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Introduction
Biotechnological advances have contributed to the diversification of the use of proteases and to favoring their demand (Ramli, Aznan, & Illias, 2017), which has generated the need to explore new sources. Proteases of plant origin, such as papain and bromelain, are widely accepted in the cosmetics, food, pharmaceutical and alcoholic beverage industries (Arshad et al., 2014; de Lencastre et al., 2016). However, there are currently proteases that have not yet been studied in depth, but with possible biotechnological applications, such as those contained in species of the family Bromeliaceae (Natalucci et al., 2009), specifically in Bromelia karatas L. (synonym: Bromelia plumieri [E. Morren] L. B. Smith) (Espejo-Serna, López-Ferrari, & Ramírez-Morillo, 2005).
Bromelia karatas is distributed in the Neotropics from the Caribbean islands to Ecuador and Brazil, including Central America. Distribution in Mexico covers 12 of its 32 states, including Yucatán (Espejo-Serna & López-Ferrari, 2010). Bromelia karatas plants grow in the types of tropical rainforests described for the Yucatán peninsula (Miranda, 1958) and even in strips of secondary vegetation near roads, where soils are shallow. This species has anatomical and morphological adaptations that allow it to tolerate periods of drought (González-Salvatierra, Andrade, Orellana, Peña-Rodríguez, & Reyes-García, 2013). The fruit is a bittersweet, juicy berry with fibrous skin and yellowish white, pink or dark brown colors; average production is 77 fruits per plant, representing a total weight of approximately 1.4 kg (Montes, Terán, Zúñiga, & Caldón, 2014).
In the state of Yucatán, the fruits of B. karatas are consumed, usually accompanied by salt and chili and previously boiled; the latter in order to avoid mouth ulcers caused by cysteine protease. This protease has been named and described as karatasin (Montes, Amador, Cuevas, & Córdoba, 1990), which has kinetic characteristics equivalent to those contained in Bromelia pinguin L. (Meza-Espinoza et al., 2017), and is analogous to bromelain and papain (Montes et al., 1990); however, it is still unknown whether there are more proteases that contribute to proteolytic activity. There are also no records of B. karatas accessions, which would be useful for their cultivation and use as a source of proteases. For this reason, it is important to identify fruit producing plants with high proteolytic contents for breeding purposes.
In this study, the effect of temperature, pH and NaCl on the proteolytic activity of wild fruits of B. karatas was evaluated, the thermal stability of proteases was determined and their molecular weights and isoelectric points were estimated by means of two-dimensional zymography, with the purpose of generating information for biotechnological processes.
Materials and methods
Sampling of plant material
In December 2017 and January 2018, B. karatas fruits were collected in three locations in the state of Yucatán, Mexico: Oxkutzcab (collection 1), Espita (collection 2) and Mérida (collection 3). In each location, 10 fruits were obtained from each of seven plants; the fruits contained 12 °Brix at the time of cutting. Honey pineapple (Ananas comosus [L.] Merr. variety comosus), acquired in a local supermarket, was used as a control.
Evaluation of proteolytic activity
Of the 70 fruits collected per location, 10 fruits were randomly selected to make a homogeneous pulp in a food processor; subsequently, the pulp was centrifuged at 14 000 g for 15 min at 4 °C. The protein concentration was determined by the Bradford method using the commercial Quick StartTM Bradford Protein Assay kit from the BIO-RAD company, according to the manufacturer's instructions. The fruit juice was frozen at -80 °C until use.
Proteolytic activity was quantified following the methodology proposed by Natalucci, Brullo, Lopez, Hilal, and Caffini (1996). The reaction mixture consisted of 1.1 mL of a 1 % (w/v) casein solution in 0.1 M phosphate buffer at pH 7, 5 mM of cysteine and 0.1 mL of fruit juice. This mixture was incubated at 37 °C for 20 min and 1.8 mL of 5 % trichloroacetic acid (TCA) were added to stop the reaction; it was then centrifuged at 7 000 g for 20 min and the absorbance of the supernatant was measured at 280 nm. The blank was a non-incubated reaction mixture plus 1.8 mL of 5 % TCA. An enzyme unit (U) was defined as one millimole of tyrosine per minute under the conditions of the described assay.
Evaluations of the effect of pH, incubation temperature, thermal stability and NaCl concentration on proteolytic activity, as well as the estimation of molecular weights and isoelectric points of proteases, were performed only for the collection with the most activity.
