INTRODUCTION
The wide range of biotechnological applications of proteolytic enzymes have
generated in recent years increased efforts to search for new sources that provide
activity profiles adaptable to current demands. This, coupled with the convenience
of using natural resources in each region, has turned much of the interest of
researchers towards the native flora.
The plant proteolytic enzymes have received particular attention due to the
property to maintain its activity in a wide range of temperature and pH (Devaraj
et al., 2008). They play an important role in regulating the biological
processes in plants, such as stress response, recognition of pathogen, mobilization
of storage protein during germination, senescence, etc. (Singh
et al., 2008).
The latex is considered a major source of plant proteases and is well-known
its use in folk medicine as well as in different industrial processes (Liggieri
et al., 2009); for example, latices from Asclepias spp. are
used in wound healing and the treatment of some digestive disorders (Obregon
et al., 2009) and it has been reported a variety of traditional uses
and pharmacological properties of Ficus religiosa (Singh
et al., 2011). This vegetal secretion is a milky fluid that contains,
either in solution or suspension, a mixture of substances which can be enzymes
such as glycosidases, proteases, phosphatases, amylases, chitinases, glucanases,
etc., which would be involved in the mechanisms of plant defense against pathogens
and insects (Pereira et al., 1999).
The first aim of this study was the proteolytic activity characterization of
the crude extract obtained from the latex de Ficus lushnathiana (Miq.)
Miq., Moraceae family laticiferous tree, indigenous to subtropical America (Argentina,
Brasil, Paraguay and Uruguay). The genus Ficus contains more than
1800 named species making them one of the largest genuses in the Moraceae family.
The ficins (name used to describe proteolytic enzymes of latex of the genus
Ficus) isolated from different species possess different characteristic
properties (Devaraj et al., 2008). Inhibitors
were used to elucidate the mechanistic nature of the occurring enzymes, the
optimal reaction conditions (pH and temperature) were determined and stability
studies at different storage conditions were performed.
The second aim of the present study consisted of determining of the molecular
features of the proteases present in the extract. Using molecular exclusion
chromatography, fractions enriched in different proteic components of the extract
were obtained. The molecular weight profile as well as the tendency to form
aggregates depending on the acidity of the medium were determined for this fractions.
Finally, controlled hydrolysis of whey proteins using the proteolytic activity
of the crude extract of F. lushnathiana (Miq.) Miq. were done. Wheys
are considered co-products of the dairy industry due to the potential use in
the food industry as protein sources of excellent nutritional value and as functional
food ingredients. Wheys derived from Mozzarella cheese and casein production
were used. Due at that the majority of reported bioactive peptides are derived
from milk proteins and have special relevance those with antimicrobial properties
(Pellegrini, 2003; Clare et
al., 2003), at the obtained peptidic fractions it was evaluated the
ability to inhibit the growth of a S. aureus strain.
MATERIALS AND METHODS
Materials: All the reagents used were purchased from Sigma-Aldrich.The
Ficus luschnathiana (Miq.) Miq. specimens (Herbarium n°MVJB28429)
belong to the Museo y JardínBotánicoAtilio Lombardo (Montevideo,
Uruguay). Whey derived of Mozzarella cheese production (Wm) was supplied by
a national dairy industry (CONAPROLE). Whey derived from casein production (Wc)
was prepared by precipitating the casein at pH 4.5 by addition of 2 N HCl.
Crude enzyme extract preparation: Latex, obtained by superficial incisions
of stems, was collected on 0.2 M sodium phosphate buffer (pH 7.5). This suspension
was centrifuged at 15493xg for 1 h at 10°C in order to discard gums and
other insoluble materials.
Preliminary purification of crude extract: About 0.3 mL of crude extract
was applied to a column packed with Sephadex G-75 (10.0x1.5 cm), equilibrated
with 50.0 mM sodium acetate buffer (pH 5.5), 50.0 mM sodium phosphate buffer
(pH 6.4) or 50.0 mM Tris-HCl buffer (pH 8.4), depending on the pH required for
subsequent experiments. Protein elution was performed with the same buffer and
1.5 mL fractions were collected and screened for proteolytic activity. The protein
profile was measured at 280 nm.
