scholarly journals Exploring the Synergy between Cellobiose Dehydrogenase from Phanerochaete chrysosporium and Cellulase from Trichoderma reesei

2016 ◽  
Vol 7 ◽  
Author(s):  
Min Wang ◽  
Xuefeng Lu
Keyword(s):  
Trichoderma Reesei ◽  
Phanerochaete Chrysosporium ◽  
Cellobiose Dehydrogenase

scholarly journals Heterologous Expression of Phanerochaete Chrysosporium Cellobiose Dehydrogenase in Trichoderma Reesei

2020 ◽  
Author(s):  
Lena Wohlschlager ◽  
Florian Csarman ◽  
Hucheng Chang ◽  
Elisabeth Fitz ◽  
Bernhard Seiboth ◽  
...  
Keyword(s):  
Electrochemical Properties ◽  
Trichoderma Reesei ◽  
Phanerochaete Chrysosporium ◽  
Catalytic Properties ◽  
Detailed Comparison ◽  
Biofuel Cells ◽  
Cellobiose Dehydrogenase ◽  
Efficient Production ◽  
Homologous Expression ◽  
High Mannose

Abstract 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 basidiomycetous 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-b-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.

scholarly journals Heterologous expression of Phanerochaete chrysosporium cellobiose dehydrogenase in Trichoderma reesei

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Lena Wohlschlager ◽  
Florian Csarman ◽  
Hucheng Chang ◽  
Elisabeth Fitz ◽  
Bernhard Seiboth ◽  
...  
Keyword(s):  
Electrochemical Properties ◽  
Trichoderma Reesei ◽  
Phanerochaete Chrysosporium ◽  
Catalytic Properties ◽  
Detailed Comparison ◽  
Biofuel Cells ◽  
Cellobiose Dehydrogenase ◽  
Efficient Production ◽  
Homologous Expression ◽  
High Mannose

Abstract 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-β-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.

Substrate specificity of cellobiose dehydrogenase from Phanerochaete chrysosporium

1998 ◽  
Vol 1383 (1) ◽  
pp. 48-54 ◽  
Author(s):  
Gunnar Henriksson ◽  
Veljo Sild ◽  
István J Szabó ◽  
Göran Pettersson ◽  
Gunnar Johansson
Keyword(s):  
Substrate Specificity ◽  
Phanerochaete Chrysosporium ◽  
Cellobiose Dehydrogenase

The 1,4-β-d-glucan cellobiohydrolases from Phanerochaete chrysosporium. I. A system of synergistically acting enzymes homologous to Trichoderma reesei

1991 ◽  
Vol 19 (2-3) ◽  
pp. 271-285 ◽  
Author(s):  
Edgar Uzcategui ◽  
Alberto Ruiz ◽  
Raquel Montesino ◽  
Gunnar Johansson ◽  
Göran Pettersson
Keyword(s):  
Trichoderma Reesei ◽  
Phanerochaete Chrysosporium

scholarly journals Expression, purification and characterization of cellobiose dehydrogenase mutants from Phanerochaete chrysosporium in Pichia pastoris KM71H strain

2020 ◽  
Vol 85 (1) ◽  
pp. 25-35
Author(s):  
Ana Balaz ◽  
Marija Blazic ◽  
Nikolina Popovic ◽  
Olivera Prodanovic ◽  
Raluca Ostafe ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Phanerochaete Chrysosporium ◽  
Sodium Dodecyl ◽  
Cellobiose Dehydrogenase ◽  
Yeast Cells ◽  
Soluble Form ◽  
Kinetic Characterization ◽  
Methanol Induction ◽  
Ph Optimum ◽  
Mutant Proteins

Production of soluble cellobiose dehydrogenase (CDH) mutant proteins previously evolved on the surface of S. cerevisiae yeast cells was established for use in biosensors and biofuel cells. For this purpose, mutant cdh genes tm (D20N, A64T, V592M), H5 (D20N, V22A, A64T, V592M) and H9 (D20N, A64T, T84A, A261P, V592M, E674G, N715S) were cloned to pPICZ? plasmid and transformed into Pichia pastoris KM71H strain for high expression in a soluble form and kinetic characterization. After 6 days of expression under methanol induction, the CDHs were purified by ultrafiltration, ion- -exchange chromatography and gel filtration. Sodium dodecyl sulfate electrophoresis confirmed the purity and presence of a single protein band at a molecular weight of 100 kDa. Kinetic characterization showed that the H5 mutant had the highest catalytic constant of 43.5 s-1 for lactose, while the mutant H9 showed the highest specificity constant for lactose of 132 mM-1 s-1. All three mutant proteins did not change the pH optimum that was between 4.5 and 5.5. Compared to the previously obtained wild types and mutants of CDH from Phanerochaete chrysosporium, the variants reported in this article had higher activity and specificity that together with high protein expression rate in P. pastoris, makes them good candidates for use in biotechnology for lactobionic acid production and biosensor manufacture.

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