Evaluation of the presence of aspartic proteases from Centaurea calcitrapa during seed germination

Enzyme and Microbial Technology 38 (2006) 893–898

Evaluation of the presence of aspartic proteases from Centaurea calcitrapa during seed germination Sofia Matos Salvador, Carlos Novo, Ana Domingos ∗ UTPAM, Departamento de Biotecnologia, Instituto Nacional de Engenharia Tecnologia e Inova¸ca˜ o, Estrada do Pa¸co do Lumiar, 22, 1649-038 Lisboa, Portugal Received 2 February 2005; received in revised form 14 June 2005; accepted 21 June 2005

Abstract Aspartic proteinases are present in a variety of organisms including plants. Common features of aspartic proteases include an active site cleft that contains two catalytic aspartic residues, acid pH optima for enzymatic activity, inhibition by pepstatin A. Plant aspartic proteinases occur in seeds and may be involved in the processing of storage proteins. Many of them have been purified and characterized. The presence of aspartic proteases in seeds of Centaurea calcitrapa during germination was investigated by measuring the activity on enzyme extracts. The aspartic proteases are present mainly in the beginning of seed germination suggesting that they could initiate the degradation of protein reserves in germinating seeds. These proteases were purified by salt precipitation followed by anion-exchange chromatography. Purified aspartic proteases have an optimal pH between 3.5 and 4.5, using FTC-hemoglobin as substrate and an optimal temperature at 52 ◦ C. The ability of seed extracts for milk clotting was tested and the clotting time that was achieved is in the same range found for flower extracts appropriated for special cheeses in which weak clotting agents are required. © 2006 Elsevier Inc. All rights reserved. Keywords: Centaurea calcitrapa; Aspartic proteases; Seed germination; Milk clotting

1. Introduction Proteinases play an important role in biotechnology since proteolysis modifies the chemical, physical, biological and immunological properties of proteins. Some plant proteinases are used in the food industry, in manufacturing cheeses and drinks, meat tenderizing, cookie baking and the production of protein hydrolysates [1]. Almost all enzymes employed commercially in milk coagulation are aspartic proteinases (APs; EC 3.4.23); they are most active at acidic pH, are specifically inhibited by pepstatin A and contain two aspartic residues indispensable for catalytic activity [2]. APs are widely distributed in a variety of organisms such as viruses, some bacteria, yeast, fungi, plants and animals [2,3]. According to the MEROPS database (http://www. merops.ac.uk), created by Rawlings and Barrett [4], APs are grouped into 14 different families, based on their amino acid

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sequence homology, which in turn are assembled into 6 different clans based on their evolutionary relationship and tertiary structure. Plant APs have been distributed among families A1, A3, A11 and A12 of clan AA, and family A22 of clan AD. The majority of plant APs belongs to the A1 family, together with pepsin-like enzymes from many different origins. The three-dimensional structure of two plant aspartic proteinases has been determined, sharing significant structural similarity with other known structures of mammalian aspartic proteinases [5,6]. With few exceptions, the majority of plant aspartic proteinases identified so far are synthesized with a prepro-domain and subsequently converted to mature enzymes. A characteristic feature of the majority of plant aspartic proteinase precursors is the presence of an extra protein domain of about 100 amino acids known as the plant-specific insert [7]. Plant APs have been detected and purified from many different plant species. However, their biological functions are not as well assigned or characterized as those of their mammalian, microbial or viral counterparts that were shown to perform many different and diverse functions. For the great majority of plant APs, no definitive role has been defined and the biological functions are still hypothetical. In general, plant APs have been

