Covalent immobilization of benzoylformate decarboxylase from Pseudomonas putida on magnetic epoxy support and its carboligation reactivity

Abstract

Epoxy attached magnetic nanoparticles were prepared and used as solid support for covalent immobi-lization and stabilization of benzoylformate decarboxylase (BFD, E.C. 4.1.1.7) fromPseudomonas putida.A three-step immobilization/stabilization procedure is applied. The enzyme is firstly covalently immobi-lized under mild experimental conditions (e.g. pH 7.0, no added MgSO4and 20◦C). Secondly, the enzymeis immobilized under more drastic conditions (higher pH values, higher ionic strengths, etc.) to facili-tate an increase in effective concentration of the enzyme on the support near the epoxide reactive sites.Thirdly, the remaining epoxy groups are blocked to stop any additional interaction between the enzymeand the support. With more drastic conditions, the loading of enzyme can be increased from 1.25 to6.70 mg enzyme per gram of support. The covalently bounded enzyme was characterized in terms ofits activity and stability for the formation of (S)-2-hydroxypropiophenone (2-HPP). The activity of theimmobilized BFD was determined to be 53.0% related to the activity of the free enzyme. The immobilizedbiocatalyst retained 95% of its original activity after five reaction cycles.