MicroGelzymes: pH-Independent Immobilization of Cytochrome P450 BM3 in Microgels

Abstract

Microgels are an emerging class of “ideal” enzyme carriers because of their chemical and process stability, biocompatibility, and high enzyme loading capability. In this work, we synthesized a new type of permanently positively charged poly­(N-vinylcaprolactam) (PVCL) microgel with 1-vinyl-3-methylimidazolium (quaternization of nitrogen by methylation of N-vinylimidazole moieties) as a comonomer (PVCL/VimQ) through precipitation polymerization. The PVCL/VimQ microgels were characterized with respect to their size, charge, swelling degree, and temperature responsiveness in aqueous solutions. P450 monooxygenases are usually challenging to immobilize, and often, high activity losses occur after the immobilization (in the case of P450 BM3 from Bacillus megaterium up to 100% loss of activity). The electrostatic immobilization of P450 BM3 in permanently positively charged PVCL/VimQ microgels was achieved without the loss of catalytic activity at the pH optimum of P450 BM3 (pH 8; ∼9.4 nmol 7-hydroxy-3-carboxy coumarin ethyl ester/min for free and immobilized P450 BM3); the resulting P450-microgel systems were termed P450 MicroGelzymes (P450 μ-Gelzymes). In addition, P450 μ-Gelzymes offer the possibility of reversible ionic strength-triggered release and re-entrapment of the biocatalyst in processes (e.g., for catalyst reuse). Finally, a characterization of the potential of P450 μ-Gelzymes to provide resistance against cosolvents (acetonitrile, dimethyl sulfoxide, and 2-propanol) was performed to evaluate the biocatalytic application potential.

Harnessing P450 Enzyme for Biotechnology and Synthetic Biology

“…immobilized P450BM3 in a new type of positively charged microgel named as PVCL/VimQ (poly( N ‐vinylcaprolactam) microgel with 1‐vinyl‐3‐methylimidazolium) as a comonomer via electrostatic adsorption [107] . The activity of the P450 microgel showed comparable activity with the free enzyme, high tolerance to organic solvent and excellent recyclability [107] . The hybridization of materials and P450s demonstrates potentials to improve enzymes’ stability and activity at different reaction environments, which opens up novel possibilities in process design of multienzyme involved tandem catalysis and overcome the incompatibility of chemo‐ and biocatalytic steps to facilitate the synthetic application.…”

“…Nöth et al . immobilized P450BM3 in a new type of positively charged microgel named as PVCL/VimQ (poly( N ‐vinylcaprolactam) microgel with 1‐vinyl‐3‐methylimidazolium) as a comonomer via electrostatic adsorption [107] . The activity of the P450 microgel showed comparable activity with the free enzyme, high tolerance to organic solvent and excellent recyclability [107] .…”

Microgels in Tandem with Enzymes: Tuning Adsorption of a pH‐ and Thermoresponsive Microgel for Improved Design of Enzymatic Biosensors

“…However, simple adsorption seems to be the easiest and the most efficient approach for the incorporation of enzymes into microgels, a high level of enzymatic activity being, moreover, reported for such systems. [ 21,44,45 ]…”

Rational design yields molecular insights on leaf binding of the anchor peptide Macaque Histatin

“…Anchor peptides (APs) are short amphiphilic peptides with sizes ranging from 20 to 100 amino acids that bind from aqueous solutions to natural 9-10 and synthetic surfaces 11-14 including metals 15-16 . Tailor-made APs are thus applicable in many fields, including biotechnology, 17 catalysis, 18 nanoparticles, 19 medicine, 20 and agriculture 9 .…”