Enzyme and Microbial Technology 40 (2007) 409413

Characterization of news proteolytic enzymes from ripe fruits of Bromelia antiacantha Bertol. (Bromeliaceae)
D. Vall s a , S. Furtado a , A.M.B. Cantera a,b, e
a

Laboratorio de Enzimas Hidrolticas, Facultad de Ciencias, Igu 4224, Uruguay a b C tedra de Bioqumica, Facultad de Qumica, Gral. Flores 2124, C.C. 1157, a Universidad de la Rep blica (UdelaR), Montevideo, Uruguay u

Abstract Crude extracts were partially puried by organic solvents fractionation: best results (96% of proteins, 91% of total caseinolytic activity) were obtained by adding four volumes of cold acetone to the crude extract. This preparation (redissolved acetone precipitate, RAP) showed maximum activity (>80%) at pH 59, and exhibited high thermal stability (>90% of residual activity after heating for 60 min at 60 C). The enzyme was completely inhibited by E-64 trans-epoxysuccinyl-leucyl-amido(4-guanidino)-butane and iodoacetic acid and activated by the addition of cysteine or -mercaptoethanol; these results strongly suggest that the isolated protease should be included within the cysteine group, as all the other studied proteases belonging to the family Bromeliaceae. IEF-zymogram of RAP showed ve bands (pI 7.3 to <9.3), most of them proteolytically actives, but only three of which (pI 7.6, 8.2 and 8.8) proved to be important. Ion exchange chromatography in DEAE-Sephadex, was selected to separate this active bands. 2006 Elsevier Inc. All rights reserved.
Keywords: Bromelia antiacantha; Fruit proteinases; Cysteine protease

1. Introduction Most plant cysteine peptidases belong to the papain family, including those of Bromeliaceae, the botanical family of pineapple. In recent years, a number of proteases from species belonging to Bromeliaceae have been isolated and characterized: stem and fruit bromealin, ananain and comosain, obtained from Ananas comosus [14], as well as proteases from fruits of Bromelia pinguin [5] B. balansae [6], Pseudonanas macrodontes [7,8] and B. hieronymi Mez [9]. The precise biological role of these cysteine proteases still remains uncertain, but by virtue of the broad substrate specicity they show, it is supposed that they might protect ripening fruits against plant pathogens, especially fungi and insects [10,11]. Much of this proteinases, are extensively used in many industrial processes. They have been exploited commercially in food industry for meat tenderizing (to separate partially connective tissues), brewing (to solubilize grain proteins and stabilize beer), and cookie baking (to improve crispness), as well as the produc-

Corresponding author. E-mail address: acantera@fq.edu.uy (A.M.B. Cantera).

tion of protein hydrolysates [12]. Such preparations are currently employed to produce many foods in which enzymes can replace potentially carcinogenic or otherwise harmful chemicals. Other applications are in tanning, in leather and textile industries, to remove hair, wool, and to soften skins [13]. Pharmaceutical applications of bromelain as a therapeutic compound have been made in 1957: its actions include antitumor properties, immunity modulation, digestive assistance, enhanced wound healing, and cardiovascular and circulatory improvement, among others, even when most of its mechanisms of action are still not completely resolved [14,15]. By their action as anti-inammatory agents and increasing the permeability of the bloodbrain barrier to nutrients and therapeutic agents, plant cysteine proteases, especially bromelain and papain, have shown certain advantages for a perspective application in vivo to Alzheimers disease patients [16]. It supported in a marked interest by the available regional vegetable biodiversity, is sought to obtain proteolytic catalysts that permit their application in different biotechnological processes, as well as the development of new undertakings. In this paper proteolytic enzymes of mature fruits of Bromelia antiacantha Bertol (Bromeliaceae) were isolated and partially characterized.

