Journal: The Febs Journal

Article Title: A modular enzyme with combined hemicellulose‐removing and LPMO activity increases cellulose accessibility in softwood

doi: 10.1111/febs.17250

Figure Lengend Snippet: Thermostability and thermal unfolding of Cc LPMO10‐Man5. Panel (A) shows temperature‐dependent activity of Cc LPMO10‐Man5. Oxidized products generated from 0.2% (w/v) PASC were quantified by HPAEC‐PAD at four time points for reactions incubated at six different temperatures, ranging from 30 °C to 80 °C. All reactions were carried out in 50 m m sodium phosphate buffer (pH 6.0) with 1 μ m LPMO and 1 m m ascorbic acid. For product quantification, solubilized cello‐oligosaccharides were first hydrolyzed with Tf Cel6A, yielding oxidized products with a DP of 2 and 3 [GlcGlc1A, Glc2Glc1A], the quantities of which were summed up to yield the concentration of oxidized products. The error bars show ±SD ( n = 3). Note that product formation over time is affected by enzyme inactivation, as explained in the main text. The plot in (B) shows the melting curve and apparent melting temperatures ( T m(app) ) for copper‐saturated Cc LPMO10‐Man5. The derivative of the fluorescence signal (−dRFU/dT, where “RFU” stands for “relative fluorescence units”) is plotted as a function of the temperature. The reaction contained 0.1 g·L −1 protein and was heated from 25 °C to 95 °C, at a rate of 1.5 °C·min −1 , in the presence of SYPRO orange (a fluorescent dye). The scan was performed four times and the figure shows a typical scan. Both apparent T m values had standard deviations below ±0.4 °C.

Article Snippet: Each binding reaction contained 1% (w/v) substrate and 4 μ m protein (i.e. Cc CBM3‐a or Cc CBM3‐b) in 50 m m sodium phosphate buffer (pH 6.0) and was carried out at 22 °C in an Eppendorf Thermomixer set to 1000 r.p.m.

Techniques: Activity Assay, Generated, Incubation, Concentration Assay, Fluorescence

Journal: The Febs Journal

Article Title: A modular enzyme with combined hemicellulose‐removing and LPMO activity increases cellulose accessibility in softwood

doi: 10.1111/febs.17250

Figure Lengend Snippet: Degree of synergy between different versions of Cc LPMO10‐Man5 and three different cellulases in reactions with Avicel or spruce wood. Different combinations of the Cc LPMO10‐Man5 versions and three cellulases (mgCel48A, mgCel6A, and mgCel6B) were incubated at 60 °C, 800 r.p.m. with 1% (w/v) Avicel (A) or ball‐milled spruce wood (B) in 50 m m sodium phosphate buffer (pH 6.0). The enzyme concentration was 0.5 μ m for each and 1 m m ascorbic acid was added to all reactions in panel (A). The degree of synergy and error bars (representing the propagated error) were calculated based on data shown in Figs and . The horizontal red line indicates a degree of synergy of 1, where a degree of synergy above 1 is indicative of synergism between two enzymes. Note that some of the Caldibacillus cellulovorans enzyme variants alone had no activity on some of the substrates, either because mannan is not present (A) or because mannan protects the cellulose from enzymatic attack (B); these instances are indicated by a red asterisk. The error bars show ±SD ( n = 3).

Article Snippet: Each binding reaction contained 1% (w/v) substrate and 4 μ m protein (i.e. Cc CBM3‐a or Cc CBM3‐b) in 50 m m sodium phosphate buffer (pH 6.0) and was carried out at 22 °C in an Eppendorf Thermomixer set to 1000 r.p.m.

