Isolation and partial characterization of basic proteinases from stem bromelain

Abstract

Crude bromelain extracts from pineapple stems (Ananas comosus) were fractionated by two-step FPLC-cation-exchange chromatography. At least eight basic proteolytically active components were detected. The two main components F4 and F5 together with the most active proteinase fraction F9 were characterized by SDS-PAGE, mass spectroscopy, multizonal cathodal electrophoresis, partial amino acid sequence, and monosaccharide composition analysis. F9 amounts to about 2% of the total protein and has a 15 times higher specific activity against the substrate L-pyroglutamyl-l-phenylanalyl-l-leucine-p-nitroanilide (PFLNA) than the main component F4. The molecular masses of F4, F5, and F9 were determined to 24,397, 24,472, and 23,427, respectively, by mass spectroscopy. Partial N-terminal amino acid sequence analysis (20 amino acids) revealed that F9 differs from the determined sequence of F4 and F5 by an exchange at position 10 (tyrosine-->serine) and position 20 (asparagine-->glycine). F4 and F5 contained fucose, N-acetylglucosamine, xylose, and mannose in ratio of 1.0:2.0:1.0:2.0, but only 50% of the proteins seem to be glycosylated, whereas F9 was found to be unglycosylated. Polyclonal antibodies (IgG) against F9 detected F4 and F5 with tenfold reduced reactivity. The pH optimum of F4 and F5 was between pH 4.0 and 4.5 and for F9 close to neutral pH. The kinetic parameters for PFLNA hydrolysis were similar for F4 (Km 2.30 mM, kcat 0.87 sec-1 and F5 (Km 2.42 mM, kcat 0.68 sec-1), and differed greatly from F9 (Km 0.40 mM, kcat 3.94 sec-1).

Bromelain: an overview of industrial application and purification strategies

“…Further fractionation by successive use of gel filtration using Sephadex G-75 and ion exchange using carboxymethyl (CM)-Sephadex and diethylaminoethyl (DEAE)-Sephacel successfully separated stem bromelain into six proteolytic components (SBA and SBB 1-5) and two active components of fruit bromelain fraction A (FBA) and B (FBB) (Ota et al 1985). Likewise, several studies on stem bromelain purification using cation-exchange and affinity chromatography yielded nonglycosylated components of stem bromelain, referred to as ananain (Napper et al 1994;Rowan et al 1990) and F9 (Harrach et al 1995), as well as the glycosylated components comosain (Napper et al 1994), F4 andF5 (Harrach et al 1995), and SBA/a and SBA/b (Harrach et al 1998). Meanwhile, Ko et al (1996), Devakate et al (2009), andGautam et al (2010) had employed additional steps of ammonium sulfate precipitation and dialysis prior to ion-exchange chromatography purification to remove most of the impurities in the stem and fruit bromelain samples.…”

“…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.…”

Bromelain: biochemistry, pharmacology and medical use

“…Today bromelain is prepared from cooled pineapple juice by centrifugation, ultrafiltration and lyophilization. The process yields a yellowish powder, the enzyme activity of which is determined with different substrates such as casein (FIP units), gelatine (gelatine digestion units) or chromogenic tripeptides [1,5,6]. In aqueous solution, bromelain rapidly deteriorates through self-digestion.…”

“…They mainly comprise glycosylated multiple enzyme species of the papain superfamily with different proteolytic activities, molecular masses between 20 and 31 kDa, and isoelectric points between > 10 and 4.8. Two major basic proteinases, F4 and F5, were further characterized and showed molecular masses of 24,397 and 24,472 Da, respectively [6] (Table 1). In addition, numerous, different protein fractions were obtained by means of various biochemical methods [SDS-polyacrylamide gel electrophoresis (PAGE), isoelectric focusing (IEF), multicathodal-PAGE].…”

Selective Modulation of Cell Adhesion Molecules on Lymphocytes by Bromelain Protease 5

“…a cysteine proteinase extracted from pine apples [2,3], as well as other proteolytic enzymes [4], Clinical trials have examined the therapeutic efficacy of both single and combined application of these enzymes in individuals with a number of different conditions, includ ing trauma, inflammatory and autoimmune diseases, mastopathy and cancer [1.5, 6]. There is accumulating evidence that these enzymes have clinical utility [1], yet the mechanisms responsible for the pharmacologic effects of these enzymes are not yet fully understood.…”