Biodegradation Of Cellulose In Novel Recyclable Aqueous Two-Phase Systems With Water-Soluble Immobilized Cellulase

Cellulosic material is one of the most abundant renewable natural resource on the earth which can be converted into glucose and soluble sugars by chemical or enzymatic process. The production of biofuels from cellulosic feedstocks has much more economic and environmental advantages than traditional fossil fuels, so scientists pay more and more attention on these green routes to obtain ethanol. However, cellulase is expensive, and it is difficult to be recycled. Free cellulase biodegradation of cellulose has been difficult for large-scale industrial production, and the application of immobilized cellulase technology successfully solves this problem successfully. In the experiment, cellulase was immobilized on reversibly soluble-insoluble polymer PMDB3.1by using1-Ethyl-3-(3-dimethyllaminopropyl) carbodiimide as cross-linker. PMDB3.1was synthesized with Methacrylic acid (MAA),2-Dimethylamino ethyl methacrylate (DMAEMA) and Butyl methacrylate (BMA) as monomers. This kind of support is soluble during the catalyzing reaction and insoluble by adjusting the pH to its isoelectric point (3.1) to recover the enzyme easily. The recovery of PMDB3.1was95.6%. When the cellulase content was2.4%(v/v)(containing150mg protein), EDC content was300mg/g polymer PMDB3.1, solution pH was6, immobilization reaction time was4hours, the reserved activity of immobilized cellulase could reach81.2%.Aqueous two-phase systems have many advantages over conventional extraction using organic solvent since the bulk of both phases consist of water. ATPS form a gentle environment for biomaterials. The interfacial tension is extremely low (0.0001-0.1dyne/cm) compared with that of water-organic solvent systems (1-20dyne/cm). The simplicity, biocompatibility, and easy scaleup operations make the use of ATPS very attractive for large-scale bioseparations. Unfortunately, recoveries of traditional phase-forming copolymers can not be achieved to result in high cost and environmental pollution. Recently, scientists have attempted to look for new phase-forming copolymers that can be recycled by changing pH, temperature, ionic strength and so on. Biodegradation of cellulose in recyclable ATPS has obvious advantages. On one hand, phase-forming polymers and cellulase could be recycled at a very low cost, and it will decrease the industrial cost and environmental pollution. On the other hand, phase transfer bioconversion of cellulose has been carried out in this novel ATPS and product inhibition and substrate inhibition will be removed. In the experiment, thermo-pH recyclable aqueous two-phase systems composed by pH-response copolymer PMDB3.1and thermo-response copolymer PNB. In PNB/PMDB3.1aqueous two-phase systems, PNB was partitioning to the top phase and PMDB3.1was partitioning to the bottom phase. The optimized partition coefficient of glucose was3.68in the presence of50mM KCl. Insoluble substrate and immobilized cellulase was biased to bottom phase, while product was partitioned to top phase. Microcrystalline cellulose was catalyzed into reducing sugar, then the product entering into the top phase. In the end, inhibition of product was removed, and the yield of reducing sugar in ATPS was increased10.94%compared with the reaction in the single aqueous phase. The saccharification in ATPS could reach40.16%when the reaction reached equilibrium.In this study, cellulose biodegradation reaction was carried out in pH-pH recyclable aqueous two-phase systems composed by two pH-response copolymers PADB38and PMDB3.1-Copolymer PADB3.8was synthesized with Acrylic Acid (AA),2-Dimethylamino ethyl methacrylate (DMAEMA) and Butyl Methacrylate (BMA) as monomers (AA:DMAEMA: BMA=23:7:1). In PADB3.8/PMDB3.1ATPS, cellulase was immobilized on pH-response copolymer PMDB3.1by using1-Ethyl-3-(3-dimethyllaminopropyl)-carbodiimide hydrochloride (EDC) as cross-linker. Optimized partition coefficient of product in PADB3.8/PMDB3.1ATPS was2.45, in the presence of40mM (NH4)2SO4. Insoluble substrate and immobilized enzyme were biased to the bottom phase, while the product was partitioned to the top phase. Microcrystalline cellulose was catalyzed into reducing sugar, and the product entered into the top phase. The yield of reducing sugar in ATPS could reach70.57%at the initial substrate concentration of0.5%(w/v),9.3%higher than that in the single aqueous phase. Saccharification yield could reach66.15%after immobilized cellulase was recycled five times in ATPS.Cellulose saccharification is catalyzed by complex cellulase systems mainly including cellobiohydrolases (EC3.2.1.91), endoglucanases (EC3.2.1.4) and β-glucosidases (EC3.2.1.21). However, the content of β-glucosidases in cellulase systems is always very low. In the experiment, β-glucosidases was supplemented into cellulase systems, and the optimum volume ratio of cellulase systems and β-glucosidases (V Celluclast1.5L FG:Vβ-Glucosidases) immobilized on polymer PMDB3.1was10:1. In PADB3.8/PMDB3.1ATPS, the concentration of glucose was4.331mg/ml after the reaction reached equilibrium (108hours). In PADB3.8/PMDB3.1ATPS, yield of glucose after108hours decreased to50.25%after reusing immobilized cellulase systems and glucosidases (V Celluclast1.51. FG=Vβ-Glucosidases=10:1) five times.