Protein determination: Protein content was determined by the method
of Lowry (Layne, 1957) using bovine serum albumin as
standard. During chromatographic purification steps, protein concentration of
each fraction was estimated firstly by measuring absorbance at 280 nm, wavelength
at which the aromatic amino acid residues absorb. Fractions with proteolytic
activity were also measured by Lowry method.
Proteolytic activity determination: It was determined as described by
Andrews and Asenjo (1986), using azocasein as substrate.
The Enzyme Unit (EU) was defined as the amount of enzyme necessary to increase
in 1 min one unit the A337 at pH 7.5 and 37°C.
Milk clotting activity: Was performed by visual evaluation of the occurrence
of the first clotting flakes as reported by Castro and
Cantera (1995). The Milk Clotting Unit (MCU) was defined as the amount of
enzyme needed to form the first detectable coagulated milk in 1 min under the
experimental conditions.
Effect of inhibitors: Crude extract was incubated with each of the inhibitors
solutions for 1 h at 25°C and the remaining proteolytic activities were
determined. The inhibitors used and their final concentrations were L-trans-epoxysuccinyl-leucylamide-(4-guanidino)-butane
(E-64) 5.0 and 10.0 μM, pepstatin A 1.0 μM, phenylmethylsulphonyl
fluoride (PMSF) 0.5 and 1.0 mM, ethylenediaminetetraacetic acid (EDTA) 5.0 and
10.0 mM and iodoacetamide 100.0 μM. In the case of pepstatin A, pH of the
crude extract was adjusted to 6.0 with glacial acetic acid while for the others
pH 7.5 was used. Inhibition percentages were calculated considering a control
without inhibitor.
Optimal conditions of pH and temperature: To determine the optimum pH
proteolytic activities were measured at 37°C at pH 5.2, 6.0, 6.4, 6.7, 7.1,
7.6, 7.9, 8.8 and 9.2 using the following buffer solutions: 0.5 M sodium acetate
buffer (pH 5.0 and 5.5), 0.5 M sodium phosphate buffer (pH 6.0, 6.5, 7.0, 7.5
and 8.0) and 0.5 M Tris-HCL buffer (pH 8.5 and 9.0). To determine the optimum
temperature proteolytic activities were measured at pH 7.5 at 8, 24, 30, 37,
50, 60, 70, 75, 80, 85 and 90°C.
Stability studies: To determine the effect of temperature on the stability
of protease preparation at pH 7.5, crude extract was incubated at -20, 7, 22,
37, 60 and 100°C, respectively. Aliquots were taken at different intervals
and the remaining activities were determined at 37°C. Intervals between
the aliquots were counted in days for the lower temperatures (-20 to 37°C),
in hours for 60°C and in minutes for 100°C due to increased protein
destabilization speed with increasing temperature. To determine the effect of
pH, crude extract was incubated at pH 5.2, 6.5, 7.5 and 9.9, respectively, at
60°C. Glacial acetic acid was used to achieve the pH 5.2 and 6.5. The pH
9.9 was obtained adding 2 N NaOH. Aliquots were taken at different intervals
and the remaining activities were measured at pH 7.5 according the activity
assay described before.
pI measurement: The pI of the proteins was determined by isoelectric
focusing using Phast System equipment (Pharmacia, Uppsala, Sweden) IEF 3-9.
Gels were revealed by silver staining according to the manufacturer's instructions.
Native electrophoresis: Samples were prepared by adding 10% glycerol
and 0.25% (w/v) bromophenol blue and then loaded on 11% polyacrylamide gel.
Electrophoresis was performed in a vertical gel apparatus (Hoefer SE 250 Mighty
Small II) using 25 mMTris, 0.2 M glycine (pH 8.6), as reservoir buffer. Current
was kept constant at 50 mA. The gels were stained with Coomassie Brilliant Blue
R 250 in methanol/acetic acid/water (1/1/8, v/v/v) solution.
