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

20/11/2020
  Maximilian Nöth and Larissa Hussmann Copyright: © BIO VI

Maximilian Nöth, Larissa Hussman, Thomke Belthle, Islam El-Awaad, Mehdi D. Davari, Felix Jakob, Andrij Pich*, and Ulrich Schwaneberg* Biomacromolecules, 2020, DOI: doi.org/10.1021/acs.biomac.0c01262

‡ These authors contributed equally to the work

Project: SFB 985

Funding: DFG: Collaborative Research Centre 985 “Functional Microgels and Microgel Systems; BMBF: Next Generation of Biotechnological Processes – Biotechnology 2020+

Subgroup: Biohybrid Systems and Computational Biology

 

The electrostatic immobilization of cytochrome P450 BM3 monooxygenase was achieved in permanently positively charged poly(N-vinylcaprolactam) microgels with 1-vinyl-3-methylimidazolium as comonomer without loss of catalytic activity.

Microgels are an emerging class of enzyme carriers due to 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) microgel with 1-vinyl-3-methylimidazolium (quaternization of nitrogen by methylation of N-vinylimidazole moieties) as 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. Cytochrome P450 monooxygenases are usually challenging to immobilize, and often high activity losses occur after the immobilization (in the case of P450 BM3 up to 100% loss of activity). The electrostatic immobilization of P450 BM3 in permanently positively charged PVCL/VimQ microgels (P450 µ-Gelzymes) was achieved without loss of catalytic activity at the pH optimum of P450 BM3. In addition, P450 µ-Gelzymes could be employed for reversible ionic strength-triggered release and re-immobilization of P450 BM3 as well as catalyst recycling for multiple reuse cycles. Finally, a characterization of the potential of P450 µ-Gelzymes to provide resistance against organic cosolvents (acetonitrile, dimethyl sulfoxide, 2-propanol) was performed to evaluate the biocatalytic application potential of P450 µ-Gelzymes.

  pH-independent electrostatic immobilization of P450 BM3 Copyright: © American Chemical Society  

Figure The pH-independent electrostatic immobilization of P450 BM3 could be achieved in permanently positively charged poly(N-vinylcaprolactam) microgels with 1-vinyl-3-methylimidazolium as comonomer. The permanent positive charge was introduced by quaternization of 1-vinylimidazole through methylation of N-vinylimidazole moieties. Reproduced with permission from Nöth et al. (2020).