Cuticle-degrading enzymes of entomopathogenic fungi: Cuticle degradation in vitro by enzymes from entomopathogens

Extracellular fluids from Metarhizium anisopliae, Beauveria bassiana, and Verticillium lecanii grown on cuticle as the sole carbon source released amino acids and N-acetylglucosamine from protein and chitin, respectively, in comminuted locust cuticle. An endoprotease, chitinase, and N-acetyl-β-glucosaminidase were each purified from culture filtrates of M. anisopliae until free of other cuticle-degrading enzymes and tested singly, in combination, or in sequence against “whole cuticle” (containing tanned and untanned proteins) and exuviae (tanned cuticle). The protease hydrolyzed ca. 25–30% of cuticle proteins (w/w), releasing peptides (mean chain length, 4.7) containing all 15 amino acids found in locust cuticle. Small amounts of amino sugars were also liberated following protein solubilization. Chitinase tested separately released monomeric N-acetylglucosamine (equivalent to 3–4% of cuticle chitin); however, when combined simultaneously with protease, N-acetylglucosamine release was increased × 1.5. Pretreatment with protease considerably enhanced chitinase activity (ca. × 3.5) compared to controls (preincubated with autoclayed protease). This implies that cuticular chitin is shielded by protein. N-acetyl-β-glucosaminidase showed no detectable activity against cuticle either alone or in combination with protease or chitinase. Exuvia was comparatively resistant to both proteolytic and chitinolytic attack; pretreatment with protease had no effect on subsequent chitinase activity. The results are discussed in relation to cuticle structure and the role of host and fungal enzymes in degrading cuticle during molting or infection.