Protein Oligomerization Based on Brønsted Acid Reaction

Oligomeric proteins are abundant in nature. However, their synthesis remains a significant challenge. A controlled oligomerization process can provide important insights into the evolution of modern proteins and the development of more efficient biocatalysts. Here, we propose a pathway for producing glucose oxidase (GOx) homooligomer (Ol-GOx), a redox enzyme with extensive biotechnological applications. We obtained Ol-GOx from the one-pot reaction of the native protein with a Brønsted acid, trifluoromethanesulfonic acid (TFMS). Ol-GOx had a hydrodynamic radius of 96 nm and a molecular mass of 2 MDa. Ol-GOx exhibited higher thermal stability in comparison to the native protein, as well as increased hydrophobicity, which is a primary characteristic needed for its stabilization in the solid state for applications in bioenergy, heterogeneous catalysis, and biomedicine. Furthermore, there were remarkable improvements in redox properties and protein stabilization, suggesting that this approach for Ol-GOx synthesis could be used to design more efficient oligomeric biocatalysts

Analytical HPLC used LiChrospher 60 RP column and eluted with potassium phosphate

<p>. (A) Standard EPI (20 µg/mL), (B) Standard pilocarpine (50 µg/mL), (C) “cultivated jaborandi leaves” solution, resulted from first extraction step, (D) “cultivated jaborandi acid” solution, obtained EPI under salt form, (E) Solution of “crude EPI” with some impurities as pilocarpine and other alkaloids, (F) last step of isolation showing EPI >98% purity.</p

Scheme of all necessary steps in obtaining Epiisopiloturine with >98% purity from Jaborandi leaves.

<p>Scheme of all necessary steps in obtaining Epiisopiloturine with >98% purity from Jaborandi leaves.</p

Mass spectrum obtained from ESI+/Ion Trap.

<p>(A) free EPI with a pseudo molecular ion m/z 287.1 Da [M+H]⁺, (B) MS² with characteristic fragment at m/z 269.1 Da [M – H₂O + H]⁺, (C) MS³ with fragments at m/z 251.0 Da [M – 2H₂O + H⁺] and 168.06 Da with proposed chemical structure.</p

EPI bond distances obtained through x-ray diffraction (Experimental) and DFT results (Calculated).

<p>Atom labels accordingly to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066702#pone-0066702-g004" target="_blank">Figure 4</a>.</p

Isolated Epiisopiloturine molecular structure.

<p>Isolated Epiisopiloturine molecular structure.</p

EPI bond angles obtained through x-ray diffraction (Experimental) and DFT results (Calculated).

<p>Atom labels accordingly to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066702#pone-0066702-g004" target="_blank">Figure 4</a>.</p

Infrared (IR) and Raman wavenumbers (cm⁻¹) of solid state EPI.

<p>Calculated vibrational wavenumbers (cm-1) for the isolated EPI molecule. A tentative assignment of the observed vibrational modes is also shown. See text for theoretical details. ν =  stretching, δ =  bending, β =  bending in plane, γ =  bending out of plane, r =  rocking, τ =  twist, sc =  scissoring, ω =  wagging, νs =  symmetric stretching, νa =  antisymmetric stretching, sh  =  shoulder.</p

Epiisopiloturine FT-IR spectra: A) experimental and B) calculated.

<p>Epiisopiloturine FT-IR spectra: A) experimental and B) calculated.</p

Epiisopiloturine FT-Raman spectra: A) experimental and B) calculated.

<p>Epiisopiloturine FT-Raman spectra: A) experimental and B) calculated.</p