Styrenic resins immobilized enzyme

A series of close-to-spherical styrene/DVB resins of varying particle size and pore diameter were employed as supports for non-covalent adsorptive attachment of CALB by hydrophobic interaction. The effect of matrix particle and pore size on CALB i) adsorption isotherms, ii) fraction of active sites, iii) distribution within supports, and iv) catalytic activity for s-CL ring-opening polymerizations and adipic acid/l,8-octanediol polycondensations is reported. Important differences in the above for CALB immobilized on methyl methacrylate and styrene/DVB resins were found. The lessons learned herein provide a basis to others that seek to design optimal immobilized enzyme catalysts for low molar mass and polymerization reactions. 

Table 2. Candida antartica Lipase B (CALB) immobilization on styrene resins of differing particle size and pore size enzyme loading, fraction of active lipase, and catalytic activity.

At enzyme-loadings of 7.9 to 8.7%, the fraction of active sites for immobilized CALB ranged from 57.7 to 64.2%. Thus, the fraction of active CALB molecules is independent of resin size for supports ranging from about 600 to 35 pm. Also, increase in the pore size of 35 pm supports from 300 to 1000 A (resins 4 and 5, respectively) at similar enzyme loadings ( 8 %) had no effect on the fraction of active CALB molecules. Compared to the fraction of active CALB molecules (40 to 45%) when CALB was immobilized on polymethyl methacrylate resins with similar particle and pore size values, styrenic resins not only have high affinity for CALB adsorption but also provide CALB with a surface environment that enables CALB to orient and take on conformations that retain a high degree of catalyst active site reactivity. 

The oxazolidine-2,5-dione heterocycle, perhaps better known as the N-carboxyanhydride of an amino acid, has been incorporated employing a modification of chloromethylated poly(styrene) (192) (76USP3985715). The reaction sequence involved utilization of a masked amino acid, ethyl acetamidocyanoacetate (205). The amino acid was liberated in a subsequent hydrolysis/decarboxylation step (Scheme 98). The cyclized, IV-carboxyanhydride-functional resins (206) were reported to be useful in solid phase peptide synthesis and as supports for enzyme immobilization. 

From the above results on CALB activity as a function of particle size for polystyrene and PMMA resins, we believe %-surface area occupied by CALB is a critical factor that can be used to improve immobilized CALB activity. Increased %-accessible surface area will increase the probability of collisions between substrates and CALB. As %-accessible surface area for CALB increased for PMMA resins a corresponding increase in polyester synthesis reaction rates was observed (see above). CALB immobilized on styrenic particles of variable size showed little differences in both %-accessible surface area and polyester synthesis catalyst activity. The potential benefit of decreasing bead particle size is to decrease diffusion constraints that lead to productive collisions between enzyme and substrate. However, for polystyrene resins, as particle size decreased, the percent of resin area in which reactions can occur does not change. In contrast, decreasing PMMA particle size dramatically... 

It is important that the support material be stable under operational conditions with respect to mechanical forces, solvent temperature, pH, etc. A wide variety of different supports have been used for enzyme immobilization including natural polymers (cellulose, agarose diitosan), synthetic polymers (acrylic resins, styrene-divinylbenzene, polypropylene), and inorganic materials (silica, porous glass, Celite). Each offers advantages and disadvantages, and the optimal support for a given enzyme and reaction must be chosen individually. 

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