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

A biopolymer epsilon-poly-L-lysine (epsilon PL) exists as a fully protonated form (epsilon PLHn+) in solutions at pH < 4. Around pH 3.5, at which the tetraphenylborate (TPB-) anion is not decomposed immediately, the epsilon PLHn+ cation associates with the TPB-anion to form a precipitate of 1:n stoichiometry, epsilon PLH(TPB)(n). By the addition of NaTPB to a culture broth containing epsilon PL and NH4+ and K+ ions, not only the polycationic epsilon PLHn+ but also the monovalent cations would be precipitated with the TPB- anion. However, epsilon PLH(TPB)(n) was purified by washing the mixed precipitate with acetone, in which NH4TPB and KTPB are soluble. By mixing the epsilon PLH(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, epsilon PL was precipitated as the hydrochloride salt. Thus, epsilon PL has been separated and purified from the culture broth. Also, the method has been successfully applied to the separation of oligomeric epsilon PL 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.