Peroxidation reactions catalyzed by sol-gel immobilized chloroperoxidase

Chloroperoxidase (CPO) from Caldariomyces fumago is a very versatile heme enzyme that catalyzes different reaction types, including peroxidation reactions of various substrates. Several of these reactions are of industrial interest. To create a re-usable biocatalyst, CPO was immobilized in a sol-gel matrix made from tetramethoxysilane (TMOS). The catalytic efficiency of sol-gel entrapped and free CPO was compared by measuring the initial rates of the peroxidation reactions with the substrates pyrogallol, N,N,N’,N’-tetramethyl-p-phenylenediamine (TMPD), and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). Hindered substrate and product diffusion inside the sol-gel host was a major cause for loss of catalytic performance. The release of methanol during the sol-gel process might be another factor as methanol and ethanol both cause a decline of CPO activity in solution. In addition, some catalytic activity was lost due to enzyme leakage which was most pronounced during the maturation phase of the gel. Depending on the substrate the catalytic efficiency of the immobilized CPO ranged between 4% (for ABTS), 8% (for TMPD), and 13 % (for pyrogallol) in comparison to free CPO in solution. The reusability of the CPO sol-gel beads was tested in up to 10 reaction cycles. A further decline in catalytic efficiency was detected for all three substrates, most notably for ABTS. Our findings are of importance for the optimization of protein sol-gel hybrid materials.