RESUMEN
Crude pineapple proteases extract (aka stem bromelain; EC 3.4.22.4) is an important proteolytic mixture that contains enzymes belonging to the cysteine proteases of the papain family. Numerous studies have been reported aiming at the fractionation and characterization of the many molecular species present in the extract, but more efforts are still required to obtain sufficient quantities of the various purified protease forms for detailed physicochemical, enzymatic and structural characterization. In this work, we describe an efficient strategy towards the purification of at least eight enzymatic forms. Thus, following rapid fractionation on a SP-Sepharose FF column, two sub-populations with proteolytic activity were obtained: the unbound (termed acidic) and bound (termed basic) bromelain fractions. Following reversible modification with monomethoxypolyethylene glycol (mPEG), both fractions were further separated on Q-Sepharose FF and SP-Sepharose FF, respectively. This procedure yielded highly purified molecular species, all titrating ca. 1 mol of thiol group per mole of enzyme, with distinct biochemical properties. N-terminal sequencing allowed identifying at least eight forms with proteolytic activity. The basic fraction contained previously identified species, i.e. basic bromelain forms 1 and 2, ananain forms 1 and 2, and comosain (MEROPS identifier: C01.027). Furthermore, a new proteolytic species, showing similarities with basic bomelain forms 1 and 2, was discovered and termed bromelain form 3. The two remaining species were found in the acidic bromelain fraction and were arbitrarily named acidic bromelain forms 1 and 2. Both, acidic bromelain forms 1, 2 and basic bromelain forms 1, 2 and 3 are glycosylated, while ananain forms 1 and 2, and comosain are not. The eight protease forms display different amidase activities against the various substrates tested, namely small synthetic chromogenic compounds (DL-BAPNA and Boc-Ala-Ala-Gly-pNA), fluorogenic compounds (like Boc-Gln-Ala-Arg-AMC, Z-Arg-Arg-AMC and Z-Phe-Arg-AMC), and proteins (azocasein and azoalbumin), suggesting a specific organization of their catalytic residues. All forms are completely inhibited by specific cysteine and cysteine/serine protease inhibitors, but not by specific serine and aspartic protease inhibitors, with the sole exception of pepstatin A that significantly affects acidic bromelain forms 1 and 2. For all eight protease forms, inhibition is also observed with 1,10-phenanthrolin, a metalloprotease inhibitor. Metal ions (i.e. Mn2+, Mg2+ and Ca2+) showed various effects depending on the protease under consideration, but all of them are totally inhibited in the presence of Zn2+. Mass spectrometry analyses revealed that all forms have a molecular mass of ca. 24 kDa, which is characteristic of enzymes belonging to the papain-like proteases family. Far-UV CD spectra analysis further supported this analysis. Interestingly, secondary structure calculation proves to be highly reproducible for all cysteine proteases of the papain family tested so far (this work; see also Azarkan et al., 2011; Baeyens-Volant et al., 2015) and thus can be used as a test for rapid identification of the classical papain fold.