Polymyxin Acylase: Purification and Characterization, with Special Reference to Broad Substrate Specificity

Abstract

Polymyxin acylase, which produces deacylated polymyxins by hydrolyzing only the fatty acyl groups of polymyxin antibiotics without affecting the peptide moiety, was purified from acetone-dried cell powder of Pseudomonas sp. M-6–3. The cell-free enzyme, solubilized by Triton X-100, was further purified on successive DEAE-cellulose, hydroxyapatite, and Sephacryl S-300 columns to a homogeneous state. This purified enzyme (Type I) had a single band with an MW of 62,000 in SDS- polyacrylamide gel electrophoresis. Gel filtration on a calibrated Sephacryl S-300 column also gave an estimated MW of 62,000. The isoelectric point of the enzyme was 5.7. Enzyme activity was optimal at pH 9.0 for colistin A (polymyxin E^ and was stable up to 50°C for 24 hr. The observed V_max and Km values were 1750nmol/min/mg and 3.85 mm, respectively. This enzyme was almost not affected by metal chelators and various thiol-enzyme inhibitors (other than /7-chloromercuribenzoate), and showed high tolerance for organic modifiers; for example, half of the activity remained in 50 % ethylene glycol buffer. This enzyme deacylated not only polymyxins but also various /V-fattyacyl compounds (peptides and amino acids). Among several fattyacyl groups (C₂-C₁₆) of /V-acyl DL-methionines, the caprinoyl (C₁₀) group was most easily liberated, and the benzyloxycarbonyl (Z) group was also slightly susceptible. When the enzyme was solubilized in 0.2 m KCl-containing buffer by Triton X-100, the enzyme (Type II) showed a slightly different substrate specificity and an increased activity for some Z-derivatives. With this enzyme, it is possible to remove the Z group from several Z-peptides under mild conditions.