International Food Research Journal 23(1): 340-349 (2016)

Journal homepage: http://www.ifrj.upm.edu.my

Characterization of partially extracellular proteases from bekasam-isolated

Lactobacillus plantarum S31 and its application to hydrolyze
skimmed-milk with antibacterial property

Budiarto, B.R., *Mustopa, A.Z. and Idarmawan, T.

Research Center for Biotechnology-Indonesian Institute of Science (Lembaga Ilmu Pengetahuan

Indonesia), Jl. Raya Bogor Km.46 Cibinong 16911 Bogor, Indonesia

Article history Abstract

Received: 21 January 2015 The extracellular proteases secrected by L. plantarum S31 has been isolated and purified
Received in revised form: from supernatant of 20-day cultivation when the cells was at the stationary phase. The
4 May 2015 isolated proteases showed two distinct proteolytic activity, named LPS31.18 and LP31.37,
Accepted: 10 June 2015
on zymogram assay after sequentially purified using 80% ammonium sulphate ammonium
sulphate saturation and sephadex G-50 with overall specific activity of 2 IU/mg and purity fold
Keywords of 8.01. Furthermore, the partially purified enzymes exhibited thermostable characteristic and
were considerably active at pH 5. The activity of the enzymes enzymes was mostly inhibited
Bioactive peptides in the presence in the presence of PMSF, EDTA and Tween-20 meanwhile cofactors (Mg2+
L. plantarum S31 and Ca2+), SDS and DTT tend tended to increase caseinolytic activity. These enzymes were
Extracellular proteases also usable to generate bioactive peptides from skimmed-milk as proven by bacterial-killing
Skimmed-milk property of skimmed-milk hydrolysate towards food spoilage-causing bacteria such as E. coli
Zymogram assay
and L. monocytogenes.
© All Rights Reserved

Introduction to produce diverse intracellular and extracellular

enzymes. Protease, amylase, lipase, pytase, and
Lactic acid bacteria (LAB) are group of saprophytic glucosidase are the common enzymes produced by
bacteria inhabitating mostly dairy products that are these remarkable microorganism and most of them
able to produce organic acid compounds such as lactic have been extensively used in food industry mainly
acid (Ohkouchi and Inoue, 2006). The growth of these to increase the value of bioproducts and some others
bacteria anaerobically on food and feed-based organic are applied as food supplement for health purposes
matters, called fermentation, will convert complex (Aguilar et al., 2000; Elfahri et al., 2014). The
molecules such as polysaccharide, protein and lipids proteolytic enzymes place the important position
into their constituents hence enhances its nutrional in biotechnology product-based industry (detergent
value (Corsetti and Settanni, 2007). For instance, industry, food industry, and pharmacitical industry)
L. plantarum has an ability to ferment starch, major and the market demand for trading of commercially
polysaccharide that constitute pearl millet gruels, produced proteases itself is estimated 60% of the
into more ready energy molecules mainly glucose total worldwide sale of the enzymes and the trend
and maltose for complementary feeding of young seems to increase in the near future due to the tools
children in third countries (Humblot et al., 2014). improvement and the more advance metodology
Increasing in protein and starch digestibility of sorgum applied in isolating and purifrying protease from
flour after fermented by L. plantarum has also been many kinds of bioresources (Rao et al., 1998).
reported (Pranoto et al., 2013). Milk fermentation Therefore, not only the vast diversity of proteases but
done by L. pentosus has potential values for yogurt also their specificity and uniqueness of action causes
industry because of its flavor produced gives unique proteases become worldwide never ending topic to
and high degree of consumen acceptability (Pan et be exploited especially their physiological aspect and
al., 2014). Furthermore, current study also has proven biotechnological application.
the benefit of some LAB in reducing major allergens The most Interesting application of proteases
content (casein and β-lactoglobuln) in dairy milk and in food industry is in bioactive peptides production
milk products (Shi et al., 2014). originated from macroproteins hydrolysates which
The sophisticated key of LAB in degrading the are resulted from complete or partial proteolytic
complex molecules during fermentation is the ability digestion (Pasupuleti et al., 2010). Some of LAB-

