Strategic optimization of xylanase–mannanase combi-CLEAs for synergistic and efficient hydrolysis of complex lignocellulosic substrates

• Precipitant selection should be based on the activity retained after cross-linking.
• CLEAs exhibit high thermostability and operational stability.
• Synergy between bacterial xylanase and mannanase improves hydrolysis.
• Combi-CLEAs highly effective in hydrolysis of lignocellulose substrates.
• Accessory enzymes pivotal for increasing concentration of monomeric sugars.

Cost-effective application of lignocellulolytic enzymes holds the key towards commercialization of enzymatic hydrolysis of lignocellulosic biomass. Carrier free immobilization of enzyme(s) offers a lucrative prospect. Combined-cross linked enzyme aggregates (combi-CLEAs) are a novel prospective and this present study addresses the preparation, characterization and application of xylanase–mannanase combi-CLEAS on lime-preteated sugarcane bagasse and milled corn stover. X6-CLEAs, X7-CLEAs, L1-CLEAs and L7-CLEAs were prepared after elaborative optimization of the precipitating agent and glutaraldehyde concentration. The highest activity after precipitation was observed with acetone but following cross-linking with glutaraldehyde less than 60% activity was retained, while more than 60% activity was retained after precipitation with ammonium sulphate and cross-linking with glutaraldehyde. Accessory enzyme activities including α-arabinofuranosidase, β-xylosidase, esterases, β-mannosidase, α-galactosidase and β-glucosidase were also determined. More than an 1.5 fold increase in thermostability compared to the free enzyme was observed over a broad temperature range (50–70 °C). Tri-synergy studies and quad synergy studies were used to generate combi-CLEAs with different protein ratios. Hydrolysis of lime pre-treated bagasse with combi-CLEAs at protein ratios corresponding to X6 (33.0%):X7 (17.0%):L1 (17.0%):L7 (33.0%) resulted in a 1.68 fold higher sugar release compared to the quad synergy model using free enzymes. Similarly, hydrolysis of corn stover with combi-CLEAs at protein ratios corresponding to X6 (40.0%):X7 (10.0%):L1 (10.0%):L7 (40.0%) resulted in an 1.58 fold higher sugar release compared to the sugar release observed with the quad synergy model using free enzymes. Monomeric sugars constituted 70–75% of reducing sugars released during hydrolysis. The role of accessory enzymes in improving enzyme synergy was clearly shown. The efficiency of combi-CLEAs compared to free enzymes makes them ideal candidates for the prudent and cost-effective commercialization of lignocellulolytic enzymes.

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