Effect of incubation temperature, pH and NaCl on proteolytic activity
The effect of incubation temperature (30, 40, 50, 60 and 70 °C) at a constant pH of 7 and the effect of pH (6, 7, 8, 9, 10 and 12) at a constant temperature of 37 °C were compared. To determine the effect of pH, the following buffers were used: 0.1 M 2-morpholinoethanesulfonic acid (MES) for pH 6; 0.1 M phosphates for pH 7 and 12; and 0.1 M Tris-HCl for pH 8, 9 and 10. Proteolytic activity was measured according to the methodology of Natalucci et al. (1996). The effect of NaCl was evaluated at 5, 10 and 20 % (w/v). The result was expressed as the percentage related to the proteolytic activity observed in the absence of NaCl (Moreno-Hernández et al., 2017).
Thermal stability of proteases
The thermal stability of the proteases of B. karatas fruit juice was evaluated at 30, 50 and 70 °C; residual proteolytic activity under standard conditions (Natalucci et al., 1996) was determined at 0, 30, 60, 120, 180 and 240 min, and expressed as the relative percentage to the proteolytic activity without prior incubation.
Statistical analysis
In the comparison of the three collections, the proteolytic activity response variable was measured as enzyme units per mL of juice and enzyme units per mg of protein, expressed as the mean ± standard deviation. Proteolytic activity was evaluated at five temperatures, six pH levels, and three NaCl concentrations. Thermal stability was analyzed graphically. Experimental data were compared with the one-factor analysis of variance (n = 4 and α = 0.05); subsequently, Tukey's multiple comparison test (α = 0.05) was performed (Zar, 1999). The data analysis was done with the R program (R Core Team, 2017).
Two-dimensional zymography with casein
Proteases in B. karatas extract were detected by two-dimensional zymography with casein under non-reducing conditions. The protein concentration was determined according to the method described by Bradford (1976), using bovine serum albumin as the protein standard.
The isoelectrofocus (IEF) was performed with strips prefabricated with ampholines (Inmobiline Dry-Strips, GE Healthcare) of 7 cm with a gradient from pH 3 to 10. The strips were rehydrated for 16 h with DeStreak solution (GE Healthcare) with 0.5 % ampholine carrier (IPG buffer, GE Healthcare) plus 100 µg of proteins of the fruit, in a final volume of 150 µL. The IEF was carried out in the Ettan IPGphor 3 Isoelectric Focusing System (GE Healthcare, USA) under the conditions indicated in Table 1. After IEF, the strips were balanced for 15 minutes in 10 mL of equilibrium buffer (6 M urea, 75 mM tris-HCl, 29.3 % glycerol, 2 % SDS [sodium dodecyl sulfate], 0.002 % bromophenol blue) with 100 mg of DTT (dithiotreitol), followed by a second 15-minute incubation in 10 mL of equilibrium buffer with 250 mg of iodoacetamide.
Table 1 Sequence and conditions to perform isoelectrofocus on Bromelia karatas fruit proteins.
| Voltage (V) | Duration (min) | Voltage (V·h-1) | |
|---|---|---|---|
| 1 | 400 | 90 | 300 |
| 2 | 1 000 | 60 | 300 |
| 3 | 5 000 | 60 | 4 000 |
| 4 | 5 000 | 30 | 3 000 |
In the second dimension, the proteins were separated into 12 % polyacrylamide gel copolymerized with 0.1 % (w/v) casein; the run was carried out in a vertical electrophoresis chamber (BIO-RAD) at 130V. Precision Plus Protein 161-0373 (BIO-RAD) was used as the molecular weight marker. After electrophoresis, the polyacrylamide gel was washed with a 2.5 % Triton X-100 solution for 1 h, followed by three washes with distilled water. Subsequently, the gel was incubated for 1 h at 37 °C in development buffer (1 % [w/v] Triton X-100, 100 mM phosphate buffer, pH 7.0, 4 mM DTT and 10 mM cysteine). The gel was stained with Coomassie blue (0.75 g-L-1 Coomassie R-250, 0.5 g-L-1 Coomassie G-250, 10 % acetic acid, 20 % methanol and 10 % ethanol) for 1 h, then it was destained in methanol (45 %) with acetic acid (10 %) and water. Proteolytic activity was detected as unstained light areas on a blue background. Finally, the gel was scanned in a GS-900 Calibrated Densitometer (BIO-RAD) and the analysis of the image for estimating its molecular weight and isoelectric point was done by 2D PDQuest Advanced software (BIO-RAD).
Results and discussion
Bromelia karatas is a wild plant with a diversity of shapes and colors in its fruits (Montes et al., 2014), which was recently typified by Monteiro and Forzza (2016). Figure 1 shows some B. karatas fruits collected in the state of Yucatán, belonging to three groups of plants selected according to fruit color.
