*4.8. Purification of the C. Thermocellum Secretome*

*C. thermocellum* DSM1313 was grown on GS-2 medium (0.5 g/L K2HPO4, 0.5 g/L MgCl2·6H2O, 0.5 g/L KH2PO4, 1.3 g/L (NH4)2SO4, 0.002 g/L resazurin, 10.5 g/L MOPS buffer, 5 g/L yeast extract, 1.25 mg/L iron(II) sulfate and 0.5 mM CaCl2, adjusted with 10 M NaOH to a final pH of 7.2) with 0.5% microcrystalline cellulose (Avicel, Sigma Aldrich, St. Louis, MO, USA) in batch culture. Nitrogen flushing was used to achieve anaerobic conditions. After 48 h of incubation at 60 ◦C, growth medium was centrifuged (10,808× *g*, 10 min). Soluble proteins were precipitate by 80% ammonium sulfate and re-suspended in Tris-buffered saline (TBS) buffer, pH 7.4. Protein concentration was measured by Bradford assay, using Bio-Rad protein assay solution (Bio-Rad) [54].

### *4.9. Cellulose Hydrolysis Assay*

A quantity of 0.6 mg/mL of *C. thermocellum* secretome solution was applied to a suspension of 250 mg/mL of microcrystalline cellulose in 20 mM citrate buffer, pH 6.1, with or without the addition of 2 μg/mL BglA (wild-type or mutant) in a reaction volume of 2 mL. Samples were incubated at 60 and 70 ◦C for 48 h, and centrifuged (16,100× *g*, 5 min). Released soluble sugars were analyzed by high-pressure liquid chromatography (HPLC, Agilent Infinity 1260 system, Agilent Technologies, Santa Clara, CA, USA) using an Aminex®HPX-87H Ion Exclusion column (Bio-Rad, Hercules, CA, USA) with a guard column, mobile phase of 5 mM H2SO4 (flow-through of 0.6 mL/min at 45 ◦C) in an Agilent 1260 Infinity LC system with RID detector (G1362A). Experiments were performed in triplicate.
