Separation and Purification of ε-Poly-L-lysine from the Culture Broth Based on Precipitation with the Tetraphenylborate Anion

Abstract

A biopolymer ε-poly-L-lysine (εPL) exists as a fully protonated form (εPLHⁿ⁺) in solutions at pH < 4. Around pH 3.5, at which the tetraphenylborate (TPB⁻) anion is not decomposed immediately, the εPLHⁿ⁺ cation associates with the TPB⁻ anion to form a precipitate of 1:n stoichiometry, εPLH(TPB)_n. By the addition of NaTPB to a culture broth containing εPL and NH₄⁺ and K⁺ ions, not only the polycationic εPLHⁿ⁺ but also the monovalent cations would be precipitated with the TPB⁻ anion. However, εPLH(TPB)_n was purified by washing the mixed precipitate with acetone, in which NH₄TPB and KTPB are soluble. By mixing the εPLH(TPB)_n precipitate and a high-concentration HCl solution, the TPB⁻ anion was decomposed immediately to hydrophobic molecules. By the addition of a much larger volume of acetone to the reaction mixture, the decomposition products dissolved in the solvent. Simultaneously, εPL was precipitated as the hydrochloride salt. Thus, εPL has been separated and purified from the culture broth. Also, the method has been successfully applied to the separation of oligomeric εPL species. The present chemical separation method is rapid, simple, and easy to carry out, and can be utilized in bioengineering studies of such basic peptides or polyamines.