Separation and Purification of ε-Poly-L-lysine with Its Colorimetric Determination Using Dipicrylamine

Abstract

A separation method for a biopolymer, ε-poly-L-lysine (εPL), with its colorimetric determination using a yellow anionic dye, dipicrylamine anion (DPA⁻), is presented. The εPL-producing culture broth was mixed with a NaDPA solution to precipitate the εPL in polycationic form with the DPA⁻ anion. The precipitate was dissolved into acetonitrile (AN). The AN solution was yellowish, and gave an absorption maximum at around 420 nm. Thus, εPL in the culture broth can be assayed colorimetrically a with multiwell microtiter plate. By the addition of bis(triphenylphosphoranylidene)ammonium chloride, the polycationic εPL was precipitated from the AN solution as the hydrochloride salt. On the other hand, the DPA⁻ anion remained in the AN solution as the quaternary ammonium salt. Thus, εPL larger than tetramer can be separated and purified after the high-throughput screening of the synthetic enzyme.