Figure 1 Bromelia karatas fruits collected in the state of Yucatán. A) Collection 1 (Oxkutzcab): bone-colored and brown fruit, B) collection 2 (Espita): reddish fruit, C) collection 3 (Mérida): light pink to darker pink fruit.
Table 2 contains the results of the proteolytic activity of B. karatas collections. Collection 2 (Espita) had greater activity (8.59 U·mg-1) than collections 1 (6.84 U·mg-1) and 3 (7.2 U·mg-1) from Oxkutzcab and Mérida, respectively. The same pattern of differences was observed in proteolytic activity per mL of juice, where collection 2 was approximately 50 % higher than the others. In contrast, the protein content was similar in all three cases, so it is ruled out that the level of maturity is the determinant of the variation in proteolytic activity (Moyano et al., 2012). Proteolytic activity in B. karatas was always higher than that of A. comosus var. comosus. In this regard, it is pertinent to consider B. karatas as a source of proteases for the food industry (Arshad et al., 2014; de Lencastre et al., 2016). Based on the results, subsequent evaluations were made only for collection 2 (Espita), which had the greatest proteolytic activity.
Table 2 Proteolytic activity of the extract of Bromelia karatas fruits collected in the state of Yucatán, Mexico.
| Proteolytic activity | Colection 1 (Oxkutzcab) | Collection 2 (Espita) | Collection 3 (Mérida) | Control |
|---|---|---|---|---|
| Protein (mg·mL-1) | 1.49 ± 0.41 b | 1.45 ± 0.16 b | 1.50 ± 0.57 b | 0.35 ± 0.02 a |
| Specific activity (U·mL-1) | 10.02 ± 0.47 b | 15.20 ± 1.04 c | 10.08 ± 0.49 b | 1.20 ± 0.44 a |
| Specific activity (U·mg-1 of protein) | 6.84 ± 0.31 b | 8.59 ± 0.72 c | 7.20 ± 0.32 b | 3.42 ± 1.26 a |
Mean ± standard deviation. Control = Ananas comosus var. comosus. Different letters represent significant differences among collections, according to Tukey's test (α = 0.05, n = 4).
Figure 2 represents the effect of pH on the proteolytic activity of B. karatas, which was higher at pH 6 and 7, decreased slightly at pH 8, 9 and 10, and was nil at pH 12. Thus, the pH 6-10 range can be considered propitious for proteolytic activity. This pattern coincides with the pH 6-8 range for B. karatas and B. pinguin of Meza-Espinoza et al. (2017), who indicate that proteolytic activity for both species decreases when the pH is greater than 8.5.
Figure 2 Effect of pH on the proteolytic activity of Bromelia karatas fruits collected in the state of Yucatán. Different letters indicate statistically significant differences according to Tukey’s test (α = 0.05). The bars represent the standard deviation of the mean.
Figure 3 shows that proteolytic activity increases as the temperature of the enzyme reaction increases from 30 to 70 °C. This temperature range would allow enzymes to adapt to the requirements of the food processes where they are used (Guadix, Guadix, Paéz-Dueñas, González-Tello, & Camacho, 2000; Li, Yu, Goktepe, & Ahmedna, 2016); however, as the incubation temperature increases, the useful life of an enzyme decreases presumably due to the denaturation of proteases (Daniel, Dines, & Petach, 1996).
Figure 3 Effect of incubation temperature on proteolytic activity of B. karatas fruits. Different letters indicate statistically significant differences according to Tukey's test (α = 0.05). The bars represent the standard deviation of the mean.
The proteolytic activity of B. karatas juice was reduced under exposure to various temperatures and periods (Figure 4); after 210 min of incubation at 30 and 50 °C, the residual activity was around 90 %, while at 70 °C, the residual activity was about 40 %. The time that proteolytic activity is maintained at these temperatures is sufficient for protein hydrolysis processes (Guadix et al., 2000; Li et al., 2016). Meza-Espinoza et al. (2017) reported lower thermal stability; after an incubation of 37 to 60 °C for 60 minutes, proteolytic activity increased from 68 to 95 %, but decreased gradually after 120 min. The differences may be due to the fact that, in the cited work, proteins partially purified by precipitation were used, while in the present work the direct juice was used.
Figure 4 Evaluation of the thermal stability of proteases of Bromelia karatas fruits at 30, 50 and 70 °C.
On the other hand, the proteolytic activity of the juice is affected by NaCl when the concentration is higher than 10 % (Figure 5). Residual activity decreases as the percentage of NaCl increases, possibly due to the interaction of salt ions with water, which induces a loss of protease solubility (Moreno-Hernández et al., 2017). However, in concentrations of 5 to 10 % NaCl, the proteolytic activity of B. karatas juice is high (80 to 96 %), so its use is possible in products such as meat softeners, where marinating is done with a less than 5 % NaCl concentration (Peña, Duran, & Baleta, 2015).
