Design of smart hydrogels for use as support matrices for immobilisation of cellulases in saccharification of lignocellulose

Abstract

Smart hydrogels could facilitate immobilisation of cellulases to allow recovery and decrease enzyme cost in the biofuel industry, as they have a soluble-gel transition. The aim of the study was to design and evaluate the use of smart hydrogels for immobilisation of cellulase system that can be recovered after hydrolysis of cellulosic biomass. Cellulases from Aspergillus niger FGSC A733 produced under solid state fermentation and commercial cellulases were used in immobilisation. Various support matrices prepared were poly-N-isopropylacrylamide (p-NIPAAm), poly-N isopropylacrylamide-co-Methacrylic acid (p-NIPAAm-co-MAA) and supermacroporous poly-crosslinked-Acrylamide-co-N,N’-Methylenebisacrylamide (p-crosslinked-AA-co MBA). Cellulases were coupled onto the support matrices by covalent attachment method through reactive groups of N-acryloxysuccinimide (NAS) or Methacrylic acid N-hydroxysuccinimide (NMS). The low critical solution temperature (LCST) of formed p-NIPAAm-co-MAA copolymer was determined by the inflection point method. The shrinking and swelling kinetics and pH sensitivity of p-NIPAAm-co-MAA copolymer and conjugates were characterised using a cloud point method. Hydrolysis of CMC using cellulase-microbeads-p-NIPAAm and cellulase-crosslinked-p-NIPAAm with different percentage gel showed activity trend of 0.05>1>10>5>0.1% and 5>2>10% respectively. HPLC analysis showed that supplementation of β-glucosidase in cellulase-crosslinked-p-NIPAAm conjugates increased glucose by 12 and 14-fold at 30 and 50 °C respectively in the avicel hydrolysate in comparison with no β glucosidase supplementation. In the hydrolysis of avicel using cellulase-crosslinked p-NIPAAm-co-MAA conjugate a total of 13.6 g/L of reducing sugar was liberated after three cycles. In comparison a total of 21.4 g/L of reducing sugars were released from avicel hydrolysis using cellulase-crosslinked-p-AA-co-MBA conjugate after 3 cycles. In contrast, reducing sugars released in thatch grass hydrolysis using free enzyme were 8 times greater than in cellulase-crosslinked-p-AA-co-MBA conjugate. Cellulase crosslinked-p-NIPAAm-co-MAA conjugates were more stable than free enzyme at 50 and 60 °C after 24 hour and 120 minutes of incubation respectively, but lost activities at 65 °C after 120 minute. Therefore the activity loss in the immobilised enzymes was more due to thermal inactivation during precipitation and recovery than incomplete recovery during precipitation cycles. The results show that cellulases immobilised on smart polymers with sol-gel transition could be used in hydrolysis of cellulose due to ease of recovery. Hydrolysis kinetics was efficient for both immobilised enzyme system (cellulase-crosslinked-p-AA-co-MBA and cellulase-crosslinked-p-NIPAAm-co MAA conjugate) since were re-used in hydrolysis of avicel. Therefore the use of these smart polymers for cellulase immobilisation can contribute in cost reduction of the enzymatic hydrolysis process in the biofuel industry.