Fluorescent Imaging of Extracellular Fungal Enzymes Bound onto Plant Cell Walls.

Lignocelluloytic enzymes are industrially applied as biocatalysts for the deconstruction of recalcitrant plant biomass. To study their biocatalytic and physiological function, the assessment of their binding behavior and spatial distribution on lignocellulosic material is a crucial prerequisite. In this study, selected hydrolases and oxidoreductases from the white rot fungus Phanerochaete chrysosporium were localized on model substrates as well as poplar wood by confocal laser scanning microscopy. Two different detection approaches were investigated: direct tagging of the enzymes and tagging specific antibodies generated against the enzymes. Site-directed mutagenesis was employed to introduce a single surface-exposed cysteine residue for the maleimide site-specific conjugation. Specific polyclonal antibodies were produced against the enzymes and were labeled using N-hydroxysuccinimide (NHS) ester as a cross-linker. Both methods allowed the visualization of cell wall-bound enzymes but showed slightly different fluorescent yields. Using native poplar thin sections, we identified the innermost secondary cell wall layer as the preferential attack point for cellulose-degrading enzymes. Alkali pretreatment resulted in a partial delignification and promoted substrate accessibility and enzyme binding. The methods presented in this study are suitable for the visualization of enzymes during catalytic biomass degradation and can be further exploited for interaction studies of lignocellulolytic enzymes in biorefineries.

Expression and characterization of a family 45 glycosyl hydrolase from Fomitopsis pinicola and comparison to Phanerochaete chrysosporium Cel45A.

To efficiently decompose biomass, fungi have developed various enzymatic and non-enzymatic strategies and are a source of versatile biocatalysts. The endoglucanases in glycosyl hydrolase CAZy family 45 (GH45) are known for their small size, a high thermostability and a broad substrate specificity that has been employed in textile and detergent industries. Here we report the heterologous expression and characterisation of an GH45 endoglucanase from the brown rot Fomitopsis pinicola and its direct comparison to an already characterised GH45 from the white rot Phanerochaete chrysosporium. Both enzymes were recombinantly expressed in Pichia pastoris and purified by two chromatographic steps. The biochemical characterisation highlighted the acidophilic character, with an optimal pH of 4, and a preference for amorphous substrates as carboxymethyl cellulose (CMC) and substrates containing ß-1,4-glucans rather than the previously reported ß-1,3/1,4-glucans lichenan and ß-glucan. The dominating products from ß-1,4-glucans were C3-C6 oligosaccharides, whereas from ß-1,3/1,4-glucans glucose was the main reaction product. From the characterisation no differences between the brown rot and the white rot GH45 was evident.

Non-productive binding of cellobiohydrolase i investigated by surface plasmon resonance spectroscopy.

Future biorefineries are facing the challenge to separate and depolymerize biopolymers into their building blocks for the production of biofuels and basic molecules as chemical stock. Fungi have evolved lignocellulolytic enzymes to perform this task specifically and efficiently, but a detailed understanding of their heterogeneous reactions is a prerequisite for the optimization of large-scale enzymatic biomass degradation. Here, we investigate the binding of cellulolytic enzymes onto biopolymers by surface plasmon resonance (SPR) spectroscopy for the fast and precise characterization of enzyme adsorption processes. Using different sensor architectures, SPR probes modified with regenerated cellulose as well as with lignin films were prepared by spin-coating techniques. The modified SPR probes were analyzed by atomic force microscopy and static contact angle measurements to determine physical and surface molecular properties. SPR spectroscopy was used to study the activity and affinity of Trichoderma reesei cellobiohydrolase I (CBHI) glycoforms on the modified SPR probes. N-glycan removal led to no significant change in activity or cellulose binding, while a slightly higher tendency for non-productive binding to SPR probes modified with different lignin fractions was observed. The results suggest that the main role of the N-glycosylation in CBHI is not to prevent non-productive binding to lignin, but probably to increase its stability against proteolytic degradation. The work also demonstrates the suitability of SPR-based techniques for the characterization of the binding of lignocellulolytic enzymes to biomass-derived polymers. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10570-021-04002-6.

Spectroelectrochemical investigation of the glyoxal oxidase activation mechanism.

