polymers. Kracher et al. examined several potential...
Lytic polysaccharide monooxygenases (LPMOs) are a class of copper-containing enzymes that oxidatively degrade insoluble plant polysaccharides and soluble oligosaccharides. Upon reductive activation, they cleave the substrate and promote biomass degradation by hydrolytic enzymes. In this study we employed LPMO9C from Neurospora crassa, which is acti...
Background
Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent redox enzymes that cleave recalcitrant biopolymers such as cellulose, chitin, starch and hemicelluloses. Although LPMOs receive ample interest in industry and academia, their reaction mechanism is not yet fully understood. Recent studies showed that H2O2 is a more efficient...
Large-scale protein domain dynamics and electron transfer are often associated. However, as protein motions span a broad range of time and length scales, it is often challenging to identify and thus, link functionally relevant dynamic changes to electron transfer in proteins. It is hypothesized that large-scale domain motions direct electrons throu...
Cellobiose dehydrogenase (CDH) is applied as a bioelectrocatalyst in biosensors because its mobile cytochrome domain is capable of direct electron transfer. This study investigates the electron transfer mechanism of CDH molecules embedded in the polycation polyethyleneimine (PEI), which has been reported as a current-boosting component of CDH-based...
Enzymatic decarboxylation of biobased hydroxycinnamic acids gives access to phenolic styrenes for adhesive production. Phenolic acid decarboxylases are proficient enzymes that have been applied in aqueous systems, organic solvents, biphasic systems, and deep eutectic solvents, which makes stability a key feature. Stabilization of the enzyme would i...
Oxygenase and peroxygenase enzymes generate intermediates at their active sites which bring about the controlled functionalization of inert C-H bonds in substrates, such as in the enzymatic conversion of methane to methanol. To be viable catalysts, however, these enzymes must also prevent oxidative damage to essential active site residues, which ca...
Biocatalytic decarboxylation of hydroxycinnamic acids yields phenolic styrenes, which are important precursors for antioxidants, epoxy coatings, adhesives and other polymeric materials. Bacillus subtilis decarboxylase (BsPAD) is a cofactor‐independent enzyme that catalyzes the cleavage of carbon dioxide from p‐coumaric‐, caffeic‐, and ferulic acid...
Cold-active enzymes maintain a large part of their optimal activity at low temperatures. Therefore, they can be used to avoid side reactions and preserve heat-sensitive compounds. Baeyer-Villiger monooxygenases (BVMO) utilize molecular oxygen as a co-substrate to catalyze reactions widely employed for steroid, agrochemical, antibiotic, and pheromon...
Lytic polysaccharide monooxygenase (LPMO) supports biomass hydrolysis by increasing saccharification efficiency and rate. Recent studies demonstrate that H 2 O 2 rather than O 2 is the cosubstrate of the LPMO-catalyzed depolymerization of polysaccharides. Some studies have questioned the physiological relevance of the H 2 O 2 -based mechanism for p...
The textile industry is one of the largest water-polluting industries in the world. Due to an increased application of chromophores and a more frequent presence in wastewaters, the need for an ecologically favorable dye degradation process emerged. To predict the decolorization rate of textile dyes with Lytic polysaccharide monooxygenase (LPMO), we...
Lytic polysaccharide monooxygenases (LPMOs) are widely distributed in fungi, and catalyze the oxidative degradation of polysaccharides such as cellulose. Despite their name, LPMOs possess a dominant peroxygenase activity that is reflected in high turnover numbers but also causes deactivation. We report on the influence of small molecules and ions o...
Oxygenase enzymes generate reactive intermediates at their active sites to effect controlled functionalizations of inert C–H bonds in substrates, such as in the enzymatic conversion of methane to methanol. To be viable catalysts, however, these enzymes must also prevent oxidative damage to essential active site residues, which can occur during turn...
Uniting the unparalleled stereoselectivity of enzymes with the efficiency of chemical catalysis offers high product yields and atom economy while limiting downstream processing efforts. However, despite an impressive series of proof-of-concept studies, the field has not yet achieved broad applicability, especially in regard to industrial requiremen...
Glyoxal oxidase (GLOX) is an extracellular source of H2O2 in white-rot secretomes, where it acts in concert with peroxidases to degrade lignin. It has been reported that GLOX requires activation prior to catalytic turnover and that a peroxidase system can fulfill this task. In this study, we verify that an oxidation product of horseradish peroxidas...
