Characterization of cuticle-degrading proteases produced by the entomopathogen Metarhizium anisopliae.
Two chymoelastases and three trypsinlike proteases were separated from culture filtrates of the entomopathogen Metarhizium anisopliae. A chymoelastase (Pr1) (pI 10.3 Mr 25,000) and trypsin (Pr2) (pI 4.42, Mr 28,500) were purified to homogeneity by ammonium sulphate precipitation, isoelectric focusing, and affinity chromatography. Inhibition studies showed that both enzymes possessed essential serine and histidine residues in the active site. Pr1 shows greater activity than Pr2 or mammalian enzymes against locust cuticle and also possesses activity vs elastin. Pr1 shows a broad primary specificity toward amino acids with hydrophobic side groups in synthetic ester and amide substrates. The kinetic properties of Pr1 demonstrate a preference for extended peptide chains with the active site recognising at least five substrate residues. The S5 and S4 subsites show a preference for negatively charged succinyl and hydrophobic acetyl groups, respectively. The S3 and S2 subsites both discriminated in favor of alanine and against proline. Pr2 rapidly hydrolyzed casein and synthetic substrates containing arginine or lysine. It possessed little or no activity vs cuticle, elastin, or synthetic substrates for chymotrypsin and elastase. Specific active site inhibitors confirmed the similarities between Pr2 and trypsin.
D000818:Animals; D001665:Binding Sites; D002918:Chymotrypsin / Q000378:metabolism; D005658:Fungi / Q000201:enzymology* / Q000472:pathogenicity; D006863:Hydrogen-Ion Concentration; D019476:Insect Proteins; D007313:Insecta / Q000382:microbiology; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D010196:Pancreatic Elastase / Q000378:metabolism; D010447:Peptide Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D011506:Proteins; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism
D019476:Insect Proteins; D011506:Proteins; C047663:cuticle proteins, insects; D010447:Peptide Hydrolases; D002918:Chymotrypsin; D010196:Pancreatic Elastase; D014357:Trypsin