Molecular mass determination: Electrophoresis was performed in a vertical
gel apparatus (Hoefer SE 250 Mighty Small II) on a 12% polyacrylamide gel, in
the presence of sodium dodecyl sulphate (SDS-PAGE). Protein samples were prepared
by adding 50% (v/v) of sample buffer (0.19 M Tris-phosphate buffer, 0.4 M β-mercaptoethanol,
6% SDS, 30% glycerol, 0.4% bromophenol blue), followed by heating for 5 min
in boiling water bath. Current was kept constant at 50 mA. The gels were stained
with Coomassie Brilliant Blue R 250 in methanol/acetic acid/water (1/1/8, v/v/v)
solution.
Zymogram: Unstained gels from PAGE were contacted for 10 min at 50°C
with an agarose gel imbibed with a 1.0% casein solution. After incubation, the
agarose gel was dehydrated and stained using Coomassie Brilliant Blue R-250
in methanol/acetic acid/water (1/1/8, v/v/v) solution. Unstained bands revealed
proteolytic activity. To detect active proteases in the presence of SDS and
reducing agent, SDS-PAGE was performed treating samples as described above in
2.12 but without heating and using a 12% polyacrylamide gel supplemented with
0.2% casein instead. After each electrophoresis, gels were incubated for 2 h
at 22°C before staining with Coomassie solution. Unstained bands revealed
proteolytic activity.
Mass spectrometry: The protein profile was performed by Matrix Assisted
Laser Desorption Ionization (MALDI) and Time of Fly (TOF) analysis (Voyager
DE-Pro, AbiSciex.).
pH-dependentoligomeric states of the proteins: After the preliminary
purification of crude extract with Sephadex G-75 at pH 5.5 and pH 8.4, fractions
with highest proteolytic activity obtained at each pH were, respectively lyophilized
and then redissolved in 0.30 mL of distilled water. Both resulting samples were
applied on HiLoad 16/60 Superdex 75 preparative grade column equilibrated with
50 mM sodium acetate buffer (pH 5.5) or 50 mMTris-HCl buffer (pH 8.4), respectively,
at a flow rate of 1.0 mL min-1. Column was calibrated with thyrogloblin
(670 kDa), aldolase (158 kDa), albumin (67 kDa), ovalbumin (44 kDa), chymotrypsinogen
A (25 kDa), myoglobulin (17 kDa) and ribonuclease A (13.7 kDa). The resulting
equation was log MW = -0.025, EV+3.382; R2 = 0.99 (MW = molecular
weight and EV = elution volume).
Hydrolysis of whey proteins: Whey was incubated with the enzymatic preparation
under defined pH and temperature conditions (pH = 7.2, 50°C), at specified
enzyme/substrate ratio (0.01 EU mg-1). Aliquots were taken at different
times (0, 15, 30, 60, 90 and 120 min) and placed in boiling water bath for 5
min to stop the reaction. The progress of hydrolysis was evaluated using TNBS,
as described by Spadaro et al. (1979). The degree
of hydrolysis (DH%) was defined as the percentage of peptide bonds cleaved,
according to the equation:
where, h is the hydrolysis equivalent formed during enzymatic action and htot
is the hydrolysis equivalent after acid hydrolysis in 6 N HCl, at 110°C,
for 24 h in sealed tubes. Hydrolysis equivalents were calculated using a calibration
curve with valine as standard: y = 0.2995x+0.0215, R2 = 0.993.
Fractionation of the products of hydrolysis: Hydrolysis products obtained
at different times were fractionated using AMICON Ultra-4 centrifugal filter
devices (3 and 10 kDa).
Evaluation of antimicrobial activity: Antimicrobial activity against
S. aureus was screened by bioautography applying 50 μL of each fraction
obtained in 2.17 on alumina thin-layer plates. Quantification of inhibition
was done in microtiter plates with an inoculum of 106 colony forming
units per mililiter (CFU mL-1) (Hadacek and Greger,
2000). Bacitracin was used as a positive control.
Determination of the peptidic concentration: The peptidic concentration
was determined using o-phthaldialdehyde as described by Church
et al. (1983).
Statistical analysis: Reported data was the average values of standard
deviations of triplicate assays. Existence of statistically significant differences
between results was evaluated by analysis of variance (ANOVA).