Fungal lytic polysaccharide monooxygenases (LPMOs) depolymerise crystalline cellulose and hemicellulose, supporting the utilisation of lignocellulosic biomass as a feedstock for biorefinery and biomanufacturing processes. Recent investigations have shown that H2O2 is the most efficient cosubstrate for LPMOs. Understanding the reaction mechanism of...
The natural function of cellobiose dehydrogenase (CDH) to donate electrons from its catalytic flavodehydrogenase (DH) domain via its cytochrome (CYT) domain to lytic polysaccharide monooxygenase (LPMO) is an example of a highly efficient extracellular electron transfer chain. To investigate the function of the CYT domain movement in the two occurri...
Lytic polysaccharide monooxygenases (LPMOs) are industrially important oxidoreductases employed in lignocellulose saccharification. Using advanced time-resolved mass spectrometric techniques, we elucidated the structural determinants for substrate-mediated stabilization of the fungal LPMO9C from Neurospora crassa during catalysis. LPMOs require a r...
Pyranose oxidase (POx) catalyzes the oxidation of d-glucose to 2-ketoglucose with concurrent reduction of oxygen to H2O2. POx from Trametes ochracea (ToPOx) is known to react with alternative electron acceptors including 1,4-benzoquinone (1,4-BQ), 2,6-dichlorophenol indophenol (DCPIP), and the ferrocenium ion. In this study, enzyme variants with im...
The catalytic function of lytic polysaccharide monooxygenases (LPMOs) to cleave and decrystallize recalcitrant polysaccharides put these enzymes in the spotlight of fundamental and applied research. Here we demonstrate that the demand of LPMO for an electron donor and an oxygen species as cosubstrate can be fulfilled by a single auxiliary enzyme: a...
This review aims to present current knowledge of the fungi involved in lignocellulose degradation with an overview of the various classes of lignocellulose‐acting enzymes engaged in the pretreatment and saccharification step. Fungi have numerous application and biotechnological potential for various industries including chemicals, fuel, pulp and pa...
CAZypedia was initiated in 2007 to create a comprehensive, living encyclopedia of the carbohydrate active enzymes (CAZymes) and associated carbohydrate-binding modules involved in the synthesis, modification and degradation of complex carbohydrates. CAZypedia is closely connected with the actively curated CAZy database, which provides a sequence-ba...
Wheat bran, a side product of the milling industry, can serve as valuable food component, feed ingredient or feedstock for biorefineries. However, all these applications bear drawbacks of sensory, physiological and technological challenges. The present study investigates an alternative utilization strategy of wheat bran as substrate for mushroom pr...
This paper describes the stabilization of flavin‐dependent monooxygenases under reaction conditions, using an engineered formulation of additives (the natural cofactors NADPH and FAD, and superoxide dismutase and catalase as catalytic antioxidants). This way, a 10 ³ ‐ to 10 ⁴ ‐fold increase of the half‐life was reached without resource‐intensive di...
Multigenicity is commonly found in fungal enzyme systems, with the purpose of functional compensation upon deficiency of one of its members or leading to enzyme isoforms with new functionalities through gene diversification. Three genes of the flavin-dependent glucose-methanol-choline (GMC) oxidoreductase pyranose dehydrogenase (AmPDH) were previou...
Background
Cellobiose dehydrogenase (CDH) is an extracellular enzyme produced by lignocellulolytic fungi. cdh gene expression is high in cellulose containing media, but relatively low CDH concentrations are found in the supernatant of fungal cultures due to strong binding to cellulose. Therefore, heterologous expression of CDH in Pichia pastoris wa...
The flavin and heme cofactor containing enzyme cellobiose dehydrogenase (CDH) is ubiquitously distributed in wood-degrading fungi. Current research provides compelling evidence that CDH is an activator for cellulolytic monooxygenases, which enhance the accessibility of crystalline cellulose surfaces for hydrolases. Such oxidative cellulose degradat...
Lytic polysaccharide monooxygenases (LPMOs) represent a recent addition to the carbohydrate-active enzymes and are classified as auxiliary activity (AA) families 9, 10, 11 and 13. LPMOs are crucial for effective degradation of recalcitrant polysaccharides like cellulose or chitin. These enzymes are copper-dependent and utilize a redox mechanism to...