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implicated in protein processing and/or degradation in different plant organs, as well as in plant senescence, stress responses, programmed cell death and reproduction [7]. APs have been purified and characterized from a number of seeds including rice [8], barley [9], cucumber, squash [10] and sunflower [11], and from leaves of the tomato plant [12], and flowers of thistle [13,14]. Participation of plant APs in storage protein degradation during the mobilization of reserve proteins in seed germination has been proposed for rice and wheat. The involvement of plant APs in proteolytic processing and maturation of storage proteins has also been proposed. It was suggested that castor bean and barley APs might be involved in the maturation process of proprotein precursors in concert with the vacuolar processing enzyme [15,16]. Similar results were also obtained with AP from Brassica napus seeds [17]. In some countries like Portugal and Spain, APs of Asteraceae flowers are used in the production of homemade cheeses with organoleptic features different from those obtained with bovine chymosin or microbial rennins. APs of those flowers have been thoroughly studied from the biochemical, kinetic, molecular and structural points of view [5,13,14,18–23] as on casein proteolysis or during cheese ripening [24–29]. We have previously reported studies on milk-clotting activity of flowers and cell suspensions of the Asteraceae, Centaurea calcitrapa [24,28]. C. calcitrapa is a plant widely distributed in Portugal that accumulates APs in all parts of the plant, particularly in its flowers [14]. Its milk-clotting activity has been exploited, in a similar way as in Cynara species, for the manufacture of cheese [25–27,29]. The purpose of this work was to characterise the presence of APs from C. calcitrapa during seed germination and evaluate their capability to be used as a milk-clotting agent as it was done before for other tissues. 2. Materials and methods 2.1. Plant material Seeds of C. calcitrapa were obtained from dried flowers collected in Portugal during flowering season.

2.2. Seeds germination Seeds were soaked in water prior to washing in a 10% (v/v) detergent solution for 10 min, followed by surface sterilisation with 70% (v/v) ethanol for 30 s and 6% (v/v) sodium hypochloride for 5 min. After rinsing four times with sterile water, the seeds were germinated on solid Schulz medium [30], without growth regulators, at 25 ◦ C in the dark. Seeds were sampled 1–5 days after the start of germination.

2.3. Enzyme extraction Seeds were finely ground with liquid nitrogen and suspended in 50 mM Tris, 5% (w/v) polyvinylpyrrolidone, pH 8.1. For purification purposes inhibitors of proteases were added to the extraction buffer: 0.1 mM PMSF (inhibitor of serine proteases), 1 mM EDTA (inhibitor of metalloproteases) and 10 ␮M iodoacetamide (inhibitor of cysteine proteases). After homogenisation, extracts were cleared by ultracentrifugation at 80,000 × g, for 20 min, at 15 ◦ C and stored at −70 ◦ C, for long-term storage.

2.4. Protease assay Proteolytic activity was measured according to the method of Twining [31] using ␬-casein fluorescein isothiocyanate labelled (FTC-casein) as substrate. Substrate solution (containing 8.5 ␮g FTC-casein and 0.65 FTC residues per casein molecule) was added to 0.2 M citrate, pH 5.1. The reaction was started by adding the enzyme solution; the assay mixture was incubated at 37 ◦ C, and after 30 min incubation, 5% (w/v) trichloroacetic acid was added to stop the reaction. The relative fluorescence of assays was measured in a Hitaschi 3000 spectrofluorometer (excitation at 495 nm; emission at 525 nm) calibrated (100% relative fluorescence) with an assay mixture in which the enzyme solution had been substituted with buffer and TCA substituted with H2 O. As a standard procedure, different sample dilutions were done to give an appropriate enzyme activity in the standard assay. One unit of enzyme was defined as that giving an increment of 1.0 fluorescence unit, under the described conditions.

2.5. Enzyme purification Aspartic proteases from seeds of C. calcitrapa were purified by a multistep procedure. The protocol purification used was based in the work previously described by Domingos and co-workers in 2000 [14]. Enzyme extracts were cleared by ultracentrifugation (80,000 × g for 20 min at 4 ◦ C) and the supernatant further subjected to fractionation by increasing the concentration of ammonium sulphate. The ammonium sulphate was added to the supernatant to give a saturation concentration of 30%. After removing the precipitate by centrifugation, salt concentration was increased stepwise to 100% saturation. The pellet was resuspended in extraction buffer and dialysed at 4 ◦ C, overnight, against the same buffer. The resulting extract was applied to a Q-Sepharose 16/10 column (Amersham Biosciences) pre-equilibrated with buffer A. Elution was achieved with a linear gradient of buffer A to 1 M NaCl, in the same buffer. The flow rate was 1 mL/min and fractions (1 mL) were assayed for proteinase activity and pooled. After dialysis, sample was loaded on a Mono Q HR 5/5 column (Amersham Biosciences). Elution was achieved using a 0–1 M NaCl gradient in buffer A. Eluted fractions were collected and assayed for protease activity as above. In parallel with this purification procedure, an affinity chromatography step was also assayed. The enzyme extracts were diluted 1:2 in equilibrating buffer before loaded (0.1 mL/min) onto the pepstatin–agarose column (Sigma, 1 mL) equilibrated with 50 mM citrate buffer, pH 4.2. The column was washed with 3 volumes 50 mM citrate buffer, 0.5 M NaCl, pH 4.2, 15 volumes 50 mM citrate buffer, 1.5 M NaCl, pH 4.2, and 6 volumes 0.1 M potassium phosphate, 0.5 M NaCl, pH 7.5. Elution was performed with 0.1 M sodium bicarbonate, pH 10, containing 0.5 M NaCl (0.5 mL/min) and the collected fractions (1 mL) were assayed for proteinase activity. Elution was also tested using a higher salt concentration (1.5 M NaCl) in the same buffer.