RESULTS
Milk clotting activity: The milk clotting activity of the crude extract
was compared with that obtained using a trypsin solution. Both enzyme solutions
were prepared so as to have the same activity by the azocasein method. Clotting
times obtained for both are shown in Table 1. Crude extract
showed higher milk clotting activity than the reference solution.
Inhibition studies: Results obtained in inhibition studies are summarized
in Table 2. The result obtained after the action of PMSF was
the same for the two concentrations tested. The total inhibition obtained suggests
that the proteolytic activity of the extract is due to serine proteinases.
| Table 1: |
Clotting Times (CT) and Milk Clotting Units (MCU) of a trypsin
solution and crude extract (both with 4.4 EU mL-1) |
 |
| aEach value is the mean of three independent determinations,
±: Indicate the standard deviation |
| Table 2: |
Inhibition percentages of the proteolytic activity |
 |
| aEach value is the mean of three independent
determinations, ±: Indicate the standard deviation |
|
| Fig. 1(a-b): |
Effect of (a) pH and (b) Temperature
on the hydrolysis of 1.0% azocasein in the presence of the crude extract
obtained from the latex of Ficus luschnathiana |
Although this compound can also act on cysteine enzymes, the lack of inhibition
observed for E-64and that the activity assay was not affected by the presence
of mercaptans confirms the absence of these type of proteinases. Negative results
obtained both with EDTA and Pepstatin A indicate a lack of metal and aspartic
proteases activity respectively. The percentage of inhibition achieved with
iodoacetamide can be explained by the fact that its action is not specific for
cysteine enzymes, but is also capable of inhibiting certain serine proteinases
(Salvesen and Nagase, 2001).
Optimal conditions of pH and temperature: Figure 1a
shows the results of the hydrolyzing activity as a function of pH. Optimal activity
was observed at pH 8.0 and each of the results obtained for pH in the range
5.5-9.0 are at least a 60% of the maximum value. Results of temperature-activity
are shown in Fig. 1b. The maximum value obtained was at 75°C.
Even at 8°C proteolysis was observed (24% compared to that observed in the
standard conditions of pH 7.5 and 37°C).
|
| Fig. 2: |
Inactivation kinetics at 60°C of crude extract at pH 7.5 |
| Table 3: |
Percentagesa of remaining activity of the extract
after incubation at different temperatures |
 |
| aEach value is the mean of three independent determinations,
±: Indicate the standard deviation |
| Table 4: |
Remaining activity (%)a of the crude extract after
incubation at different pH (60°C) |
 |
| aEach value is the mean of three independent determinations,
±: Indicate the standard deviation |
Stability at different temperatures: The results of determinations of
stability of the extract at different temperatures are summarized in Table
3. Freezer storage (-20°) adversely affects activity of the extract.
Moreover, the results suggest the possibility of storage for prolonged times
(upto 5 months) at 7°C. Even at room temperature (22°C) the remaining
activity values obtained were quite high for two months and after 15 days only
3% of the activity was lost. The behaviour at 60°C is illustrated in Fig.
2. It can be seen that the remaining activity did not change after 1 h of
incubation and decreased to 79% after 5 h. The experimental half life observed
was 14 h. As at 100°C the extract was completely inactivated after 1 min
of incubation.
Stability at different pH: The results of the stability determinations
at different pH are summarized in Table 4. In order to visualize
promptly the decrease of the activity remaining, the temperature selected for
the study was 60°C. The extract was more stable at moderately acidic conditions
and the highest values of remaining activity were obtained at pH 6.5. The lowest
values on the other hand were obtained in the most alkaline medium. Differences
in the slopes of these curves occurred in the first 5 h of incubation, after
that all the inactivation rates were similar for all the pH tested.
|
| Fig. 3: |
Chromatographic elution profile of crude extract on Sephadex
G-75 column |
|
| Fig. 4(a-b): |
Native electrophoresis of the most active
fractions obtained after the preliminary purification of crude extract
by (a) Gel filtration and (b) Zymogram showing four active bands |
Molecular characteristics of the extract proteins: Molecular analysis
of protein species present in the extract revealed a mixture of great complexity.
