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

摘要

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 puritled 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 deg C with salt. The K_m of the deacylase for echinocandin B was 50 #mu#M and its V_(max) was 14.6 #mu#mole cyclic hexapeptide min~(-1) mig~(-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.