*Corresponding author.
Email: azmustopa@yahoo.com
341 Budiarto et al./IFRJ 23(1): 340-349

produced proteases have been reported to exhibit This method is based on the tyrosine formation
promising result in production of bioative peptides during casein hydrolysis by protease and the tyrosine
for medicine purposes (antimicrobial peptides, released was monitored at wavelength 540 nm. A
antidegenerative peptides, anticancer peptides), food total of 6 µL of test sample was firstly mixed with
industry (biopreserpative peptides) and agriculture 6 µL phosphate buffer (pH 7.4, 25 mM), then added
application (biopesticides). Ueno et al. (2004) have with 6 µL of 1% (w/v) casein solution. The reaction
succeeded applying L. helveticus CM4-purified mixture was incubated at 37°C for 30 min. Following
endopeptidase to produce anti-hypertensive peptides incubation, 12 µL of trichloroacetic acid was added
using synthetic pro-peptides as substrate. Application to the mixture. After centrifugation at 12000 r/min
of proteases isolated from L. lactis ssp. cremoris for 1 min, reaction mixture was added with 143
AM2 in reducing bitterness of peptides has been µL of reagent A [a mixture of Na2CO3 solution and
reported by Bouchier et al. (2001). Furthermore, CuSO4.5HO solution (5:1)], followed by adding 29
generating antimicrobial peptides from β casein µL of Folin Ciocalteau reagent, and then incubated for
using protease isolated from L. helveticus PR4 have 15 min before measured at 540 nm. A negative control
been succesfully done by Minervini et al. (2003) with was prepared by adding tricarboxylicacidcycle acid
promising result. To our knowledge until now there to the reaction mixture to prevent proteolytic reaction
is no report regarding the application of extracellular prior to addition of the substrate. One unit of protease
proteases produced by L. plantarum in generating activity is defined as 1 µmol of tyrosine released
antimicrobial peptides from complex macroprotein during enzymatic reaction per mL reaction mixture
hydrolysis such as skimmed-milk. The aims of this per minute under the experimental conditions.
study are divided into two major goals which are
(1) to isolate and purify the extracellular enzymes Determination of cell growth curve and its
produced by L. plantarum and (2) its application extracellular proteolytic activity
to generate bioctive peptides from skimmed milk 5 mL of fresh MRS medium was inoculated
substrate as source of natural antibacterial agents at 0.1% L. plantarum S31 taken from stock liquid
which is active mainly against food spoilage bacteria. culture then let stand for over night at 37oC without
shaking. Over night culture of L. plantarum S31
Materials and Methods (approximatly 250 μL culture) then transfered into
250 mL MRS medium incubated at 37oC without
Materials shaking. Sampling of culture was done by taking
Culture of L. plantarum S31 was kindly obtained 1.5 mL of culture at interval 2 hrs of cultivation
from Dr. Apon Zaenal Mustopa, Research Center then the cell density (OD600nm), pH medium and
for Biotechnology-Indonesian Institute for Science, extracellular protease activity was measured.
Indonesia. Sulphopropyl (SP)-sepharose was from
Sigma Chemical Company, St. Louis, Missouri, Screening of protease using plate assay
USA. Sephadex SG-50 was from GE Heathcare Bio- Plate assay described by Hassan et al. (2013)
Science, Uppsala, Sweden. MRS (de Man, Rogosa, and More et al. (2011) was adapted with minor
Sharpe) media and skimmed- milk were from Oxoid modification. Protease activity in each purification
LTD, England. Pierce bicinchoninicacid (BCA) steps was monitored through clear zone formation
protein assay kit and Pierce silver staining kit were on gelatin-added agarose. About 30 mL of 0.5%
from Thermo Scientific,USA. All other chemicals agarose medium containing 0.2% of gelatin in 25
were purchased from Sigma Chemical Co. mM Tris-HCl buffer (pH 7.4) was placed on Petri
disc. Sample (50 µL) was loaded into well then
Cultural condition incubated for 24 h at 37 C for enzymatic reaction.
L. plantarum S31 was maintained on MRS broth The development of clear zone around the wells was
medium. For the experimental purposes 0.1% of L. detected by applying Coomassie Blue (0.25% w/v) in
plantarum S31 suspension was subcultured into new methanol: acetic acid: water 5:1:4 (v/v/v) for 15 min
5 mL MRS medium incubated at 37oC for overnight at room temperature, followed by destaining step to
prior to use. remove staining solution using destain solution (66
mL methanol: 20 mL acetic acid: 114 mL H2O bidest)
Protease activity assay until the clear zone could be seen visually.
Activity of extracellular protease produced
by L. plantarum S31 was measured using method Purification of extracellular proteases
developed by Cupp-Enyard (2008) with modification. Protein isolation was performed according to
 Budiarto et al./IFRJ 23(1): 340-349 342