In order to remove several non-proteins components that absorb at 280 nm including
the polyphenols, preliminary purification of crude extract by exclusion chromatography
with G-75 was performed at pH 6.4 which resulted in the elution profile shown
in Fig. 3. The A337 values are the results of proteolytic
activity assay performed on each fraction. Fractions 4, 5 and 6 were pooled,
lyophilized and then redissolved in 0.25 mL of distilled water. The resulting
sample was checked by native electrophoresis (Fig. 4a) and
zymogram (Fig. 4b). Coomassie staining showed at least 11
proteins bands.
|
| Fig. 5: |
Mass spectroscopy of the pooled 4-6 fractions obtained by
exclusion chromatography |
Those with the lowest charge to radius ratio showed significant proteolytic
activities evidenced as two broad undyed bands and each of them was closely
accompanied by another very faint band. With the aim to determine the molecular
weight profile of the pooled fractions mass spectrometry (MALDI-TOF) was carried
out. The spectrum obtained is shown in Fig. 5. It can be seen
that it is a very complex sample with a great diversity of molecular size compounds
of about 10-40 kDa. Compounds with molecular masses lower than 20 kDa probably
could have been generated by autolytic processes as was previously reported
for ficins of F. carica (Azarkan et al.,
2011).
SDS-PAGE of each of the 4, 5 and 6 fractions obtained by exclusion chromatography
with G-75 column, with and without a previous treatment of heating for 5 min
in boiling water bath (Fig. 6a) provided additional data and
apparently contrasting with that obtained by MALDI. These fractions had the
same molecular weight profile differing only in the relative amounts of each
molecular species. By comparing with molecular weight standards, molecular species
were predicted having higher molecular weight values than those found by MALDI.
Heat treated samples exhibited a band corresponding to approximately 60 kDa
and also bands of lower molecular weight with values equivalent to those determined
with MALDI. Samples without thermal treatment exhibited even higher molecular
weights, greater than 80 kDa but the 60 kDa band is not present.
Electrophoresis in polyacrylamide gel supplemented with 0.2% casein (Fig.
6b) for non-heat treated samples indicated active proteases in presence
of SDS and reducing agent, mainly higher molecular weight species. Also there
is a less clearly unstained area, corresponding to a lower molecular weight
of about 20 kDa, plus several other very faint bands of molecular sizes in between.
Oligomeric states: The facts that for a single fraction eluted from
a gel filtration (pH 6.4) was obtained a broad range of molecular sizes (20
to about 100 kDa) in a SDS-PAGE (pH 8.9) as well as the observed discrepancy
between the molecular weight values obtained by MALDI and by electrophoresis
(above 60 kDa only for the latter) can not be explained only by quaternary structures.
|
| Fig. 6(a-b): |
(a) PAGE-SDS of fractions 4, 5 and 6
without heat treatment for Lanes: 1, 3 and 5, respectively and with a
heat treatment of 5 min in boiling water for Lanes: 2, 4 and 6, respectively
and (b) Electrophoresis in polyacrylamide gel supplemented with 0.2% casein.
Lane 1: Standards, Lanes 2, 3: Fractions 5 and 6, respectively, without
thermal treatment. Arrows indicate unstained areas more evident |
A possible explanation is that the molecular species present in the crude
extract are grouped in different ways depending on the pH of the medium and
that the largest clusters are formed at alkaline pH. Heat treatment is probably
capable of breaking the largest aggregates but fails to eliminate the forces
that hold together the grouping of 60 kDa or may be that this grouping is reorganized
during the electrophoretic run. In order to confirm these hypotheses, an exclusion
chromatography of the crude extract at pH 5.5 and 8.4 with a HiLoad 16/60 Superdex
75 preparative grade column pre-calibrated with molecular weight standards was
perfomed. The respective elution diagrams are shown in Fig. 7a,
b. Three peaks were obtained at pH 5.5, a small one that eluted
in a volume corresponding to dead volume (MW = 80 kDa) and two major peaks corresponding
to 67 and 44 kDa. At pH 8.4 the resolution of the sample decreased: a large
peak eluted in a volume corresponding to MW = 80 kDa, followed by a small plateau
(62-70 kDa).
