Cellulose is the principal component of biomass, a renewable source of energy derived from woody plants, agricultural residues, and other similar forms of biological matter. It is a polymer that can be hydrolyzed to yield glucose, and subsequently transformed by fermentation to yield fuels and chemicals. The enzyme cellulase, a biological catalyst composed of several proteins, is required to achieve rapid cellulose hydrolysis; however, use of this catalyst is not practical at present because it is very expensive and no satisfactory method has been developed to recover it from the hydrolysate mixture.
The enzymatic hydrolysis of cellulose could become a more economical process if the enzyme could be recovered from the reaction mixture in active form and reused several times. This could be achieved if an immobilized cellulase were used to hydrolyze lignocellulosic substrates. However, the use of an immobilized enzyme to catalyze the hydrolysis of an insoluble substrate is difficult because effective interaction between enzyme and substrate would be greatly impaired by the enzyme's immobility.
There are, however, some reports describing the use of immobilized cellulase to hydrolyze insoluble cellulose. For example, cellulase has been immobilized in a collagen fibril matrix. Although details on the recovery of activity after immobilization were not reported, the immobilized enzyme in a fluidized bed reactor reportedly hydrolyzed insoluble microcrystalline cellulose, tradename Avicel, manufactured by F.M.C. Corporation of Philadelphia, Pa., (0.55% w/v) circulating through the bed. Greater than 80% hydrolysis of the substrate was achieved after 160 H at 30.degree. C. Furthermore, no leakage of the enzyme was reported to occur, which suggests that intimate association between the immobilized enzyme and insoluble cellulose had to occur, even during constant circulation of substrate.
The cellulase complex of Trichoderma reesei C30 has been immobilized by covalent coupling to cyanogen bromide-activated Sepharose after aminolylation of the carbohydrate side chains of the enzyme with ethylenediamine. However, more than 90% of the Avicelase activity was lost when the cellulase was immobilized by this method. Cellulase from Aspergillus terreus has been attached to controlled pose glass and claimed 72% retention of activity. However, enzyme leakage from the support was the cause of the high activity in this case. Generally, covalently immobilized cellulases are not useful because of their low reactivity towards cellulose.
In the Soviet Union cellulose has been enzymatically converted to glucose in columnar reactor, and released cellulase protein is readsorbed on excess or newly added cellulose. Although the use of cellulose as an adsorbent improved the economy of enzyme utilization, the .beta.-glucosidase component of cellulase is poorly adsorbed on cellulose and the affinity of different endoglucanases from the same and different species differs widely. Therefore key cellulase enzyme components which poorly adsorb on cellulose, but nevertheless are essential for complete hydrolysis of cellulose to glucose, could be lost if cellulose were used as the adsorbent.
Other examples of adsorbents that have been used in an attempt to bind cellulase include Dowex Anion Exchange Resin, Ceramic-SiO.sub.2 ZrO.sub.2 /MgO PSZ (partialy stabilized zirconia), FSZ (fused stabilized zirconia), Macrosorb titania and Macrosorb calcium phosphate; however, no attempts resulted in success. There is no clear way to predetermine which adsorbent may be useful in separating cellulase from a reaction solution. Therefore there is a need to develop a process to recover the cellulase and reuse it in subsequent hydrolyses processes.
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