0141-0229/$ see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2006.07.011

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D. Vall s et al. / Enzyme and Microbial Technology 40 (2007) 409413 e the absorbance value at 337 nm, by modication of the method of Andrew and Asenjo [19]

2. Experimental 2.1. Chemicals

2.6. Protein content determination

Biolyte 310 carrier ampholytes, bovine serum albumin (BSA), Coomassie Brilliant Blue R-250 (CBB), casein (Hammarsten type), cysteine, ethylendiaminetetraacetic acid (EDTA), glycine, iodoacetic acid (IAA), isoelectric point markers (IEF mix 3.69.3), phenylmethylsulfonyl uoride (PMSF), sodium dodecyl sulfate (SDS), trans-epoxysuccinyl-l-leucylamido-(4guanidino)butane (E-64), trichloroacetic acid (TCA), and Tris were purchased from Sigma Chemical Company (St. Louis, USA). Acrylamide, mercuric chloride (HgCl2 ), N,N -methylenbisacrylamide and sulfanilamide-azocasein were purchased from Fluka (Bunchs, Switzerland). DEAE-Sephadex were purchased from Pharmacia Biotech (Uppsala, Sweden). All other chemicals were obtained from other commercial sources and were of the highest purity available. Proteins present in the crude extract and in partial puried fractions were measured according to Bradfords method [20] using BSA as standard. The protein content of chromatography fractions was estimated by absorbance at 280 nm.

2.7. Optimum pH
Redissolved acetone precipitate (RAP) caseinolytic activity was measured at 37 C using different pH values. The buffers used were: citric acidcitrate buffer 0.1 M, pH 4; phosphate buffer 0.1 M pH 56 and TrisHCl 0.1 M, pH 711.

2.2. Plant material

B. antiacantha Bertol. (commonly called gravat or banana-do-mato), is a a terrestrial plant that arrives at 2 m height. It has short stem, long leaves with abundant protective thorns ready in rosettes. Flowers emerge in the center of the leaves, which acquire an intense reddish tone in the internal part. Mature fruits have an orange yellow color and the form is like a small banana. They are commonly found in Rocha department (Uruguay), in humid grounds of sandy regions and in recovery degraded vegetation.

2.8. pH stability
To measured pH stability, the RAP was diluted (1/10) in the appropriated buffers (pH 311). Samples (0.1 ml) were incubated at 10 C for 0209 h and the residual caseinolytic activity was assayed according to the method used for proteases described above.

2.9. Thermal stability

For testing heat stability, RAP was incubated at different temperatures ranging from 25 to 70 C for 2, 5, 10, 20, 40, 60 and 120 min. The residual caseinolytic activity was measured under standard assay conditions.

2.3. Crude extract preparation

Crude extracts (CE) were obtained by chopping and homogenizing frozen fruits (50 g) for 1 min in an Mixer with 250 ml of cold 0.1 M sodium phosphate buffer pH 6.0 contained 5 mM EDTA and 5 mM cysteine (nal concentration) in order to avoid phenoloxidase activity and oxidation, respectively. Homogenates were ltered through a two-folded piece of gauze to remove plant debris, and then centrifuged for 30 min at 16,000 g. Supernatants were collected, ltered when needed, and immediately frozen at 20 C until analysis. All operations were carried out at 04 C.

2.10. Optimum temperature

In order to determine optimum temperature, the caseinolytic activity of the RAP was measured in the temperature range 3785 C.

2.11. Ion strength stability

The ability of the redissolved acetone precipitate (RAP) to retain its activity under growing ionic strength, was tested by exposing it at different concentrations of sodium chloride (0.42.5 M) during 1 h at 37 C with casein as substrate as previously described.

2.4. Preliminary purication of crude extract

Crude extract (CE) was treated with one to ve volumes of cold (20 C) acetone with gentle agitation and left to settle for 20 min at 20 C, before centrifugation at 16,000 g for 30 min. The nal acetone precipitate was redissolved with one volume of 0.1 M phosphate buffer, pH 6.0, and frozen until further use [17].

