Covalent immobilization of catalase onto spacer-arm attached modified florisil: Characterization and application to batch and plug-flow type reactor systems
Introduction
Industrial applications for catalase (EC 1.11.1.6) include removal of hydrogen peroxide after sterilization steps in dairy product processing [1] or from bleaching batches [2], [3]. Catalase has also been used in coupled enzyme systems to prevent oxidase inactivation or product destruction by hydrogen peroxide [4], [5], [6]. Catalase has been covalently immobilized on carrier materials such as alumina [2], chitosan [7], magnesium silicate (florisil) [8], poly(ethylene terephthalate) [9], carbon nanotubes (CNTs) [10], eggshell [11], controlled pore glass (CPG) [12] and Eupergit C [13]. Since the recovery yield and reusability of immobilized catalase in these applications are quite limited, much attention has been paid to obtain a better immobilized catalase preparation. Immobilization via covalent bonds is a preferable method. However, the selection of conditions for immobilization by covalent binding which involve more complicated and less mild conditions is more difficult than in the cases of the other carrier binding methods. On the other hand, since covalent bonds are being formed, stable immobilized enzyme preparations which do not leach enzyme into solution in the presence of substrate or high ionic concentration solutions are formed in almost all cases [14]. The disadvantage of covalent immobilization is that this method leads to significant inactivation of enzymes. The presence of an appropriate spacer may mitigate the influence of the solid surface and convey flexibility to the enzyme, and alteration of the enzyme microenvironment. A popular spacer used to create a terminal carboxylic group on a matrix is 6-aminohexanoic acid (6-AHA) [15], [16]. This compound provides a primary amine coupling functionality on one end and a carboxylic group on the other. Florisil which contains 15% MgO and 85% SiO2 shows good mechanical properties, thermal stability and resistance against microbial attack and organic solvents was tested by many immobilization studies chosen as support because of its hydrophilic character helping to minimize the partitioning of substrate between carrier and the bulk aqueous solution, which adversely affects the global reaction rate [4], [17].
Our previous work has confirmed the enhancement of activity retention of the immobilized catalase by using 3-aminopropionic acid (3-APA) as a spacer arm [8]. As far as we know, there is not any detailed study on the performance of immobilized catalase considering the effect of spacer arm. The present study was designed to investigate the effects of 6-AHA as a spacer arm which may create some mild hydrophobic interactions because of its 5-carbon linear structure on the activity and stability of immobilized catalase. Immobilization conditions of catalase immobilized onto florisil via glutaraldehyde (CIG) and glutaraldehyde + 6-amino hexanoic acid (CIG-6-AHA) were optimized and immobilized catalase preparations were characterized. In order to decide the effect of the reactor type on the performance of CIG and CIG-6-AHA in terms of the total amounts of H2O2 decomposed (TAD-H2O2), batch and plug-flow type reactors were used.
Section snippets
Materials
Hydrogen peroxide was obtained from Merck AG (Darmstadt, Germany). Bovine liver catalase (44,500 U mg protein−1), florisil (particle size 150–250 μm, pore size 6–8 nm and specific surface area 170–300 m2 g−1), 3-aminopropyltriethoxysilane (3-APTES), glutaraldehyde (GA) solution (aqueous solution, 50% (w/w)), 6-AHA, 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate (CDI), ninhydrin reagent (2% solution) and all the other chemicals were obtained from Sigma (St. Louis, MO).
Modification of florisil
The
Optimization of immobilization conditions
The surfaces of supports such as silica gel, quartz and glass are chemically modified to develop the functional groups that can bind enzymes. In the surface modification reactions, the hydroxyl group can be used as a functional group on the particle surface. Surface amino groups are introduced on the silica surface by the reaction between the surface hydroxyl group and 3-APTES. In this study, after modification with 3-APTES the amount of –NH2 groups on the surface of the carrier was determined
Conclusions
Bovine liver catalase covalently immobilized onto modified florisil via GA and GA + 6-AHA. The optimum immobilization conditions of CIG and CIG-6-AHA were different because of the differences of binding reactions of catalase onto modified florisils. Catalytic efficiency of CIG-6-AHA is 40 fold higher than that of CIG. The increasing length of hydrophobic tail of spacer arm enhanced the catalytic efficiency and also performance in batch and plug-flow type reactors of immobilized catalase onto
Acknowledgements
This work was supported by TUBITAK (The Scientific and Technical Research Council of Turkey) with the Project number of “104T411” and Research Grants FEF2005D11 from Cukurova University.
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