Combinatorial optimization of the hybrid cellulase complex structure designed from modular libraries

Abstract

Cellulase selectively recognizes cellulose surfaces and cleaves their β-1,4-glycosidic bonds. Combining hydrolysis using cellulase and fermentation can produce alternative fuels and chemical products. However, anaerobic bacteria produce only low levels of highly active cellulase complexes so-called cellulosomes. Therefore, we designed hybrid cellulase complexes from 49 biotinylated catalytic domain (CD) and 30 biotinylated cellulose-binding domain (CBD) libraries on streptavidin-conjugated nanoparticles to enhance cellulose hydrolysis by mimicking the cellulosome structure. The hybrid cellulase complex, incorporating both native CD and CBD, significantly improved reducing sugar production from cellulose compared to free native modular enzymes. The optimal CBD for each hybrid cellulase complex differed from that of the native enzyme. The most effective hybrid cellulase complex was observed with the combination of CD_6-4 from Thermobifida fusca YX and CBD₄₆ from the Bacillus halodurans C-125. The hybrid cellulase complex/CD_6-4-CBD₄₆ and -CD_6-4-CBD_2-5 combinations showed increased reducing sugar production. Similar results were also observed in microcrystalline cellulose degradation. Furthermore, clustering on nanoparticles enhanced enzyme thermostability. Our results demonstrate that hybrid cellulase complex structures improve enzyme function through synergistic effects and extend the lifespan of the enzyme.

Fig. 1

Hybrid cellulase complex design in this study.

Fig. 2

Producing reducing sugar from phosphoric-acid swollen cellulose (PSC, 3.5 mg mL^–1) in a 50 mM acetate buffer (pH 5.0, 200 mM NaCl) at 45 °C for 96 h in the presence of free CD, a CD and CBD mixture (CD + CBD), and a native modular enzymes (native) CD and CBD cluster on streptavidin-conjugated CdSe nanoparticles (hybrid cellulase complex), made from five module enzymes, (a) CD_5-6, (b) CD_6-4, (c) CD₇, (d) CD₂₆, and (e) CD₄₄. Native CBDs used in the experiments were CBD_1-4, CBD_2-3, CBD_1-5, CBD₁₁ and CBD₄₄, respectively. All experiments were performed at a CD concentration of 40 nM.

Fig. 3

Producing reducing sugar from 3.5 mg mL^–1 of PSC in a 50 mM acetate buffer (pH 5.0, 200 mM NaCl) at 45 °C for 96 h in the presence of (a) CD_5-6, (b) CD_6-4, (c) CD₇, (d) CD₂₆, and (e) CD₄₄, made from modular enzymes, and different CBD clusters on streptavidin-conjugated CdSe nanoparticles (hybrid cellulase complexes). Producing reducing sugar from PSC (3.5 mg mL^-1) in a 50 mM acetate buffer (pH 5.0, 200 mM NaCl) at 45 °C for 96 h in the presence of (f) CD_8-2, (g) CD_9-3, (h) CD₇₄, and (i) CD₁₂₄, made from non-module enzymes, and different CBD clusters on hybrid cellulase complexes. All experiments were performed at a CD concentration of 40 nM. The free CD was used as a reference. N and NC show native enzymes and native clustering enzymes, respectively.

Fig. 4

The reducing sugar production from PSC (3.5 mg mL⁻¹) in a 50 mM acetate buffer (pH 5.0, 200 mM NaCl) at 45 °C at 96 h with (a) CD_6-4 (CD), a native module structure CD_6-4-CBD_2-3 (N) or hybrid cellulase complexes with native CBD (NC). (b) Reducing sugar production using a hybrid cellulase complexes with different CBDs (blue bar), (c) CD_6-4-CBD₄₆ and CD_6-4-CBD_x clusters on separate streptavidin-conjugated CdSe nanoparticles mixed at an equal molar ratio (1:1) (red bar), and with (d) CD_6-4-CBM₄₆, CD_6-4-CBM_2-5, and another CD_6-4-CBD_x cluster on separate streptavidin-conjugated CdSe nanoparticles (green bar) mixed at an equal molar ratio (1:1:1). All experiments were performed at a total CD concentration of 40 nM, and the CD/CBM ratios were 7:23. Each experiment was conducted thrice, and the average values are plotted with error bars representing standard variations.

Fig. 5

Synergistic effect of the reducing sugar production from PSC (3.5 mg mL⁻¹) in a 50 mM acetate buffer (pH 5.0, 200 mM NaCl) at 45 °C for 96 h on CD_6-4-CBD₄₆ and CD_6-4-CBD_2-5 clusters on separate streptavidin-conjugated CdSe nanoparticles. All the experiments were performed at a CD concentration of 40 nM, and the CD/CBM ratios were 7:23. Each experiment was conducted thrice, and the average values are plotted with error bars representing standard variations.

Fig. 6

Producing reducing sugar from Avicel (10 mg mL^-1) in a 50 mM acetate buffer (pH 5.0, 200 mM NaCl) at 45 °C for 96 h in the presence of (a) free CD_6-4 (open black circles), native module enzyme (closed black circles), native clustering enzymes (open black square) (used as references). (b) Hybrid cellulase complexes with CD_6-4 and different CBDs indicating the top five producing reducing sugar from PSC (blue symbols) of (c) two types (red symbols) and (d) three types (green symbols). All experiments were performed at a CD concentration of 2.5 μM.

Fig. 7

Producing reducing sugar from 10 mg mL⁻¹ Avicel in 50 mM sodium acetate (pH 5.0, 200 mM NaCl) at 45 °C for 96 h by equally mixing the CD_6-4-CBD₄₆ and CD_6-4-CBD_2-5 cluster (40 nM CD) (a) and 1, 2.5, 5, and 10 μM of the native modular enzymes estimated using the native module enzyme dosage corresponding to the hybrid cellulase complexes (2.5 μM) from the standard curve of the native module enzyme amount at 96 h (b).

Fig. 8

Thermal stability of clustered enzymes (normal line) and free (dotted line) at 50 °C (blue) and 70 °C (red). The enzymes (2.5 μM) were maintained at both temperatures without the substrate for 3 days in acetate buffer at pH 5.0. The activity determined before incubation was at 100%. Produced reducing sugars were measured at an enzyme concentration of 40 nM. The actual reducing sugar concentrations produced by these hybrid cellulase complex solutions at 0 h were 0.57 mg ml-1 at 50 ℃ at without nanoparticles, 1.96 mg ml-1 at 50 ℃ with nanoparticles, 0.55 mg ml-1 at 70 ℃ without nanoparticles, and 1.87 mg ml-1 at 70 ℃ with nanoparticles.