PAGE-SDS (with and without thermal treatment of samples) and zymogram (data
not shown) were performed to fractions 41 (F41) and 45 (F45)
of the gel filtration done at pH 5.5. For both fractions, molecular sizes profiles
evidencing new regroupings were obtained. For F41 without thermal
treatment a major band having more than 100 kDa with strong proteolytic activity
and two very faint bands of approximately 100 and 39 kDa, respectively were
observed while with thermal treatment there was only an intense band of 39 kDa.
For F45 without thermal treatment two intense bands, a wide one of
about 100 kDa and a thin band of about 80 kDa, both with proteolytic activity
while with thermal treatment there were bands corresponding to 42-49 kDa. Additionally,
isoelectric focusing was performed to these fractions and results are shown
in Fig. 8. While there is a differential enrichment for both
samples (F41 had a preponderance of higher pI molecules and in F45 there was
a greater abundance of those of lower pI), there was no net resolution, showing
both lanes a similar and very complex bands distribution.
|
| Fig.7(a-b): |
Chromatographic elution profile of crude
extract on HiLoad 16/60 Superdex 75 at pH (a) 5.5 and (b) 8.4 |
Hydrolysis of whey proteins: The degree of hydrolysis for each whey
are shown in Fig. 9. It can be seen that whey derived from
casein production was hydrolyzed to a lesser degree than that from Mozzarella
cheese reaching at 120 min of reaction values of 2.40±0.10 and 3.1±0.09%,
respectively, that represent a statistically significant difference according
to ANOVA.
|
| Fig. 8: |
Isoelectric focusing of fraction 41 (F41) and 45
(F45) of the gel filtration performed on HiLoad 16/60 Superdex
75 at pH 5.5 |
|
| Fig. 9: |
Hydrolysis curves of wheys derived from
Mozzarella cheese and casein production (Wm and Wc,
respectively). Reactions conditions were pH= 7.2, 50°C and enzyme/substrate
ratio = 0.01 EU mg-1 |
Antimicrobial activity: Growth inhibition of a S. aureus strain
was observed for both wheys with the aliquots obtained at 90 and 120 min of
hydrolysis, particularly with the fractions corresponding to peptide sizes smaller
than 10 kDa (Fig. 10a and b). It can be
seen that although at 90 min there is a marked antimicrobial action for the
fraction corresponding to sizes smaller than 10 but larger than 3 kDa, after
120 min the bioactivity is better for displaced to sizes smaller than 3 kDa.
This could be because the large bioactive peptides found in first place are
intermediate product of hydrolysis which are degraded afterwards resulting in
the antimicrobial molecules smaller than 3 kDa evidenced after 120 min of reaction.
Comparing the results for both wheys, it can be seen greater action in the case
of Wm. Figure 10c shows the results for the aliquots obtained
at 0-30 min of reaction. No significant results were obtained in that period
showingthere is no detectable antimicrobial activity prior to hydrolysis. In
none of the cases there was antimicrobial activity for fractions above 10 kDa.
Although some whey proteins, such as lactoferrin (Recio
and Visser, 2000), have antimicrobial activity, its low proportion in the
whey, as well as the dilution factors of the method, make their activity undetectable.
This shows that the hydrolysis process greatly increases the antimicrobial activity
that may have the starting material.
Inhibitionquantification was done in microtiter plates. This results as well
as the peptide concentration of fractions with the highest antimicrobial activity
from Wm, are summarized in Table 5. The greater antimicrobial
effect is observed for the fraction of lower peptidic concentration,indicating
that the observed bioactivity is mainly due to the peptides of less than 3 kDa.
| Table 5: |
Growth inhibition percentage of a S. aureus strain
for the aliquots obtained from Wm at 120 min of hydrolysis |
 |
| aEach value is the mean of three independent determinations,
±: Indicates the standard deviation |
|
| Fig. 10(a-c): |
Petri dishes containing hydrolysate fractions
applied on thin-layer plates. (a) Fractions from whey derived of Mozzarella
cheese production (Wm), after 90 and 120 min of hydrolysis. (b) Fractions
from whey derived from casein production (Wc), after 90 and 120 min of
hydrolysis. (c) Fractions corresponding to the first 30 min of hydrolysis,
from Wm (up) and Wc (below) |
But this does not rule out the possibility that also in the fraction corresponding
to sizes between 3-10 kDa antimicrobial activity is due also to the fraction
below 3 kDa, since the filtering process involves a balance between the permeate
and retentate of components of lower size than the membrane pore.