2.12. Effect of inhibitors and activators

The effect of specic inhibitors [21] on proteolytic activity was determined by measuring the residual activity of RAP at pH 8.0 after preincubation at 37 C for 30 min. The inhibitors assayed was: 1 mM PMSF; 0.1 mM iodoacetic acid; 0.01 mM E-64; 1 mM HgCl2 ; 10 mM EDTA. After preincubation period (30 min) with HgCl2 (10, 20 and 30 mM) inhibition reversion of RAP proteolytic activity was tested by the addition of -mercaptoethanol (15 mM nal concentration). To prove the effect of activators, azocaseinolytic activity was also assayed in the presence of 15 mM -mercaptoethanol.

2.5. Proteolytic activity determination

The reaction mixture contained 0.1 ml of crude extract and 1.1 ml of 1% casein in a 0.1 M TrisHCl buffer (pH 8.0) with 15 mM cysteine (nal concentration). The reaction was carried out at 37 C and stopped 20 min later by adding 1.8 ml of 5% (w/v) trichloroacetic acid (TCA). Each test tube was centrifuged at 3000 g for 30 min and the absorbance of the supernatant was measured at 280 nm. One caseinolytic unit (Ucas ) was dened as the amount of protease that produces an increment of one absorbance unit per minute in the assay conditions [18]. Azocasein was the substrate used in inhibition assays with PMSF, E-64, HgCl2 , IAA, EDTA and in the anion exchange chromatographic steps. For this determination, the reaction mixture contained 340 l of an appropriate dilution of the enzyme preparation, 340 l of azocasein solution (1%, w/v, in distilled water) and 340 l of activity buffer (0.1 M TrisHCl buffer, pH 8.0) was incubated for 20 min at 37 C. The reaction was stopped by adding 340 l of TCA (10%, w/v, in distilled water). After centrifuging for 20 min at 20,600 g, absorbance was measured at 337 nm. In this case, one enzymatic unit (EU) was dened as the quantity of enzyme required to increase one absorption unit

2.13. Isoelectric focusing (IEF) and zymogram

Isoelectric focusing (IEF) was developed in 5% polyacrylamide gels containing broad pH range ampholytes (biolyte 310, Bio-Rad) in a Mini IEF Cell (Model 111, Bio-Rad). Samples was precipitated with four volumes of cold (20 C) acetone and redissolved in deionized water twice. About 110 g of protein was loaded in each case. Focusing was carried out under constant voltage conditions in a stepped procedure: 100 V for 15 min, 200 V for the following 15 min, and 450 V for the last 60 min. One of the gels was xed and stained with Coomassie Brilliant Blue R-250, while the other, unstained, was contacted for 20 min at 56 C with an agarose

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Fig. 1. Effect of pH on proteolytic activity of RAP (range pH 411). The proteolytic activity was assayed using casein as substrate.

Fig. 2. Stability at different pH values (311) on the proteolytic activity of RAP.

gel imbibed in 1% casein solution, in order to detect the bands with proteolytic activity [22]. After incubation, the agarose gel was dehydrated and stained with Coomassie Brilliant Blue R-250.

2.14. Crude extract chromatographic prole

The column (20 mm 500 mm), packed with DEAE-Sephadex was equilibrated with 50 mM TrisHCl buffer pH 8.5 and RAP (4.5 ml) was applied. After adding 25 ml of the equilibrating buffer, chromatography was developed by elution of the bound material with a linear gradient of sodium chloride (00.3 M) in the same buffer with a ow rate of 2 ml/min. Protein concentration and enzyme activity were determined in the eluted fractions, and the corresponding proles were drawn.