Sugar beet pulp (SBP) is a promising feedstock for the production of 2nd generation biofuels, but efficient enzymatic hydrolysis remains a key challenge; therefore, new process designs and/or bioreactor designs are crucial to overcome this hurdle. In this regard, horizontal rotating tubular bioreactors (HRTB) offer the advantage of high substrate l...
Background:
Lytic polysaccharide monooxygenases (LPMOs) belong to the "auxiliary activities (AA)" enzyme class of the CAZy database. They are known to strongly improve the saccharification process and boost soluble sugar yields from lignocellulosic biomass, which is a key step in the efficient production of sustainable economic biofuels. To date,...
The tobacco-related plant species Nicotiana benthamiana has recently emerged as a versatile expression platform for the rapid generation of recombinant biopharmaceuticals, but product yield and quality frequently suffer from unintended proteolysis. Previous studies have highlighted that recombinant protein fragmentation in plants involves papain-li...
A recently proposed coenzyme regeneration system employing laccase and a number of various redox mediators for the oxidation of NAD(P)H was studied in detail by kinetic characterization of individual reaction steps. Reaction engineering by modeling was used to optimize the employed enzyme, coenzyme as well as redox mediator concentrations. Glucose...
The flavocytochrome cellobiose dehydrogenase (CDH) is secreted by wood decomposing fungi and is the only known extracellular enzyme with the characteristics of an electron transfer protein. Its proposed function is the reduction of lytic polysaccharide monooxygenase (LPMO) for subsequent cellulose depolymerisation. Electrons are transferred from FA...
The UDP-sulfoquinovose synthase Agl3 from Sulfolobus acidocaldarius converts UDP-d-glucose and sulfite to UDP-sulfoquinovose, the activated form of sulfoquinovose required for its incorporation into glycoconjugates. Based on the amino acid sequence, Agl3 belongs to the short-chain dehydrogenase/reductase enzyme superfamily, together with SQD1 from...
Polymer-multiwalled carbon nanotube (MWCNT) nanohybrids, which differ in surface charge have been synthesized to study the bioelectrocatalysis of adsorbed cellobiose dehydrogenase (CDH) from Phanerochaete sordida on gold electrodes. To obtain negatively charged nanohybrids, poly(3-amino-4-methoxybenzoic acid-co-aniline) (P(AMB-A)) was covalently li...
Lytic polysaccharide monooxygenase (LPMO) represents a unique principle of oxidative degradation of recalcitrant insoluble polysaccharides. Used in combination with hydrolytic enzymes, LPMO appears to constitute a significant factor of the efficiency of enzymatic biomass depolymerization. LPMO activity on different cellulose substrates has been sho...
The recalcitrance of lignocellulose renders the enzymatic hydrolysis of plant biomass for the production of second generation biofuels into a major challenge. This work investigates an efficient and economic approach for the enzymatic hydrolysis of sugar beet pulp (SBP), which is a difficult to degrade, hemicellulose-rich by-product of the table su...
Lignocellulosic biomass is a renewable resource that significantly can substitute fossil resources for the production of fuels,
chemicals, and materials. Efficient saccharification of this biomass to fermentable sugars will be a key technology in future
biorefineries. Traditionally, saccharification was thought to be accomplished by mixtures of hyd...
Sugar beet pulp is an abundant industrial waste material which holds a great potential for bioethanol production due to its high content of cellulose, hemicelluloses and pectin. Its structural and chemical robustness limits the yield of fermentable sugars obtained by hydrolysation and represents the main bottleneck for bioethanol production. Physic...
Background
Recent studies demonstrate that enzymes from the glycosyl hydrolase family 61 (GH61) show lytic polysaccharide monooxygenase (PMO) activity. Together with cellobiose dehydrogenase (CDH) an enzymatic system capable of oxidative cellulose cleavage is formed, which increases the efficiency of cellulases and put PMOs at focus of biofuel rese...
The genome of Neurospora crassa encodes two different cellobiose dehydrogenases (CDHs) with a sequence identity of only 53%. So far, only CDH IIA, which
is induced during growth on cellulose and features a C-terminal carbohydrate binding module (CBM), was detected in the secretome
of N. crassa and preliminarily characterized. CDH IIB is not signifi...