Abstract
Polymyxin acylase, which produces deacylated polymyxins by hydrolyzing only the fattyacyl 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 E1) and was stable up to 50°C for 24 hr. The observed Vmax 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 p-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 N-fattyacyl compounds (peptides and amino acids). Among several fattyacyl groups (C2-C16) of N-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.