3. Results and discussion The crude extract (CE) was obtained from ripe fruits, and its proteolytic activity was determined. This activity was unaffected (100% activity) by prolonged storage at 20 C (nal check carried out at 180 days). Maximum activity was 200 Ucas per gram of fruit, and maximum specic activity was 20 Ucas per mg of protein in presence of cysteine (15 mM nal concentration). As CE usually contained phenolic compounds, which could oxidize and irreversibly react with proteins, a previous treatment with different volumes of acetone (15) were done. Best yield was obtained by adding four volumes of solvent. The redissolved acetone precipitate (RAP) contained 96% of proteins and 91% of total caseinolytic activity with respect to CE. On basis of these results, the RAP was selected for enzyme purication and partial characterization. This preparation exhibited high caseinolytic activity (higher than 80%) in a broad pH range (59), with two maximum: at pH 6.0 and 9.0 (Fig. 1). The enzymes was unaffected by incubations at 10 C during 50 h at different pH range (49) (Fig. 2) As can be seen in Fig. 3, no activity loss was observed when RAP was incubated at 37 C during 180 min or at 55 C during 60 min, while at 60 C after 30 min remained 80% of initial activity. The enzyme was almost completely inactivated by heating 30 min at 65 C. In spite of this, the optimum temperature was observed at 63 C (Fig. 4). Low sodium chloride concentrations (0.2 M) does not affect caseinolytic activity, but diminishes with the increase of salt concentration (28% of residual activity at 2.5 M NaCl) (Fig. 5).

Fig. 3. Thermal stability of the proteases of RAP. The proteolytic activity was assayed at 37 C with casein as substrate.

In order to establish to which mechanistic class belonged the proteases present in the RAP, different proteolytic enzyme inhibitors were tested. The data shown in Fig. 6 reveal that the enzymatic preparation was completely inhibited by E-64 (0.01 mM), IAA (0.1 mM) and HgCl2 (1 mM) but unaffected by the action of PMSF (1 mM) and EDTA (10 mM). The mercuric chloride (10 and 20 mM) inhibition was completely reverted by addition of cysteine (15 mM), but it had no effect on inhibition provoked by 30 mM of the same one (Fig. 7). The addition of mercaptoethanol (15 mM) notably increase proteolytic activity of RAP (more than three-fold). These results strongly suggest that the enzyme preparation contains cysteine-type proteases, as

Fig. 4. Effect of temperature on proteolytic activity of RAP. Proteolytic activity were made at those temperature values with casein as substrate at pH 8.

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Fig. 5. Effect of ionic strength on the proteolytic activity of RAP using different concentrations of sodium chloride (0.02.5 M) during 1 h at 37 C.

Fig. 8. Isoelectric focusing and zymogram of RAP. (1) pH markers; (2) redissolved acetone precipitate (RAP); (3) RAP zymogram.

Fig. 6. Effect of inhibitor on the proteolytic activity of RAP incubated on each inhibitor solution during 30 min at 37 C. (1) RAP without inhibitor; (2) RAP and PMSF (1 mM); (3) RAP and EDTA (10 mM); (4) RAP and IAA (0.1 mM); (5) RAP and HgCl2 (1 mM); (6) E64 (0.01 mM).

all the other studied proteases belonging to the family Bromeliaceae [23]. Isoelectric focusing of RAP, showed ve protein bands (pI between 7.3 and 9.0). The zymogram analysis (specic devel-

oped for proteases) [22] showed only three of which (pI: 7.6; 8.2; 8.8) was proteolytically actives (Fig. 8). On the basis of these results, anion exchange chromatography was selected for the next purication step. Anion exchange chromatography on DEAE-Sephadex of RAP (Fig. 9) allowed the separation of the main proteolytic component. A non-retained fraction including the active pI 7.6 fraction eluted before the application of the saline gradient and

Fig. 7. Effect of HgCl2 on proteolytic activity of RAP and subsequent addition of -mercaptoethanol (15 mM) at the reaction mixture. (1) RAP; (2) RAP and HgCl2 (10 mM); (3) RAP and HgCl2 (20 mM); (4) RAP and HgCl2 (30 mM). The proteolytic activity was assayed with azocasein as substrate.

Fig. 9. Ion Exchange chromatography in DEAE-Sephadex (start buffer: TrisHCl 50 mM, pH 8.5 (20 C); NaCl linear gradient (0.00.3 M) in the same buffer.

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two more active fractions eluted when sodium chloride gradient reached 0.14 M and 0.24 M.
[10]

Acknowledgements
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The present work was supported by CYTED (Project IV.22) and PEDECIBA Qumica. Authors wish thank to Mariela Bruno, Laura M. L pez for very helpful technical assistance. o References
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