Proteolytic enzymes from Bromelia antiacantha as tools for controlled tissue hydrolysis in entomology

Abstract

A crude extract with high proteolytic activity (78.1 EU/mL), prepared from ripe fruit of Bromelia antiacantha was used to hydrolyze and remove soft tissues from the epigyne of Apopyllus iheringi. This enzymatic extract presented four actives isoforms which have a broad substrate specificity action. Enzyme action on samples was optimized after evaluation under different conditions of pH, enzyme-substrate ratio and time (parameters selected based on previous studies) of treatment (pH 4.0, 6.0 and 8.0 at 42°C with different amount of enzyme). Scanning electron microscopy was used to evaluate conditions resulting in complete digestion of epigyne soft tissues. Optimal conditions for soft tissue removal were 15.6 total enzyme units, pH 6.0 for 18 h at 42°C.

Keywords: Bromelia antiacantha; Epigyne; Hydrolysis tissue.

Figure 1

Mass spectrometry and determination of molecular weight of proteins present in CE ofB. antiacanthaby MALDI-TOF MS/MS.

Figure 2

Native PAGE (lane 1) and Zymogram (lane 2); 25 μg of protein of CE from B.antiacanthawere loaded. The arrows represent the proteins bands with proteolytic activity.

Figure 3

Determination of cleavage sites of papain and CE enzymes on insulin β chain determined for fragments analysis by MALDI-TOF MS and GPMAW software v6.0. All insulin β chain cleavage sites are shown with arrows, with positions indicating the P’1 cleavage site. *Hydrofobic residues. ^aBulky hydrofobic residues.

Figure 4

Photographs taken under increased ventral face ofA. iheringiepigine subjected to various conditions of pH, CE specific activity and incubation time at 42°C. Epigine treatment: A) incubated in distilled water 22 h, B) 2.4 EU_TOT, pH 4.0, 22 h, C) 2.4 EU_TOT, 0.1 M Tris–HCl, pH 6.0, 22 h, D) 2.4 EU_TOT, 0.1 M Tris–HCl, pH 8.0, 22 h, E) 15.6 EU_TOT, 0.1 M Tris–HCl, pH 8.0, 6 h, F) 15.6 EU_TOT, 0.1 M Tris–HCl, pH 6.0, 6 h and G) 15.6 EU_TOT, 0.1 M Tris–HCl, pH 6.0, 8 h.

Figure 5

SEM image ofA. iheringiepigyne treated for 8 h with 15.6 EU_TOTof CE (0.1 M Tris–HCl, pH 6.0 at 42°C). Image A shows dorsal view and image B shows ventral view. In figure B can identified two structures: a) porous zone and b) spermathecae.

Figure 6

Spermatheca and porous zone shown at higher magnification: A) right spermathecae B) right spermatheca higher magnification and C) porous zone.

Figure 7

Surface duct and pore of epigyne shown at higher magnification: A) surface duct B and C) Pore of epigyne duct.