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 peroxidase, the radical cation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), is an activator for GLOX. A spectroelectrochemical cell was used to generate the activating radical species, to continuously measure its concentration, and to simultaneously measure the catalytic activity of GLOX based on its O2 consumption. The results show that GLOX can undergo multiple catalytic turnovers upon activation and that activity increases with the activator concentration. However, we also found that the ABTS cation radical can serve as an electron acceptor which becomes visible in the absence of O2. Furthermore, GLOX activity is highly restrained by the naturally occurring, low O2 concentration. We conclude that GLOX is indeed an auxiliary enzyme for H2O2 production in white-rot secretomes. Its turnover rate is strongly regulated by the availability of O2 and the radical generating activity of peroxidases present in the secretome, which acts as a feedback loop for GLOX activity.

Real-Time Measurement of Cellobiose and Glucose Formation during Enzymatic Biomass Hydrolysis.

Enzymatic hydrolysis of lignocellulosic biomass for biofuel production relies on complex multi-enzyme ensembles. Continuous and accurate measurement of the released key products is crucial in optimizing the industrial degradation process and also investigating the activity and interaction between the involved enzymes and the insoluble substrate. Amperometric biosensors have been applied to perform continuous cellobiose measurements during the enzymatic hydrolysis of pure cellulose powders. The oxygen-sensitive mediators used in these biosensors restricted their function under physiological or industrial conditions. Also, the combined measurements of the hydrolysis products cellobiose and glucose require a high selectivity of the biorecognition elements. We employed an [Os(2,2'-bipyridine)2Cl]Cl-modified polymer and cellobiose dehydrogenase to fabricate a cellobiose biosensor, which can accurately and specifically detect cellobiose even in the presence of oxygen and the other main product glucose. Additionally, a glucose biosensor was fabricated to simultaneously measure glucose produced from cellobiose by ß-glucosidases. The cellobiose and glucose biosensors work at applied potentials of +0.25 and +0.45 V versus Ag|AgCl (3 M KCl), respectively, and can selectively detect their substrate. Both biosensors were used in combination to monitor the hydrolysis of pure cellulose of low crystallinity or industrial corncob samples. The obtained results correlate with the high-performance liquid chromatography pulsed amperometric detection analysis and demonstrate that neither oxygen nor the presence of redox-active compounds from the lignin fraction of the corncob interferes with the measurements.

Comparative characterization of glyoxal oxidase from Phanerochaete chrysosporium expressed at high levels in Pichia pastoris and Trichoderma reesei.

In the secretome of Phanerochaete chrysosporium, a white-rot fungus serving as a model organism to elucidate lignocellulose deconstruction, the copper containing metalloprotein glyoxal oxidase (GLOX) is potentially involved in the crucial production of hydrogen peroxide to fuel and initiate oxidative biomass degradation by lignin-degrading peroxidases. Its ability to oxidize a variety of aldehydes and α-hydroxy carbonyls with the concomitant reduction of dioxygen to hydrogen peroxide has attracted attention for its application as green biocatalyst in different industrial fields. Here we report and compare two efficient processes for the heterologous production of GLOX from P. chrysosporium using the well-established methanolytic yeast Pichia pastoris and the filamentous fungus Trichoderma reesei as expression hosts with subsequent purification by anion exchange and hydrophobic interaction chromatography. Both processes were shown to be suitable for the production of the target protein at high levels. GLOX produced in T. reesei carries mainly Man5 glycosylation while the enzyme produced in P. pastoris exhibits the typical high-mannose type N-glycosylation. The enzyme expressed in P. pastoris showed slightly higher specific activities which correlates with the higher copper loading of 65.5 % compared to 51.9 % for the protein from T. reesei. The pH optimum for both recombinant proteins was 6.0, however, GLOX activity was found to be highly affected by different buffer species. Both enzymes showed very similar substrate affinities and turnover numbers with the highest catalytic efficiency observed for methylglyoxal. GLOX from both expression hosts is therefore a suitable enzyme for further mechanistic characterization and application studies.

Heterologous expression of Phanerochaete chrysosporium cellobiose dehydrogenase in Trichoderma reesei.