DISCUSSION
The crude extract of F. luschnathiana (Miq.) Miq. latex has a marked
proteolytic activity with an action model corresponding to serine proteinases.
The high milk clotting activity observed opens the possibility of its use in
the cheese industry, as well as in other biotechnological industries. A similar
behaviour of high milk clotting activity was previously reported for religiosin
B, a serine protease from Ficus religiosa, by Kumari
et al. (2012).
The proteolytic action reaches its maximum at pH 8.0 and 75°C. The fact
that significant values of activity were observed in a wide range of pH and
temperature opens a broad spectrum of applications. The optimum values of pH
and temperature found in this study are similar to those reported for other
serine proteases derived from latex. Indicain, a dimeric serine protease from
Morus indica showed maximum values at pH 8.5 and 80°C (Singh
et al., 2008).
Stability studies showed the ability of the extract to remain unchanged for
long periods at refrigeration temperature which would be economically beneficial
for an industrial application because imply important economic and energy savings.
The stability at high temperatures (60°C) allows its use in processes that
require hours of continuous proteolytic processing. Brunius
and Sundbom (1987) studied the stability of a trypsin solution from bovine
pancreas at 37°C and pH 7.3. These authors reported that in these conditions
the activity decreases to less than 10% after 24 h meanwhile the serinic activity
in the crude extract from Ficus even after 48 h at 60°C at pH 7.5
still conserved 18% of its initial activity. The greater thermostability found
for the crude extract from Ficus enables the possibility of its application
in processes that require high temperatures for extended periods. Moreover,
the inactivation after 1 min at 100°C makes it possible to stop the reaction
without the addition of chemicals that may be incompatible in the food industry.
Regarding pH, acid environment affected the protein structure of enzymes to
a lesser extent than alkali.
Multiple proteases of the same family are quite often reported in latex bearing
plants. However, the cause of such multiplicity has not been extensively explained
(Sharma et al., 2012). The proteolytic activity
of crude extract of Ficusis due to several proteic components of different
molecular weight and isoelectric points, most of them still retained the activity
in presence of SDS and reducing agents, making it suitable for its use in the
detergent industry. These proteic components exhibited pH-dependant clustering
forms, with a tendency to form larger aggregates at basic pH. In summary, the
tendency to form aggregates of variable composition coupled with the possibility
that some components have quaternary structure and/or different degrees of glycosylation
resulted in an extract with a high level of analytical complexity. Anyway, current
interest of the authors of this study lies not in obtaining pure enzymes but
in evaluating the potential use of the crude extract as such and in a future
work the isolation of the present peptidases.
Enzymes present in the crude extract are capable of hydrolyzing proteins ofwheys
derived from Mozzarella cheese production and from casein production, yielding
higher degrees of hydrolysis for the former.As a result of the hydrolytic process
performed under controlled pH and temperature, fractions enriched with antimicrobial
peptides capable of inhibiting upto 94% the growth of a S. aureus strain
were obtained. These could help to solve problems of resistance to traditional
antibiotics, an ever-increasing global threat that involves the main pathogens
and drugs.
ACKNOWLEDGMENTS
This study was performed under the project CYTED IV-22.The authors acknowledge
the support provided by JulianGago of Museum and Botanical Garden Atilio Lombardo,
who contributed with their knowledge of indigenous flora and provided samples.
The MALDI-TOFF analyses were conducted with support from the Pasteur Institute
of Montevideo, Uruguay.Language corrections were done by Ph.D. Ana Acevedo.