the protocol described by Omund et al. (1990) with fluoride) and EDTA (Ethylenediaminetetraacetic acid)
slight modification. Supernatant (crude extract) at 0.5mM and 5 mM were used as protein inhibitors.
of 20-day L. plantarum S31 liquid culture was For metal ions treatment, MgCl2 and CaCl2 (0.1 mM
subjected to saturation using 80% solid ammonium and 1 mM) were used. While Dithiothreitol was used
sulphate, then resuspensed in 25 mM Tris-HCl as reducing agent. In each case, 7 μg of the enzyme was
pH 7.4. The crude extract was then applied onto a incubated in the presence of increasing concentration
Sephadex G-50 column pre-equilibrated with 25 mM of treatment agents in 25 mM Tris buffer, pH 7, for
Tris-HCl pH 7.4. Crude protein-containing Sephadex 30 min at 37oC and assayed for proteolytic activity
G-50 column was separated using the same buffer as described earlier. A control assay was done with
with flow rate was fixed at 1 mL/min. Fractions with enzyme solution without treatment agents and the
protease activity was freeze-dried and resuspended in resulting activity was considered as 100%.
the same buffer up to 25% of the original volume.
The presence of protein was determined by BCA Preparation of skimmed-milk hydrolysate using
method at the wavelength of 540 nm with mixing 10 extracellular proteases L. plantarum S31
µL of sample with working solution. Skimmed-milk hydrolysate was prepared
according method as described by Pan et al. (2004)
Gelatin zymography with minor modification. As much as 2.5% (w/v)
The method described by Raser et al. (1995) skimmed-milk was made by weighing 0.25 gram
and Kleiner and Stevenson (1994) was adapted. As skimmed-milk dissolved in distillated water and the
mentioned, this method is very powerful not only to milk suspension was adjusted to pH 7. Extracellular
detect protease activity but also to predict molecular proteases L. plantarum S31 was added at an enzyme/
mass of the target protein. Firstly, the sample was run substrate ratio of 1/100 (v/v). A time-course analysis
on 0.2% gelatin-containing gel electrophoresis. After of skimmed-milk digestion was performed by
separation of protein, the protein was reactivated incubating the mixture for 3, 6, 9, 12, 15 and 18
by incubating the gel in 2.5% Triton X-100 for 40 hrs on a shaker at the temperature and pH fixed as
min at 37°C. The third step is staining the gel in per the optimum values. After hydrolysis treatment,
0.05% Coomassie Blue solution for 2 hrs. The final milk hydrolysate solution was boiled at 100oC for 10
step is removing the excess Coomassie Blue using min and centrifuged at 13000 rpm, 4°C for 15 min.
destaining solution until clear band appeared on gel The obtained supernatant from the optimal digestion
which indicates the protease activity. treatment was stored at -18 °C until use.

