MOESM1 of Chaetomella raphigera β-glucosidase D2-BGL has intriguing structural features and a high substrate affinity that renders it an efficient cellulase supplement for lignocellulosic biomass hydrolysis
Creators: name: Mu-Rong Kao
name: Hsion-Wen Kuo
name: Cheng-Chung Lee
name: Kuan-Ying Huang
name: Ting-Yen Huang
name: Li, Chen-Wei
name: C. Chen
name: Wang, Andrew
name: Su-May Yu
name: Tuan-Hua Ho
Publisher: name: figshare
Year: 2019
Subjects: subject: Biophysics
subject: Biochemistry
subject: Space Science
subject: Medicine
subject: Biotechnology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: FOS: Chemical sciences
subject: 69999 Biological Sciences not elsewhere classified
subject: FOS: Biological sciences
subject: FOS: Biological sciences

description: Additional file 1: Figure S1. Phylogenetic analysis of microbial GH3 β-glucosidases. The closest sequence to D2-BGL, i.e. B. cinerea GH3 protein, exhibits only 72% sequence similarity to D2-BGL. Among filamentous fungi, Ascomycota phylum is highlighted in yellow and Basidiomycota phylum is highlighted in orange. Table S1. Accession number of GH3 β-glucosidases mentioned in the phylogenetic tree. Table S2. Summary of D2-BGL sequence modifications and their effect on recombinant protein productivity and purification. T1572C: silent mutation. M1-9: codon modification from CGC to AGA at positions M1:364–366, M2:496–498, M3:838-840, M4:859–861, M5:979–981, M6:1114–1116, M7:1360–1362, M8:1951–1953 and M9:2011–2013. Table S3. Activity of native D2-BGL and P. pastoris-expressed D2-BGL toward different substrates. Table S4. Purification tables of D2-BGL and Novozyme 188 (N188). Figure S2. Purification of D2-BGL by affinity chromatography. (a) The chromatograph shows that the major part of recombinant D2-BGL was eluted with 30% of elution buffer. b) SDS-PAGE analysis suggests that most D2-BGL is found in the second peak. Figure S3. Sequential purification of N188. (a) Partially purified N188 was found in elution fractions 21 to 23 after anion-exchange chromatography. (b) After site-exclusion chromatography, purified N188 was collected from elution fractions 18 and 19. Figure S4. Kinetics of D2-BGL and N188. Determination of Km and Vmax using cellobiose (a and b) or pNPG (c and d) as substrates, and determination of inhibition constant Ki using pNPG as substrate for D2-BGL (e) and for N188 (f). Table S5. Cellulase activities of different enzyme mixtures used during the experiment of biomass saccharification. Table S6. Cellulase activities in enzyme mixtures used for sugarcane bagasse saccharification. Figure S5. Variation of glycerol and ammonia concentrations during fermentation in a one-ton bioreactor. Table S7. D2-BGL crystallographic data collection and refinement statistics. Figure S6. PNGase F cannot remove N-glycans without denaturation at 100 °C. Line 1: D2-BGL with PNGase F, line 2: D2-BGL without PNGase F. Figure S7. Mutations at N255 and G257 cause loss of enzyme activity. (a) Among all amino acid residues involved in the active site pocket, N255, F256 and G257 differ between D2-BGL and β-glucosidases from Aspergillus, resulting in D2-BGL having a shorter loop. (b) Point mutation at position N255 (mutants N255D and N255S) or G257 (mutant G257D) reduces the cellobiase activity of D2-BGL.