Immobilisation of P450 BM‐3 and an NADP⁺ Cofactor Recycling System: Towards a Technical Application of Heme‐Containing Monooxygenases in Fine Chemical Synthesis

Abstract

Cytochrome P450 monooxygenases are potentially a very useful class of hydroxylation catalysts; they are able to introduce oxygen at activated and nonactivated carbon-hydrogen bonds and thus lead to regio-and/or stereochemically pure compounds. However, this potential is lowered by their intrinsic low activity and inherent instability. P450-catalysed biotransformations require a constant supply of NAD(P)H, making the process an expensive one. To render these catalysts more suitable for industrial biocatalysis, the immobilisation of P450 BM-3 (CYP 102A1)from Bacillus megaterium in a sol-gel matrix was combined with a cofactor recycling system based on NADP + -dependent formate dehydrogenase (EC 1.2.1.2) fromPseudomonas sp. 101 and tested for practical applicability. This approach was used for the conversion of β-ionone, octane and naphthalene to the respective hydroxycompounds with DMSO as cosolvent using sol-gel immobilised P450 BM-3 mutants.

Microbial P450 enzymes in biotechnology

“…A successful alternative for the laboratory-scale purification of P450s is metal-ion affinity chromatography using His 6 -tagged proteins (Delcarte et al 2003;Maurer et al 2003). However, both approaches described above, are either time-consuming or expensive and thus not suitable for the gramscale purification of P450 monooxygenase.…”

“…Maurer et al were able to show that P450 BM-3 from Bacillus megaterium binds firmly to anion exchangers such as DEAE and SuperQ (Maurer et al 2003). However, the products of the reaction also adsorbed to these matrices.…”

“…In addition, washes with higher ionic strength leached the enzyme from the carrier. The enzyme was unable to adsorb on celite, a porous silicate matrix derived from diatomaceous soil (Maurer et al 2003). Immobilization of two P450 BM-3 mutants was further examined using a sol-gel matrix derived from tetraethoxyorthosilicate (TEOS) (Gill 2001).…”

“…(Tishkov et al 1999), enabling it to be used as NADPH-regenerating enzyme in combination with P450 BM-3 (Fig. 4) (Maurer et al 2003). A further approach to overcome the problem of cofactor costs is the use of the peroxide shunt pathway with strong oxidants such as hydrogen peroxide or periodate which directly catalyze the reduction of the heme-iron (Naqui et al 1986;Coon et al 1996).…”

Cloning, expression and characterisation of CYP102A2, a self-sufficient P450 monooxygenase from Bacillus subtilis

“…Great efforts are presently being made to improve storage and operational stability of bacterial P450 enzymes (e.g. tolerance towards organic solvents or hydrogen peroxide) (Maurer et al 2003, Salazar et al 2003, Seng Wong et al 2004). …”