Determination of optimum temperature and pH for Gel electrophoresis

protease activity Sodium Dodecyl Sulphate Polyacrylamide
The effect of temperature and pH on protease Gel Electrophoresis (SDS-PAGE) with 10%
activity was evaluated following method described by polyacrylamide gel was used to analyze either
Zhang et al. (2010) with modification. The optimum molecular mass of extracellular protease or the
temperature was determined by carrying out the skimmed-milk hydrolysate as described by Laemmi
assay at different temperatures ranging from 25°C to (1970). The band of protein was compared to the
75°C with 10°C interval. Meanwhile, the optimal pH standard molecular mass of marker to determine
for protease activity was determined within the pH its molecular mass (Bio-Rad). Protein bands were
range 3-9 using the following buffers: 0.1 M citrate stained with Coomassie Brilliant Blue R250 to
buffer (pH 4-6), 0.1 M Tris-HCl (pH 7-9) and 0.1 evaluate the pattern of skimmed milk hydrolysate
M glycine-NaOH (pH 10-11).The residual enzyme or using silver staining reagents to detect molecular
activity was measured by performing the assay as mass of extracellular protease L. plantarum S31
described above. according to the manual instruction. Photo-CapMw
software (Vilber Lourmat, USA) was used to calculate
Determination of protease activity due to surfactants, molecular mass of proteins.
protein inhibitors, metal ions, and reducing agent
treatment Antibacterial activity assay of extracellular
The method to evaluate these treatments on proteases-hydrolyzed skimmed milk
protease activity was adapted from Yadav et al. (2012). The antibacterial activity was measured using
Tween-20 (non-ionic detergent) and Sodium Dodesyl Tetrazolium Blue Chloride (TBC) method as
Sulphate (anionic detergent) at 2.5% and 5% were described by Moussa et al. (2013) with modification.
used as surfactants. PMSF (phenylmethylsulfonyl As much as 0.1% bacterial suspension culture was
343 Budiarto et al./IFRJ 23(1): 340-349

transfered into 5 mL Nutrient Broth and incubated

over-night at 37oC with 150 rpm. Serial dilution was
done by taking 1 mL of over-night culture diluted
with 8 mL 0.8% NaCl to obtain 3x108 CFU/mL then
followed by taking 250 μL from the first dilution
mixed with 10 mL Nutrient Broth to obtain 1.8x106
CFU/mL. Skimmed-milk hydrolysate (75 μL) was
mixed with bacterial culture (50 μL) in microplate
wells then incubated for 24 hrs at 37oC with 150
rpm. Filtered-Tetrazolium Blue Chloride solution
(25 μL) was added into each wells and let stand for
30 min at 37oC before measuring absorbance at 540
nm using ELISA reader. Two bacterial species were
used as test microorganisms for determination of
antibacterial activity: Escherichia coli (ATCC 8739)
and Listeria monocytogenes (BTCC B693). The
inhibitory activity (%) was measured following this
equation:

