Abstract
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.
Highlights
Lignocellulosic biomass obtained from agricultural plant residues (e.g. corn stover (CS), wheat straw, sweet sorghum), dedicated energy crops and woody forest residues are considered as sustainable feedstocks to produce fuels and chemicals in a biorefinery [1,2]
Results of glucose yields for 31 enzyme combinations were further analysed by Minitab to predict the optimal enzyme ratios of five core enzymes, namely CBH I, CBH II, EG I, EX and βX, for both extractive ammonia (EA)-CS(−) and EA-CS(+), at three different enzyme mass loadings
In order to further understand the inhibitory role of alkali-soluble lignin on core cellulases and core hemicellulases at optimal enzyme ratios, we designed another set of experiments using pure Avicel and beechwood xylan and added water-insoluble, ethanol-soluble lignin fraction (WIL) isolated by EA extraction to the substrate before adding the enzyme cocktail [14]
Summary
Lignocellulosic biomass obtained from agricultural plant residues (e.g. corn stover (CS), wheat straw, sweet sorghum), dedicated energy crops (e.g. switchgrass, miscanthus, energy cane, shortrotation willow) and woody forest residues are considered as sustainable feedstocks to produce fuels and chemicals in a biorefinery [1,2]. Lignocellulosic biomass comprises two major sugar polymers, namely cellulose (a homopolysaccharide comprised of D-glucose units linked together by β-1,4-glucosidic bonds), with a degree of polymerization of 10 000 or higher, and hemicellulose (different heteropolysaccharides containing different combinations of D-glucose, D-galactose, Dmannose, D-xylose, L-arabinose, D-glucuronic acid and 4-O-methyl-D-glucuronic acid), with a degree of polymerization below 200. We carried out hydrolysis using pure substrates such as Avicel and beechwood xylan by varying the composition of purified enzyme cocktails in the presence and absence of lignin Experimental results from these studies have helped us understand which classes of enzymes lose activity most rapidly during hydrolysis when alkali-soluble lignin is present in the substrate. Fundamental understanding of enzyme activity in the presence of lignin will help to develop more stable enzymes to improve the efficiency of sugar conversion and reduce the cost of producing biofuels
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