Supplementary material from "The effect of alkali-soluble lignin on purified core cellulase and hemicellulase activities during hydrolysis of extractive ammonia-pretreated lignocellulosic biomass"

Removing alkali-soluble lignin using extractive ammonia (EA) pretreatment of corn stover (CS) is known to improve biomass conversion efficiency during enzymatic hydrolysis. In this study, we investigated the effect of alkali-soluble lignin on six purified core glycosyl hydrolases and their enzyme synergies, adopting 31 enzyme combinations derived by a five-component simplex centroid model, during EA-CS hydrolysis. Hydrolysis experiment was carried out using EA-CS(−) (approx. 40% lignin removed during EA pretreatment) and EA-CS(+) (where no lignin was extracted). Enzymatic hydrolysis experiments were done at three different enzyme mass loadings (7.5, 15 and 30 mg protein g⁻¹ glucan), using a previously developed high-throughput microplate-based protocol, and the sugar yields of glucose and xylose were detected. The optimal enzyme combinations (based on % protein mass loading) of six core glycosyl hydrolases for EA-CS(−) and EA-CS(+) were determined that gave high sugar conversion. The inhibition of lignin on optimal enzyme ratios was studied, by adding fixed amount of alkali-soluble lignin fractions to EA-CS(−), and pure Avicel, beechwood xylan and evaluating their sugar conversion. The optimal enzyme ratios that gave higher sugar conversion for EA-CS(−) were CBH I: 27.2–28.2%, CBH II: 18.2–22.2%, EG I: 29.2–34.3%, EX: 9.0–14.1%, βX: 7.2–10.2%, βG: 1.0–5.0% (at 7.5–30 mg g⁻¹ protein mass loading). Endoglucanase was inhibited to a greater extent than other core cellulases and xylanases by lignin during enzyme hydrolysis. We also found that alkali-soluble lignin inhibits cellulase more strongly than hemicellulase during the course of enzyme hydrolysis.