Results

L. plantarum S31 growth and its extracellular

proteases activity
Figure 1. Activity of L. plantarum S31 extracellular
The correlation of L. plantarum S31 growth with
proteases and Chromatogram profile of Sephadex G-50
its extracellular protease activity is shown in Figure gel filtration eluted by 25 mM Tris-HCl pH 7.4. (a)
1a. The initial protease activity (63.17 μg/mL) was Produciton of extracellular proteases during L. plantarum
detected when cells entered early stationary phase S31 growth on MRS medium. (b) Sample loaded was
at 12-day culture (at this point the cells reached protein pellet obtained from crude extract saturated with
maximum growth) and then this proteolytic activity 80% ammonium sulphate. PA means protease activity
steady increased to its maximal activtiy (276.28 μg/ measured quantitatively based on casein hydrolysis while
mL) at 22-day culture where the cells entered late GA means gelatinolytic activity obtained from zymogram
stationary phase. Meanwhile, no proteases activity assay. nt means the sample was not tested.
was detected at 25-day of culture probably due to
proteins degradation. This result clearly showed activity. Base on this result, the G1 was chosen to be
that incubation time plays a substantial role in the applied in next study. SDS-PAGE separation result
maximum enzyme production. Furthermore, the (Figure 2) showed several protein bands appeared in
extracellular proteases produced by L. plantarum sephadex G50-purified G1 in which protein bands
S31 could be harvested at early to late stationary with molecular mass of ~18 kDa (LPS31_18) and ~37
phase of cultivation that the crude extract of 20-day kDa (LPS31_37) were corresponding to extracellular
culture exhibited proteolytic activity on agar assay proteases. This was later proven from zymogram
(data not shown). result (Figure 2) that clearly showed the formation
of white zone due to proteolytic action of these two
Purification of extracellular proteases L. plantarum proteases on gelatin subtrate. In addition, the protein
S31 band with molecular mass above ~120 kDa was
Extracellular proteases from L. plantarum S31 also observed in zymogram result presumably this
has been successfully purified from supernatant protein was oligomeric state of either LPS31.18 of
of 20-day medium culture by using of ammonium LPS31.37 respectively. Furthermore, the purity and
sulphate precipitation and sephadex G-50 column protease specific activity of purified enzymes after all
chromatography. Chromatogram pattern as shown in process of purification steps was 8.01 fold and 2 IU/
figure 1b created several peaks and designed as G1 to mg respectively (Table 1).
G7 when the column was eluted by 25 mM Tris-HCl
pH 7.4. Quantitative and qualitative test then was Characteristic of extracellular proteases L.
applied to detect proteolytic activity in each group and plantarum S31
only G1 exhibited both caseinolytic and gelatinolytic Table 2 shows summary of extracellular proteases
 Budiarto et al./IFRJ 23(1): 340-349 344

kDa α-lactoalbumin rose.

Antibacterial property of extracellular proteases L.

plantarum S31-degradated skimmed-milk
The correlation between extracellular proteases
L. plantarum S31 hydrolyzed-kimmed-milk (18-hrs
hydrolysis, pH 7, and 37oC) with antibacterial activity
is summarized in Table 3. The IC50 of skimmed-milk
hydrolysate against E. coli was at around 0.3 mg/mL
while this concentration opposed to L. monocytogenes
gave only half of inhibition activity compared to both
nisin and chloramphenicol control 47.09±0.02% vs
94.14±0.04% and 100±0.005%). Increasing the
dose treatments tended to decrease bacterial-kiling
capacity of skimmed-milk hydrolysate to both
Figure 2. SDS-PAGE and zymogram assay of extracellular
patogenic bacteria tested.
proteases L. plantarum S31. (a) SDS-PAGE analysis of
protein in crude extract (CE), 80% saturated-ammonium
sulphate protein pellet (P), and group 1 of sephadex Disscusion
G50 fraction (G50). (b) Zymogram result of group 1 of
sephadex G50 fraction (G50). M was broad molecular In this present study, the extracellular proteases
weight markers (Bio-Rad). The yellow arrow indicates the L. plantarum S31 have been successfully isolated
protein coresponding to extracellular proteases while their from supernatant of 20-hrs culture. The extracellular
proteolytic enzyme location was pointed by red arrow. proteases produced by L. platarum S31 was greatly
depended on cell growth indicating tight corelation
of L. plantarum S31’s biochemical characteristic. between incubation time and proteases synthesis.
The proteases activity was low when enzymes Generally the harversting time could be done in
incubated at 25oC but its activity rapidly increased range of early to late stasionary phase of cells
when exposed to 35oC. The proteolytic activity tend growth. For instance, supernatant harvested at 20-hrs
to stable, retained more than 70% of its initial activity cultivation exhibited good proteolytic activity when
over temperature range of 45oC to 75oC indicating the tested visually on gelatin-containing agar assay. This
characteristic of thermostable enzyme. The enzymes result is relevant with other reports proving that
showed good proteolytic activity in acid to neutral most of extracellular bacterial proteases reached its
pH condition and its maximum activity was at pH 5. optimal yield at stasionary phase of growth (Ferrero
No activity was recorded at base condition. et al., 1996; Kaur et al., 1998; Bhaskar et al., 2007;
Tween-20 (non-ionic surfactant) completely Wang et al., 2008; Palsaniya et al., 2012). However,
abolished enzymes activity. In sharp contrast, SDS L. plantarum S31 in this study was only cultured on
(ionic surfactant) significanlty increased protease basal MRS medium, we predicted the yield of protease
activity up to 50%. PMSF (serine protease-type enzymes or even pattern of proteases produced may
inhibitor) and EDTA (metalloprotease-type inhibitor) be further engineered by modifying the nutritional
at 5 mM treatment greatly reduced half of proteolytic composition of growth medium as have been done
activity of the enzymes. Metal ions, MgCl2 and CaCl2, on Oenococcus oeni and Streptococcus thermophilus
effected the enzymes activity in different degree of (Folio et al., 2008; Chang et al., 2014). If the
concentration. MgCl2 increased dose-dependently differences of carbon sources, nitrogen sources and
the proteolytic activity of the enzyme. On the other micronutrients added into MRS medium would effect
hand, CaCl2 repressed enzyme activity when only significantly on yield and pattern of extracellular
high dose was applied. Somehow, DTT treatment proteases produced by L. plantarum S31 need further
enhanced proteolytic activity of extracellular clarification.
proteases L. plantarum S31 up to 30%. Two types of extracellular proteases L. plantarum
Generally, all proteins (lactoferin, albumin, S31 have been successfully identified from gel
casein, β-lactoglobulin and α-lactoalbumin) filtration-purified G1 group using zymogram assay
composing skimmed-milk were reduced time- and those proteolytic activity observed precisely
dependently and significantly occurred after 15-hrs corresponded to protein bands with molecular
hydrolysis process. Interestingly, at 18-hrs hydrolysis mass of ~18 kDa and ~37 kDa that appeared on
one new protein band appeared with molecular mass SDS-PAGE agar so we named these proteases as
of ~21 kDa and also smear protein band below ~16
345 Budiarto et al./IFRJ 23(1): 340-349

