Purification and characterization of multiple forms of the pineapple-stem-derived cysteine proteinases ananain and comosain

Abstract

A mixture of ananain (EC 3.4.22.31) and comosain purified from crude pineapple stem extract was found to contain numerous closely related enzyme forms. Chromatographic separation of the major enzyme forms was achieved after treatment of the mixture with thiol-modifying reagents: reversible modification with 2-hydroxyethyl disulphide provided enzyme for kinetic studies, and irreversible alkylation with bromotrifluoroacetone or iodoacetamide gave enzyme for structural analyses by 19F-n.m.r. and electrospray mass spectrometry respectively. Structural and kinetic analyses revealed comosain to be closely related to stem bromelain (EC 3.4.22.32), whereas ananain differed markedly from both comosain and stem bromelain. Nevertheless, differences were seen between comosain and stem bromelain in amino acid composition and kinetic specificity towards the epoxide inhibitor E-64. Differences between five isolatable alternative forms of ananain were characterized by amidolytic activity, thiol stoichiometry and accurate mass determinations. Three of the enzyme forms displayed ananain-like amidolytic activity, whereas the other two forms were inactive. Thiol-stoichiometry determinations revealed that the active enzyme forms contained one free thiol, whereas the inactive forms lacked the reactive thiol required for enzyme activity. M.s. provided direct evidence for oxidation of the active-site thiol to the corresponding sulphinic acid.

Amino‐acid sequence and glycan structures of cysteine proteases with proline specificity from ginger rhizome Zingiber officinale

“…For this reason, and because of the ambiguities discussed above, the GP-I sequence reported here must be regarded as tentative. The appearance of multiple forms of the ginger proteases is not remarkable, however, as such heterogeneity has also been seen in other species, for example, papaya [24] and pineapple stem [25]. The N-linked oligosaccharides of plant glycoproteins typically have 5±13 glycosyl units and fall into two main classes: high-mannose-type glycans and complex-type glycans.…”

Bromelain: an overview of industrial application and purification strategies

“…Stem bromelain contains 285 amino acids where the most abundant amino acids are alanine and glycine, while histidine and methionine are present in the lowest amounts (Murachi 1964;Ota et al 1964). It appeared to differ significantly in the number of lysine, arginine, and isoleucine compared to ananain and comosain (Napper et al 1994). Ananain possesses more hydrophobicity in the region near histidine-157 and sequence insert between 170 and 174 residues which cannot be found in stem bromelain (Lee et al 1997).…”

“…Moreover, using chromatographic methods, other basic proteolytic (ananain, comosain, F4, F5, and F9) and acidic components of stem bromelain fraction A (SBA/a) and fraction B (SBA/b) have been partially and fully purified (Feijoo-Siota and Villa 2011;Harrach et al 1995Harrach et al , 1998Murachi et al 1964;Napper et al 1994;Rowan et al 1990;Wharton 1974). The molecular weight of purified stem bromelain is 23.40-35.73 kDa, fruit bromelain 31.00 kDa, ananain 23.…”

“…The presence of asparagine-175 hydrogen-bonded with histidine-159 favors the catalytic function in papain. Stem bromelain is distinguished from papain by its lower value of second-order rate constant of inactivation by iodoacetate and iodoacetamide due Table 1 Amino acid composition of stem bromelain, ananain, and comosain (Napper et al 1994) Amino acid Stem bromelain Ananain Comosain Asp 18 19 18 Thr 9 8 7 Ser 17 18 17 Glu 16 13 13 Gly 22 24 25 Ala 25 20 20 Val 14 14 13 Met 3 2 3 IIe 17 14 12 Leu 6 9 9 Tyr 14 12 12 Phe 6 5 7 His 1 2 2 Lys 15 11 10 Arg 6 10 11 Cys 7 7 7 Fig. 1 Catalytic mechanism of cysteine proteinase action.…”

Molecular Cloning, Expression, and Functional Characterization of a Cystatin from Pineapple Stem

“…11) The mechanisms for these pharmacological effects are not yet fully understood. Identification of the proteins and corresponding genes in the pineapple stem is limited; only the bromelain isoforms, 12) ananain, 13) comosain, 13) and bromelain inhibitors 14) have been reported in the literature. Interestingly, no single protein could completely replace the protein extract for its pharmacological effects.…”