2.6. Milk-clotting activity Clotting activity was measured as the time spent in milk clotting at 30 ◦ C, after the enzyme addition, according to the Norma FIL-IDF 110A [32]. Protease extract was added to 10 mL of standardized milk containing 10 mM CaCl2 and incubated at 30 ◦ C in a test tube. The coagulation point was determined by manual rotating of the test tube periodically.

2.7. Determination of optimal pH The effect of pH on aspartic proteolytic activity was measured using the same procedure but with FTC-hemoglobin as substrate. Purified proteinase extracts were pre-incubated for a period of 30 min, at 37 ◦ C in 0.2 M sodium borate buffer over the pH range of 3.0–7.0 and 0.1 M Tris (pH 8–10). After this incubation time, the substrate was added. For each pH, a blank containing no enzyme was used to determine non-enzymatic release of TCA-soluble fluorescent substances.

2.8. Optimal temperature determination In order to determine the optimal temperature for aspartic proteolytic activity, purified proteinases were assayed at 25, 37, 42, 47, 54, 60 and 70 ◦ C, during

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30 min. Proteolytic activity was measured as described previously. Blanks were done for each temperature.

2.9. Inhibition assays The effect of selective inhibitors (leupeptin, PMSF, aprotinin, cystatin, iodoacetamide, o-phenantroline and pepstatin A, all from Sigma) on proteinase activity was tested. The inhibitors were added to the enzyme solution (seed extracts corresponding to the first day of germination) and the mixtures were pre-incubated for 30 min, at 37 ◦ C before proteinase activity assay. Controls were prepared by pre-incubating the enzymatic preparation with the solvent used to dissolve inhibitors.

2.10. Protein quantification Protein concentration was determined according to the method of Bradford [33], using bovine serum albumin as the standard. Samples were incubated at room temperature and absorbance read at 595 nm using a Shimadzu spectrophotometer.

2.11. SDS-polyacrylamide gel electrophoresis Samples were analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) using 15% (w/v) acrylamide, according to Laemmli [34]. The proteinase activity on gel was detected by adding heat denaturated hemoglobin (0.05% (w/v), final concentration) to acrylamide solution prior to polymerisation of the gel mixture [35]. Samples were incubated with different proteases inhibitors (0.1 mM PMSF, 1 mM EDTA and 10 ␮M iodoacetamide) as described above. As a control 10 ␮M pepstatin A was added to samples in a second gel. After electrophoresis, the gel was washed three times, during 15 min in 50 mM Tris, pH 6.8, containing 0.05% (v/v) Triton 100 X, and incubated overnight, at 40 ◦ C in 50 mM Tris, pH 6.8. Gels were stained with 0.2% (w/v) Coomassie Brilliant Blue R and destained in 50% (v/v) methanol/10% (v/v) acetic acid.

Fig. 1. Specific proteolytic activity of seeds of C. calcitrapa, measured at different time points corresponding to days 0–5 of germination. Proteolytic activity was measured using casein as substrate. The points are the means of three replicate determinations. Bars represent mean standard deviation.

aspartic proteases are present mainly in the beginning of seed germination (Fig. 2a). These data are according to the abovementioned results obtained measuring the aspartic proteolytic activity directly on the seed extracts (Fig. 1). As a control, samples were also incubated before loaded, with pepstatin A (Fig. 2b) confirming that the proteolytic bands, observed in Fig. 2a, are mainly due to aspartic proteases.

