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Covalent immobilization of cytochrome P450 BM3 (R966D/W1046S) on glutaraldehyde activated SPIONs
Author(s) -
Bahrami Atieh,
Garnier Alain,
Larachi Faïçal,
Iliuta Maria C.
Publication year - 2018
Publication title -
the canadian journal of chemical engineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.404
H-Index - 67
eISSN - 1939-019X
pISSN - 0008-4034
DOI - 10.1002/cjce.23208
Subject(s) - hydroxylation , chemistry , glutaraldehyde , bacillus megaterium , immobilized enzyme , biocatalysis , covalent bond , cofactor , context (archaeology) , active site , enzyme , combinatorial chemistry , organic chemistry , catalysis , reaction mechanism , biology , paleontology , bacteria , genetics
Selective hydroxylation of the C‐H bond of saturated hydrocarbon chains at room temperature is the signature of an invaluable biocatalyst, cytochrome P450 BM3 from Bacillus megaterium . Despite this remarkable ability, because of the enzyme's inherent low stability and dependence on electron supply by expensive NADPH, developing stable and economic BM3 systems is a challenging subject. To improve BM3 stability, facilitate its reuse, and reduce the process cost, this study suggests covalent immobilization of R966D/W1046S P450 BM3 on glutaraldehyde pre‐activated super paramagnetic iron oxide nanoparticles (SPIONs). This double mutant consumes less expensive cofactors like NADH and BNAH and its immobilization on magnetic support facilitates its separation and reuse. Free and immobilized enzyme performances were evaluated by 10‐ p NCA hydroxylation and BM3 selectivity (hydroxylation at ω (1–3) positions of a fatty acid) was confirmed in a reaction involving myristic acid. The enzyme activity recovery was up to 60 % with 100 % enzyme binding efficiency. BM3‐SPIONs were easily separated from the reaction medium by applying a magnet, and recycled for 5 times, after which they could still present half of their initial activity. The enzyme storage stability was significantly improved: after one month of storage at 4 °C, the immobilized enzyme showed 80 % residual activity toward NADH while the soluble enzyme was inactive after a week. Binding an enzyme to fabricated SPIONs is a promising technique to increase enzyme stability and prevent downstream contamination in biocatalytic processes. In this context, BM3‐SPIONs can be a practical model system in cost‐effective large‐scale applications of such enzymes.