Immobilization of P450 BM-3 monooxygenase on mesoporous molecular sieves with different pore diameters

The immobilization of the isolated heme domain of P450 BM-3 (BM3H_F87A) on two mesoporous molecular sieves, MCM-41 (pore diameter 25 Å) and SBA-15 (pore diameter 60 Å and 133 Å) was examined systematically, and the activity of the immobilized enzyme toward para-nitrophenoxydodecanoic acid (12-pNCA) and n-octane was determined. Hydrogen peroxide was utilized as source of electrons and oxygen to support the monooxygenase activity of BM3H_F87A. The mesoporous materials were characterized by X-ray diffraction and nitrogen adsorption analyses before and after immobilization. The results revealed that the immobilization efficiency of MCM-41 and SBA-15 after single immersion was strongly affected by the pH value of the enzyme solution, initial enzyme concentration and agitation conditions. By modelling the 3D structure in silico and performing electrostatic potential calculations, the pH-dependence of the enzyme immobilization could be explained and a possible orientation of the protein on mesoporous materials was predicted. The oxidizing activity of the immobilized enzyme was found to depend on pore diameter and accessibility of the substrate for the enzyme. The highest activity toward 12-pNCA of 830 nmol product/mg P450/min was observed with BM3H_F87A immobilized on SBA-15 with pore diameter 133 Å. Enzyme activity toward n-octane was similar for the enzyme immobilized on SBA-15 of 60 Å and 133 Å, and was at least two-fold higher as compared to a system with free enzyme.