Immobilization of Cellulases
As the cost of cellulase is a major factor, many researchers have fancied the idea of totally reusable cellulase, which can drastically cut the cost of cellulosic ethanol production. Immobilization of catalysts is a well-established technology in the chemical catalysis field that will allow facile recycling of catalysts. Consequently, enzyme immobilization techniques have been investigated by many groups working in this field [47-49]. In the immobilization of cellulases, pH-sensitive polyacrylate amphiphilic polymers [50], polyaniline coated polymer microspheres [51], nanofibrous PVA membranes [52], electrospun polyacrylonitrile (PAN) nanofibrous membranes [53], acrylamide grafted acrylonitrile copolymer membranes [54], and bromomethylated poly (2,6-dimethyl-1,4-phenyl — ene oxide) hollow fiber cation-exchange membranes [55] have been tested in recent years. Enzymes immobilized on soluble carriers are able to hydrolyze insoluble cellulosic substrates more efficiently; however the enzyme is difficult to be reused. Whereas enzymes immobilized on insoluble carriers are easy to recover and reuse, but enzyme catalysis efficiency is reduced due to diffusion controlled mass transfer and steric hindrance in biphasic systems with water insoluble substrates, and low geometrical congruence with protein surfaces. Therefore, immobilization of enzyme on reversibly soluble-insoluble polymeric carriers have been developed to overcome these problems [56, 57]. These smart enzyme carrying polymers can easily be dissolved and recovered by changing the physical conditions such as pH [58-60], temperature [61], and addition of certain ions [62]. The soluble-insoluble immobilized enzyme system shows excellent performance for hydrolysis of insoluble substrates in soluble state, because this type of immobilization has minimum effect on shape and flexibility of the enzyme. Further, immobilized enzyme can be easily recovered in the insoluble form from a reaction mixture by centrifugation or filtration.
Liang and coworkers have recently reported the synthesis of a promising pH-sensitive enzyme carrier for immobilizing cellulase with high and low optimum pH due to its dissolving characteristics [50]. This water soluble copolymer was synthesized with methacrylic acid, 2-(dimethylamino) ethyl methacrylate, and butyl methacrylate and used as carrier for cellulase. The immobilization of cellulase on pH-responsive co-polymer; poly(methacrylic acid — 2-(dimethylamino) ethyl methacrylate — butyl methacrylate) is shown in Figure 6.5.
The copolymer is insoluble between pH 2.5 and 4.1, and soluble below pH 2.5 or above 4.1. Its recovery in aqueous solution was 97.2% by adjusting its isoelectric point to 3.1. Cellulase was covalently immobilized on the carrier polymer with 1-ethyl-3-(3-dimeth — yllaminopropyl) carbodiimide [63]. Under optimized conditions, the activity yield of immobilized cellulase was 63.24% and its recovery was 96.8% by adjusting its isoelectric point to 3.5. Maximum activity of the immobilized cellulase was achieved at 60°C (pH 5.0), while free cellulase exhibited maximum activity at 55°C (pH 5.0). Furthermore, they reported that the immobilized cellulase retained
Figure 6.5 Synthesis and cellulase immobilization on the pH-responsive co-polymer; poly(methacrylic acid-2-(dimethylamino)ethyl methacrylate-butyl methacrylate). The functional monomers mole ratio of co-polymer is methacrylic acid: 2-(dimethylamino) ethyl methacrylate : butyl methacrylate = 19:1:1. Immobilization conditions; reaction time — 4 h, pH: 6.0, 1-ethyl-3-(3- dimethyaminopropyl)-carbodiimide hydrochloride (EDC) amount: 300 mg/g co-polymer [63].
83.1% of its initial activity after repeated five cycles of hydrolysis reaction [50]. Relative activity in reusing immobilized cellulase in five cycles is shown in Figure 6.6.