Identification of proteases produced by entomopathogenic fungi Beauveria bassiana (Bals) Vuill. strain CG432 previously activated in coffee berry borer alive (Hypothenemus hampei)

Abstract

Conidia of entomopathogenic fungi can penetrate the insect exoskeleton both by mechanic action of the germinative tube and by production of multiple proteases, chitinases, and lipases in response to the composition of the insect cuticle. Therefore the purpose of this work was to produce, to purify, and to characterize the structure of the proteases produced by the fungus Beauveria bassiana CG432, previously activated in adults of coffee berry borer (Hypothenemus hampei) grown under submerged culture conditions. A suspension containing 106 activated conidia/mL was inoculated to a culture medium at 28ºC and 150 rpm for 3 days. Protease extracts, were obtained by centrifugation at 8000g for 20 minutes, were fractionated and were concentrated by ultrafiltration using controlled porosity membranes of 100 kDa and 3 kDa, respectively. Gel filtration chromatography on Sephadex G-100 isolated one protein peak (peak II), which contained 56% of residues of the aminoacid aspartic acid, characterized by HPLC using an ODS-C18 reverse phase column. The peak protein showed specific activity 43 times superior when compared to the free-cell extract in bovine serum albumin, subtilisin-like protease activity, and a single protein band reveled by Brilliant Coomassie Blue on a gelatin zimogram PAGE electrophoresis, by native conditions. The homogeneity of peak II was confirmed by revelation of a single band during determination of the isoelectric pH 4.5, but molecular mass determination by PAGE-2D electrophoresis showed 2 bands of 23 and 26 kDa. Proteases were characterized as serine proteases with cysteine residues important to activity, since they had been inhibited by phenylmethylsulphonyl fluoride and p-chlormercuricbenzoic acid. Peak II proteases showed Km of 4x10-4 on subtilisin-like substrate.