BACKGROUND: Cellobiose dehydrogenase from Phanerochaete chrysosporium (PcCDH) is a key enzyme in lignocellulose depolymerization, biosensors and biofuel cells. For these applications, it should retain important molecular and catalytic properties when recombinantly expressed. While homologous expression is time-consuming and the prokaryote Escherichia coli is not suitable for expression of the two-domain flavocytochrome, the yeast Pichia pastoris is hyperglycosylating the enzyme. Fungal expression hosts like Aspergillus niger and Trichoderma reesei were successfully used to express CDH from the ascomycete Corynascus thermophilus. This study describes the expression of basidiomycetes PcCDH in T. reesei (PcCDHTr) and the detailed comparison of its molecular, catalytic and electrochemical properties in comparison with PcCDH expressed by P. chrysosporium and P. pastoris (PcCDHPp). RESULTS: PcCDHTr was recombinantly produced with a yield of 600 U L-1 after 4 days, which is fast compared to the secretion of the enzyme by P. chrysosporium. PcCDHTr and PcCDH were purified to homogeneity by two chromatographic steps. Both enzymes were comparatively characterized in terms of molecular and catalytic properties. The pH optima for electron acceptors are identical for PcCDHTr and PcCDH. The determined FAD cofactor occupancy of 70% for PcCDHTr is higher than for other recombinantly produced CDHs and its catalytic constants are in good accordance with those of PcCDH. Mass spectrometry showed high mannose-type N-glycans on PcCDH, but only single N-acetyl-D-glucosamine additions at the six potential N-glycosylation sites of PcCDHTr, which indicates the presence of an endo-N-acetyl-ß-D-glucosaminidase in the supernatant. CONCLUSIONS: Heterologous production of PcCDHTr is faster and the yield higher than secretion by P. chrysosporium. It also does not need a cellulose-based medium that impedes efficient production and purification of CDH by binding to the polysaccharide. The obtained high uniformity of PcCDHTr glycoforms will be very useful to investigate electron transfer characteristics in biosensors and biofuel cells, which are depending on the spatial restrictions inflicted by high-mannose N-glycan trees. The determined catalytic and electrochemical properties of PcCDHTr are very similar to those of PcCDH and the FAD cofactor occupancy is good, which advocates T. reesei as expression host for engineered PcCDH for biosensors and biofuel cells.

Cellobiose dehydrogenase.

Cellobiose dehydrogenase (CDH) is an extracellular hemoflavoenzyme secreted by fungi to assist lignocellulolytic enzymes in biomass degradation. Its catalytic flavodehydrogenase (DH) domain is a member of the glucose-methanol-choline oxidoreductase family similar to glucose oxidase. The catalytic domain is linked to an N-terminal electron transferring cytochrome (CYT) domain which interacts with lytic polysaccharide monooxygenase (LPMO) in oxidative cellulose and hemicellulose depolymerization. Based on CDH sequence analysis, four phylogenetic classes were defined. CDHs in these classes exhibit different structural and catalytic properties in regard to cellulose binding, substrate specificity, and the pH optima of their catalytic reaction or the interdomain electron transfer between the DH and CYT domain. The structure, reaction mechanism and kinetics of CDHs from Class-I and Class-II have been characterized in detail and recombinant expression allows the application in many areas, such as biosensors, biofuel cells biomass hydrolysis, biosynthetic processes, and the antimicrobial functionalization of surfaces.

Enzymatic assembly of carbon-carbon bonds via iron-catalysed sp3 C-H functionalization.

Although abundant in organic molecules, carbon-hydrogen (C-H) bonds are typically considered unreactive and unavailable for chemical manipulation. Recent advances in C-H functionalization technology have begun to transform this logic, while emphasizing the importance of and challenges associated with selective alkylation at a sp3 carbon1,2. Here we describe iron-based catalysts for the enantio-, regio- and chemoselective intermolecular alkylation of sp3 C-H bonds through carbene C-H insertion. The catalysts, derived from a cytochrome P450 enzyme in which the native cysteine axial ligand has been substituted for serine (cytochrome P411), are fully genetically encoded and produced in bacteria, where they can be tuned by directed evolution for activity and selectivity. That these proteins activate iron, the most abundant transition metal, to perform this chemistry provides a desirable alternative to noble-metal catalysts, which have dominated the field of C-H functionalization1,2. The laboratory-evolved enzymes functionalize diverse substrates containing benzylic, allylic or α-amino C-H bonds with high turnover and excellent selectivity. Furthermore, they have enabled the development of concise routes to several natural products. The use of the native iron-haem cofactor of these enzymes to mediate sp3 C-H alkylation suggests that diverse haem proteins could serve as potential catalysts for this abiological transformation, and will facilitate the development of new enzymatic C-H functionalization reactions for applications in chemistry and synthetic biology.