Membrane-associated echinocandin B deacylase of Actinoplanes utahensis: purification, characterization, heterologous cloning and enzymatic deacylation reaction

produces echinocandin B, a neutral lipopeptide. A deacylase from Actinoplanes utahensis catalyzes cleavage of the linoleoyl group from echinocandin B, a key step in generating a potential antifungal agent. Virtually all (99.8%) deacylase activity was cell-associated. The deacylase was salt-solubilized, heat-treated and purified to apparent homogeneity by a 3-step chromatographic procedure. The enzyme was a heterodimer consisting of 63- and 18-to-20-kDa subunit, optimally active at pH 6.0, and at 60°C with salt. The Km of the deacylase for echinocandin B was 50 μM and its Vmax was 14.6 μmol cyclic hexapeptide min−1 mg−1protein. The substrate specificity of the enzyme was broad with respect to both acyl and cyclic peptide analogues of echinocandin B. The two deacylase subunit genes were cloned and over-expressed in Streptomyces lividans. The recombinant deacylase was purified from the culture filtrate to apparent homogeneity by a 1-step chromatographic procedure. Using the recombinant deacylase, an enzymatic deacylation of immobilized echinocandin B resulted in the generation of cyclic hexapeptide at gram-level. Journal of Industrial Microbiology & Biotechnology (2000) 24, 173–180.