Techniques: Incubation, Concentration Assay, Activity Assay

Journal: The Febs Journal

Article Title: A modular enzyme with combined hemicellulose‐removing and LPMO activity increases cellulose accessibility in softwood

doi: 10.1111/febs.17250

Figure Lengend Snippet: Binding of the individual CBM3s of Cc LPMO10‐Man5 to Avicel or ball‐milled spruce wood. (A) Binding to Avicel shown as reduction in the concentration of soluble protein over time (measured by A 280 ). (B) SDS/PAGE analysis of Cc CBM3‐a and Cc CBM3‐b binding to Avicel and spruce wood. The binding reactions contained 1% (w/v) substrate and 4 μ m CBM in 50 m m sodium phosphate buffer (pH 6.0) and were carried out at 22 °C in an Eppendorf thermomixer set to 1000 r.p.m. The incubation time for the experiment shown in (B) was 60 min. The error bars in panel (A) show ±SD ( n = 3). The experiment in panel (B) was done only once ( n = 1).

Article Snippet: Each binding reaction contained 1% (w/v) substrate and 4 μ m protein (i.e. Cc CBM3‐a or Cc CBM3‐b) in 50 m m sodium phosphate buffer (pH 6.0) and was carried out at 22 °C in an Eppendorf Thermomixer set to 1000 r.p.m.

Techniques: Binding Assay, Concentration Assay, SDS Page, Incubation

Journal: The Febs Journal

Article Title: A modular enzyme with combined hemicellulose‐removing and LPMO activity increases cellulose accessibility in softwood

doi: 10.1111/febs.17250

Figure Lengend Snippet: Progress curves for synergy experiments with spruce wood and degree of synergy focusing on mannan degradation. Panel (A) shows mannan degradation in reactions with Cc LPMO10‐Man5 or Cc Man5‐CBM3‐b alone and combined with one of three cellulases: mgCel48A, mgCel6A and mgCel6B, in 50 m m sodium phosphate buffer (pH 6.0) containing 1% (w/v) spruce wood. The reactions were incubated at 60 °C and 800 r.p.m. The enzyme load was 0.5 μ m of each and no reductant was added to the reactions. Samples were withdrawn at different timepoints and filtrated and further diluted in sodium hydroxide (0.1 m final concentration) before HPAEC‐PAD analysis. Reactions with cellulases alone or Cc LPMO10‐Man5 variants without the Man5 domain did not yield any mannan‐derived products and are not shown. Panel (B) shows the degree of synergy, with error bars representing the propagated error, calculated based on data shown in panel (A). The horizontal red line indicates a degree of synergy of 1, where a degree of synergy above 1 is indicative of synergism between two enzymes. The error bars show ±SD ( n = 3).

Article Snippet: Each binding reaction contained 1% (w/v) substrate and 4 μ m protein (i.e. Cc CBM3‐a or Cc CBM3‐b) in 50 m m sodium phosphate buffer (pH 6.0) and was carried out at 22 °C in an Eppendorf Thermomixer set to 1000 r.p.m.

Techniques: Incubation, Concentration Assay, Derivative Assay

Journal: The Febs Journal

Article Title: A modular enzyme with combined hemicellulose‐removing and LPMO activity increases cellulose accessibility in softwood

doi: 10.1111/febs.17250

Figure Lengend Snippet: Progress curves for the generation of soluble oxidized cello‐oligomers from spruce wood with varying enzyme variants or their combinations. 1% spruce wood was incubated for up to 24 h with 1 μ m Cc LPMO10‐Man5, 1 μ m Cc LPMO10‐CBM3‐a, or 1 μ m Cc LPMO10‐CBM3‐a combined with 1 μ m Cc Man5‐CBM3‐b. All reactions were carried out in 50 m m sodium phosphate buffer pH 7.0, at 50 °C in absence of an external electron donor. Solubilized products were separated from the insoluble fraction by filtration and further hydrolyzed by Tf Cel6A (0.5 μ m ) yielding oxidized products with a degree of polymerization of 2 and 3 (GlcGlc1A, Glc 2 Glc1A), which were quantified to yield the concentration of oxidized products. The error bars show ±SD ( n = 3). Since product levels were low and differences modest, this complete experiment was repeated two times, with essentially identical results.

Article Snippet: Each binding reaction contained 1% (w/v) substrate and 4 μ m protein (i.e. Cc CBM3‐a or Cc CBM3‐b) in 50 m m sodium phosphate buffer (pH 6.0) and was carried out at 22 °C in an Eppendorf Thermomixer set to 1000 r.p.m.

Techniques: Incubation, Filtration, Concentration Assay