Covalent Binding

Covalent binding means that the enzyme is immobilized by a chemical reaction between the enzyme and the support material to form covalent bonds. Although this immobilization generally enhances enzyme stability, one major disadvantage of this method is that the activity of the immobilized enzyme is often significantly decreased because the active site maybe blocked, multiple point-binding may occur, or the enzyme may be denatured (Butterfield et al. 1994; Ganapathi et al. 1998; Zhuang and Butterfield 1992, 1993). As shown in Fig. 17a, the enzyme is directly immobilized, often through the e-amino functionality of lysine residues on the protein. Because the protein often contains multiple lysine residues, which spread over the enzyme surface, different orientations of the enzyme occur, and it may thus be difficult for the active site of the enzyme to interact with the substrate.

Fig. 16. Hydrolysis process of high (a) and low (b) poly(acrylic acid) grafting degree

Time (hour)

Fig. 16. Hydrolysis process of high (a) and low (b) poly(acrylic acid) grafting degree

Fig. 17. Schematic representation of direct graft (a) and site-specific (b) immobilization of enzymes

Piletsky et al. (2003) functionalized PPMMs with polyaniline (PANI) and immobilized horseradish peroxidase (HRP) onto the membrane surface by both physical adsorption and covalent attachment. PANI is a semiconduc-tive material that is stable in an oxygen atmosphere, contains amino groups, and is suitable for the covalent immobilization ofbiomolecules through car-bodiimide or glutaric dialdehyde chemistry. Electronactive properties of PANI polymer could be envisaged as an additional advantage over conventional polymer matrices for enzyme immobilization, due to the fact that the electron-donating ability of PANI may play an important role in the enzyme catalysis of redox reactions (Malinauskas 1999). The PANI-coated PPMMs showed a high affinity for this enzyme as a result of the combination of electronstatic and hydrophobic interactions between the PANI and proteins (Bossi et al. 2002; Chen et al. 2000a, b). PANI has three oxidation states, leukoemeraldine (reduced), emeraldine, and pernigraniline (oxidized; see Fig. 18), and these different forms possess different adsorption properties to proteins. The highest and lowest bindings were achieved by reduced and oxidized PANI, respectively. HRP immobilized on the PANI-coated PPMM was shown to retain 70% of its activity after 3 months of storage at +5 °C, suggesting that this material can be used for practical application, such as in bioreactors as enzyme membranes.

Becker and coworkers (2002) presented a process involving continuous synthesis and simultaneous product release of amylase by an enzyme-immobilized membrane (see Fig. 19). PPMMs were functionalized by pho-toinitiated graft polymerization of GMA and the enzyme, amylosucrase, was immobilized by the reaction with epoxy groups on the membrane surface. Amylosucrases are found in many Neisseria species and catalyze the reaction sucrose + {1,4-a-D-glucosyl}„ ^ D-fructose + {1,4-a-D-glucosyl}„+1

The enzyme transfers the glucose moiety of sucrose to the nonreducing end of an a-1,4-glucan chain, extending it linearly by one glucose unit and thereby releasing a fructose molecule. A sucrose solution and mal-tooligosaccharide (MOS) mixture containing from 3 to 6 glucose units with

Fig. 18. Oxidation states of polyaniline

Pernigraniline

Fig. 18. Oxidation states of polyaniline

Amylosucrase
Fig. 19. Scheme of enzymatic poly- or oligoglucan synthesis within the membrane pore

a-1,4-linkage was used as a primer to filtrate through the membrane at 37 °C. The formed products (i. e., glucose coupled to the primer molecule), was characterized by high-performance liquid chromatography analysis of the filtrates at different flow rate (Fig. 20). The initial MOS mixture contained oligosaccharides with a degree of polymerization (DP, i. e., number

Fig. 20. Chromatograms of the filtrates with flow rate of 0.2 ml/min. (a) and 0.1 ml/min (b). DP Degree of polymerization

of glucose residues) of 3, 4, 5, and 6, with the DP3 oligosaccharide being most abundant followed by DP4 and DP5 oligosaccharides. At the flow rate of 0.2 ml/min the maltooligosaccharides were enlarged by one glucose unit per membrane passage and a definite shift can be seen in the oligosaccharide distribution: the DP3 peak remains almost constant, the DP4 peak is much reduced, implying that the DP4 product may be preferentially utilized by the enzyme as an acceptor for the glucose moiety (Fig. 20a). In addition, at the slower flow rate of 0.1 ml/min, more than one glucose unit is coupled to a subset of the primer molecules at each passage (Fig. 20b).

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