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

Epoxy attached magnetic nanoparticles were prepared and used as solid support for covalent immobilization and stabilization of benzoylformate decarboxylase (BFD, E.C. 4.1.1.7) from Pseudomonas putida. A three-step immobilization/stabilization procedure is applied. The enzyme is firstly covalently immobilized under mild experimental conditions (e.g. pH 7.0, no added MgSO4 and 20 °C). Secondly, the enzyme is immobilized under more drastic conditions (higher pH values, higher ionic strengths, etc.) to facilitate 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 enzyme and the support. With more drastic conditions, the loading of enzyme can be increased from 1.25 to 6.70 mg enzyme per gram of support. The covalently bounded enzyme was characterized in terms of its activity and stability for the formation of (S)-2-hydroxypropiophenone (2-HPP). The activity of the immobilized BFD was determined to be 53.0% related to the activity of the free enzyme. The immobilized biocatalyst retained 95% of its original activity after five reaction cycles.