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

Aspergillus nidulans 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 degrees C with salt, The K-m of the deacylase for echinocandin B was 50 mu M and its V-max was 14.6 mu mol cyclic hexapeptide min(-1) mg(-1) protein, 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.