Carrier-free co-immobilization of xylanase, cellulase and beta-1,3-glucanase as combined cross-linked enzyme aggregates (combi-CLEAs) for one-pot saccharification of sugarcane bagasse

Combined cross-linked enzyme aggregates (combi-CLEAs) are an innovative prospect and a lucrative technology. The present study addresses the preparation, characterization and application of combi-CLEAs with xylanase, cellulase and beta-1,3-glucanase to achieve one-pot bioconversion of lignocellulosic biomass to fermentable sugars. A three-phase partitioning (TPP) method was used to aggregate the enzymes. Glutaraldehyde (100 mM) was employed as a cross-linker with the cross-linking time of 7.5 h. Scanning electron microscopy of the tri-enzyme biocatalyst has a coarse-grained appearance. Combi-CLEAs were more thermally stable, retaining about 70% of their initial activity at 70 degrees C compared to 30% for the free enzyme. The storage stability of combi-CLEAs was more than 97% of their activity after incubation for 11 weeks at 4 degrees C, whereas the free enzymes retained about 65% of initial activity. The residual activity of combi-CLEAs remained constant at 90% until the sixth cycle. Contrary to free enzymes that remain in the hydrolysate, which prevents their recovery, reuse of combi-CLEAs was possible. Free enzymes hydrolyze the ammonia cooked sugarcane bagasse at about 73%, whereas the combi-CLEAs resulted in maximum hydrolysis of about 83.5% in 48 h.