Figure 5 Effect of NaCl on the proteolytic activity of Bromelia karatas fruits. Different letters indicate statistically significant differences according to Tukey's test (α = 0.05). The bars represent the standard deviation of the mean.
Figure 6 shows the zymogram obtained for the detection of proteolytic activity in B. karatas. The two-dimensional zymogram of B. karatas fruit, under non-reducing conditions, showed at least 40 light areas with apparent molecular weights between 27.3 and 290 kDa, with isoelectric points between 4.6 and 9.7 (Table 3). The 40 detected light areas potentially represent proteases including karatasin (Montes et al., 1990) and most of the proteases (between 21 and 97 kDa under non-reducing conditions) reported by Meza-Espinoza et al. (2017). This variety of molecular weights may be due in part to the association of several karatasin units among their cysteine residues (Meza-Espinoza, 2017; Montes et al., 1990).
Figure 6 Two-dimensional zymogram with casein and Coomasie blue staining for the detection of proteolytic activity of Bromelia karatas juice under non-reducing conditions. Light areas indicate the presence of proteolytic activity.
Table 3 Relationship of molecular weights (MW) and isoelectric points (IP) of proteases detected by two-dimensional zymography in Bromelia karatas juice under non-reducing conditions.
| Light area | MW | IP | Light area | MW | IP | Light area | MW | IP |
|---|---|---|---|---|---|---|---|---|
| 1 | 102.5 | 4.7 | 15 | 52.6 | 6.5 | 29 | 169.6 | 7.3 |
| 2 | 68.9 | 4.6 | 16 | 56.1 | 6.8 | 30 | 184.7 | 7.6 |
| 3 | 50.0 | 4.9 | 17 | 46.5 | 6.5 | 31 | 170.4 | 7.9 |
| 4 | 46.3 | 4.9 | 18 | 41.1 | 6.6 | 32 | 141.1 | 8.5 |
| 5 | 48.9 | 5.2 | 19 | 41.0 | 6.8 | 33 | 130.1 | 8.9 |
| 6 | 50.4 | 5.6 | 20 | 187.0 | 5.2 | 34 | 114.9 | 9.3 |
| 7 | 49.6 | 6.0 | 21 | 290.5 | 5.6 | 35 | 105.9 | 9.6 |
| 8 | 38.9 | 5.3 | 22 | 141.1 | 5.6 | 36 | 52.9 | 9.6 |
| 9 | 29.8 | 4.8 | 23 | 282.5 | 5.9 | 37 | 29.6 | 7.7 |
| 10 | 93.1 | 5.2 | 24 | 142.0 | 5.9 | 38 | 34.0 | 8.2 |
| 11 | 68.4 | 5.2 | 25 | 250.7 | 6.2 | 39 | 29.6 | 9.7 |
| 12 | 81.7 | 6.7 | 26 | 142.9 | 6.1 | 40 | 27.3 | 9.7 |
| 13 | 55.6 | 7.4 | 27 | 196.4 | 6.5 | |||
| 14 | 49.1 | 6.7 | 28 | 195.1 | 6.9 |
Two-dimensional zymography can offer a complete map of the proteases present, allow their identification, including isotypes, and provide information for their isolation (Larocca, Rossano, Santamaria, & Riccio, 2010). In the case of B. karatas, there are no previous studies of 2D zymograms; however, in other Bromeliad species, the isoelectric points range from 3.1 to 8.9 (Bruno, Pardo, Caffini, & López, 2002), which is similar to what was found in this work. Variations in isoelectric points can be attributed to post-translational modifications (Kozlowski, 2016) or to changes in some amino acids, which are reflected in enzyme activity, as occurs in B. pinguin proteases (Payrol, Obregón, Trejo, & Caffini, 2008).
The results show the potential of B. karatas as an alternative source of proteases. However, characterization of the proteases of this species, by sequencing the proteins with the highest proteolytic activity, is important to determine diversity, molecular weight, and isolating points accurately.
Conclusions
The proteolytic activity of the wild fruits of Bromelia karatas of the state of Yucatan is greater than that of Ananas comosus var. comosus. This activity is due to the effect of a mixture of proteases with variable molecular weights in non-reducing conditions. Bromelia karatas proteases are stable at 30 to 50 °C for at least three hours; the optimal working pH range is 6 to 10, and enzyme activity remains high (80 %) at 10 % NaCl concentration. Given the above characteristics, B. karatas can be considered as an alternative source of proteases for the food industry.
