Engineering of biocatalytic microgels and bifunctional peptides for biohybrid systems

  • Engineering von biokatalytischen Mikrogelen und bifunktionalen Peptiden für biohybride Systeme

Nöth, Maximilian Wolfgang Stefan; Schwaneberg, Ulrich (Thesis advisor); Pich, Andrij (Thesis advisor)

Aachen : RWTH Aachen University (2021, 2022)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2021


Biohybrid materials and systems have great potential in the fields of biocatalysis and materials science, as the combination of biological and synthetic building blocks enables the design of novel biohybrid catalyst and material concepts that convey new functionalities. New strategies for integrating biocatalytic functionalities into materials and for material and surface functionalization are needed, to develop new biohybrid materials and systems. In this thesis, two novel biohybrid systems for biocatalysis and universal material and surface functionalization were developed: in the first chapter, P450 μ-Gelzymes were established by immobilizing cytochrome P450 BM3 monooxygenase in stimuli-responsive "smart" microgels, while in the second chapter, a universal surface functionalization toolbox based on bifunctional peptides was developed for the functionalization of synthetic polymers, metals, and silicon-based materials.Enzymes are biocatalysts evolved by nature to perform (bio)chemical reactions at environmentally benign reaction conditions with impressive chemo-, regio-, and stereoselectivities. P450 monooxygenases are versatile biocatalysts with high synthetic application potential, but their application is yet challenged, amongst others, by their low operational stability (e.g., low organic solvent tolerance, enzyme inactivation after a certain reaction time). Enzyme immobilization is one the most successful strategies to improve enzyme stability and enables the release, re-immobilization, and recycling of enzymes. P450 monooxygenases are challenging to immobilize, and in the case of P450 BM3 from Bacillus megaterium, the activity of the immobilized enzyme is often deteriorated or entirely lost. Microgels have attracted attention as an innovative class of "smart" and stimuli-responsive carriers for enzyme immobilization due to their chemical and mechanical stability, tuneable architecture, biocompatibility, porosity, high water content, and the ability to achieve high enzyme loadings and provide a protective environment for the immobilized enzymes. Therefore, new strategies for integrating enzymes in microgels and new microgel systems need to be developed to harness the potential of stimuli-responsive microgels as a carrier platform for "sensitive" enzymes that are challenging to immobilize. This work reports the first pH-independent immobilization of P450 BM3 in novel poly(N-vinylcaprolactam) microgels with 1-vinyl-3-methylimidazolium as comonomer without loss of catalytic activity (biohybrid P450 μ-Gelzymes) and the first systematic study of P450 μ-Gelzyme performance. Poly(N-vinylcaprolactam) microgels were synthesized with a pH-independent, positive charge by modifying 1-vinylimidazole moieties through a quaternization reaction (1-vinyl-3-methylimidazolium). The pH-independent immobilization allowed to operate biohybrid P450 μ-Gelzyme catalysts at the pH activity optimum (pH 8). In addition, P450 μ-Gelzymes enabled ionic-strength triggered release and re-immobilization of P450 BM3 as well as biocatalyst recycling for repeated use and provided initial protective effects against organic cosolvents.The biological transformation of materials science is paving the way to novel biohybrid material concepts by combining and integrating biological and synthetic building blocks in materials (e.g., biofunctionalization of synthetic polymers, metals, and silicon-based materials). Universally applicable and specific material and surface functionalization methodologies are prerequisites for the biological transformation of materials science but pose a key challenge due to the vastly different properties and chemistries of materials and surfaces. Innovative material and surface functionalization technologies have to be developed to address this challenge. Biological surface functionalization with material binding or anchor peptides is a simple strategy to endow materials with biological and synthetic functionalities and an energy-efficient and environmentally benign alternative to chemical and physical surface functionalization strategies. Following this notion, a novel toolbox concept for universal material and surface functionalization based on bifunctional peptides was developed. Bifunctional peptides were synthesized by specific modification of the universal anchor peptide LCI from Bacillus subtilis with different functional amine moieties (e.g., reactive groups for click chemistry, fluorescent dyes, antibiotics, synthetic polymers) through sortase-mediated ligation employing sortase A from Staphylococcus aureus. Sortase-mediated ligation further enabled the purification of bifunctional peptides by a negative purification strategy using Strep‐tag II affinity chromatography (purities > 90%). In general, the bifunctional peptide toolbox enabled surface functionalization either as a two or one-step strategy. In the case of the one-step strategy, the desired functionality was directly introduced to LCI, while in the case of the two-step strategy, LCI was modified with a reactive group that enabled further functionalization (e.g., employing click chemistry). For the two-step strategy, synthetic polymers (polypropylene, polyethylene terephthalate), metals (stainless steel, gold), and silicon were functionalized with reactive groups for copper-free azide-alkyne click chemistry. The one-step strategy was demonstrated by direct functionalization of polypropylene with a fluorescent dye and biotin. These results represent the first systematic combination of universal anchor peptides, like LCI, and sortase-mediated ligation in a toolbox concept for universal surface functionalization, including the first peptide-mediated functionalization of polypropylene and polyethylene terephthalate with reactive groups for copper-free azide-alkyne click chemistry.