3. Results and discussion The main purpose of this work was to study the presence of the aspartic proteases from seeds of C. calcitrapa during germination. In order to achieve this goal, extracts from non-germinated seeds and seeds collected at different germination times were analysed. Samples (seed crude extracts) were homogenised in extraction buffer containing protease inhibitors, as referred above, and assayed for proteolytic activity. Since it was used inhibitors for the other classes, this assay shows the proteolytic activity due only to proteinases from aspartic class. Obtained results suggest that the proteolytic activity is higher on the first day of germination (Fig. 1). The presence of APs during germination was also investigated by observing the activity of the enzyme extracts on an acrylamide gel containing 0.05% (w/v) hemoglobin. Samples corresponding to non-germinated seeds and samples collected after 1–5 days of germination were incubated with inhibitors for cysteine, serine and metalloproteases before loaded on the gel (Fig. 2a). After incubation, overnight at pH 6.8, proteolytic activity appeared as negative bands upon staining with Coomassie Brilliant Blue. Results suggest that proteolytic activity on the acrylamide gel was stronger for samples corresponding to seeds at the first and second days of germination, indicating that the

Fig. 2. Detection of proteolytic activity of seed extracts after 0–5 days of germination on acrylamide gel containing 0.05% (w/v) hemoglobin. The gel was stained with Coomassie Blue. Samples were incubated with: (a) inhibitors for cysteine (10 ␮M iodoacetamide), serine (0.1 mM PMSF) and metalloproteases (1 mM EDTA), and (b) inhibitor for aspartic proteases (10 ␮M pepstatin A). M: molecular weight marker (Amersham Biosciences).

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Table 1 Effect of selective inhibitors on the activity of seed extracts of C. calcitrapa assayed on the first day of germination Inhibitors None Aprotinin Cystatin Iodoacetamide Leupeptin PMSF Pepstatin A o-Phenantroline

Target proteases

Concentration

Inhibition (%)

Serine proteinases Cysteine proteinases Cysteine proteinases Cysteine and some serine proteinases Serine proteinases Aspartic proteinases Metalloproteinases

3.40 U/ml 0.50 mM 0.50 mM 0.50 mM

100 7 43 35 30

1.00 mM 0.01 mM 10.0 mM

7 92 8

Reactions were carried out at 37 ◦ C using casein as substrate. The inhibitor concentrations refer to the pre-incubation mixtures. Data are averages of three replicate determinations.

Inhibition assays using the appropriated inhibitors for the different protease classes were performed using the enzyme extracts corresponding to the first day of germination. As it can be observed in Table 1, PMSF, o-phenantroline and aprotinin had only a low effect on the enzyme activity at high concentrations, while pepstatin A (a specific inhibitor of aspartic acid proteases) showed to inhibit the proteolytic activity in a large extent (≈90%). These inhibition data indicates that the proteolytic activity is mainly due to the presence of APs. However, for cysteine proteases it was also found a quite high level of inhibition (30–40%). These results are in accordance with what was previously published by authors working with other plants, namely by Zhang and Jones [36] reporting that 42 proteases are involved in germination of barley seeds and among them, 27 are cysteine proteases. Proteases from the other classes (serine and metalloproteases) seem to be much less active at this germination stage. APs are present in most, if not all, resting seeds, but they have gained much less attention than cysteine proteinases, which are dominant in germinating seeds and play a major role in the mobilization of reserve proteins [36,37]. The presence of the APs in resting seeds led to the hypothesis that they could initiate the degradation of protein reserves in germinating seeds before the massive de novo biosynthesis of cysteine proteinases. However, the question of how the hydrolysis of storage protein is initiated and controlled at the very beginning of germination remains to be clarified [9,38,39]. Accordingly, the results here obtained, indicating that the APs from seeds of C. calcitrapa are more active in the beginning of germination, suggest that they may be involved in degradation of protein reserves. In rice seeds it was proposed that the APs could be involved in the hydrolysis of c-globulin during the initial stage of germination [8]. A similar role was suggested for wheat seeds APs based on its ability to hydrolyze, in vitro, the main wheat storage protein, gliadin. Localization of both proteins in the wheat seed endosperm also favored this hypothesis [40]. In the case of phytepsin from barley seeds and the AP purified from A. thaliana seeds, it was suggested also the concerted participation of these plant APs and other proteases in protein-storage processing mechanisms [16,41]. Most APs occurring in seeds have been purified and their enzymatic properties have been investigated [6,9,11].

For further characterization, APs from seeds of C. calcitrapa were extracted and purified in the presence of proteases inhibitors to assure that only proteases belonging to the aspartic classes were detected during the purification steps. For the purification of APs from seeds it was used a multi-step purification procedure, as above referred. Affinity chromatography on pepstatin A–agarose (Sigma) has been chosen as a technique to purify several APs from seeds [8,11,15,17,41]. However, this procedure revealed to be unsuccessful for the purification of APs from C. calcitrapa seed extracts. Even using different elution conditions it was not possible to detect any enzyme activity on eluted samples. This result was probably due to an irreversible binding between enzyme and inhibitor. In order to be sure that the absence of activity on the eluted fractions was not only due to a low concentration, samples were concentrated on Centricon-3 microconcentrators (Amicon) before assayed for proteolytic activity. The same behavior was found for APs from flowers and leaves of C. calcitrapa [14]. Thus, the purification procedure used for APs from seeds was mainly based in which was previously described [14]. Seed extracts were precipitated using ammonium sulphate; for further purification it was chosen the fraction with 30–80% ammonium sulphate concentration, since it was the fraction that showed a higher proteolytic activity. After salt precipitation, the resuspended pellet was dialyzed and applied to a Q-Sepharose column. Fractions containing proteolytic activity were eluted at 500 mM NaCl in the extraction buffer, containing proteases inhibitors. After ultrafiltration, fractions were loaded to the anion-exchange Mono Q column. Fractions showing proteolytic activity were eluted at 10 mM NaCl. The yield obtained at the end of the purification process was 63% with 37-fold purification. For pH determination hemoglobin was used as substrate instead of casein since this protein precipitates at low pH values. On these assays as well as on optimal temperature determination, were used purified extracts. The pH optimum of purified APs from seeds of C. calcitrapa was detected between pH 3.5 and 4.5 (Fig. 3), which is similar to that of other aspartic proteinases [41–43]. Experiments on pH dependence done with dried flow-

Fig. 3. Effect of pH on activity of aspartic proteinases activity. Proteolytic activity was measured using FTC-hemoglobin as substrate. Purified proteinases extracts were pre-incubated for a period of 30 min, at 37 ◦ C in 0.2 M sodium borate buffer (pH 3.0–7.0) and 0.1 M Tris buffer (pH 8–10). The points are the means of three replicate determinations. Bars represent mean standard deviation.

S.M. Salvador et al. / Enzyme and Microbial Technology 38 (2006) 893–898

Fig. 4. Effect of temperature on activity of aspartic proteinases. Proteolytic activity was measured using casein as substrate. Purified proteinases extracts were incubated during 30 min, at 25, 37, 42, 47, 54, 60 and 70 ◦ C. Blanks were done for each temperature. The points are the means of three replicate determinations. Bars represent mean standard deviation.

ers, dried leaves [14], cell cultures [44] and hairy roots [45] from C. calcitrapa showed optimal pH values in the same range of those found for seeds. As expected, the activity of purified APs from seeds of C. calcitrapa was higher at high temperatures, i.e., for 50–60 ◦ C (Fig. 4). The same behavior was found for APs from other C. calcitrapa tissues [14]. Besides the involvement of APs in fundamental processes, this class of enzymes is very interesting because of the ability of some of them to clot milk. This feature of APs from flowers of Cynara cardunculus was utilized in the manufacturing of Portuguese cheese, and their APs have been extensively studied in order to improve its use as a milk-clotting agent [29,46,47]. The degradation of caseins from milk of different species by APs from flowers and cell suspension extracts of C. calcitrapa was studied to compare degradation patterns obtained with the extracts thereof with those of a commercial rennet. Those extracts exhibit higher specificity toward ovine and caprine caseinates, suggesting that they can be used as alternative rennet to produce cheeses from caprine and/or ovine milk [29,33]. The ability of extracts from C. calcitrapa seeds for milk clotting was also tested. Crude extracts corresponding to the first day of germination were used since the most prominent proteolytic activity was detected at this stage. The clotting time for these extracts (180 U/mg total protein) was 7.5 h. This value is similar to the clotting time achieved for C. calcitrapa flowers extracts [24] and lower than the values found by some authors working, respectively, with Cynara, and buckwheat extracts [42,46]. This result allows us to suggest that the extracts studied could be used in special cheeses in which weak clotting agents are required as an alternative/substitute for animal chymosine. However, additional studies are needed before application in dairy processing. References [1] Uhlig H. Plant proteases. In: Industrial enzymes and their applications. NY: John Wiley & Sons, Inc.; 1998. p. 147–51.

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