Table 1. Summary of partially purification of extracellular proteases L. plantarum S31

Tabel 2. Characteristic of extracellular proteases L. plantarum S31

*
values are represented as mean±SD (n=3)
**
Enzymes also showed well adapted at increased temperature from 45oC to 75oC
***
No activity was found when enzymes adapted to alkaline enviroments
****
It was done at 37oC, pH 7 with 18 hours incubation as evaluated by using SDS-PAGE

LPS31.18 and LPS31.37. Application of zymogram S31 as no protein band was detected on SDS-PAGE
assay to identify extracellular proteases in our agar in which the sample was denaturated prior to
study significantly enhances the successfulness separation. In non-denaturating condition as seen
of enzyme isolation due to simplicity of detection. on zymogram agar one of extracellular proteases L.
Moreover, this technique could predict not only the plantarum S31 seems able to form dimeric structure
activity of targeted proteases but also its molecular through chemical interaction. This phenomena
mass (Kleiner and Stevenson, 1994; Raser et al., also has been observed in other proteases such as
1995). Moreover, interesting finding in this study is at Euphorbia neriifolia Linn Nerrifolin S (non-
appearing of ~120 kDa protein band on zymogram denaturating separation: 94 kDa, denaturating
agar. This protein was predicted to be oligomeric separation: 47 kDa) Arabidopsis thaliana Tripeptidyl
state of one of extracellular proteases L. plantarum Peptidase (non- denaturating separation: 5- to
 Budiarto et al./IFRJ 23(1): 340-349 346

Table 3. Antibacterial activity of skimmed-milk obtained from 18 hrs hydrolysis by

extracellular proteases L. plantarum S31 against food-born patogenic bacteria

Inhibition activity shown in the table as mean ± SD (n=2)

na means no antibacterial activity observed
9-MD complex, denaturating separation: 153 and with inhibitory value nearly 50% of untreatment
142 kD) and prokaryotic rhomboid proteases (non- control (100% protease activity). In fact that the
denaturating separation: ~40 kDa, denaturating extracellular proteases L. plantarum S31 in this study
separation: ~20 kDa) (Book et al., 2005; Yadav et al., was composed of 2 type of proteases with different
2012; Sampathkumar et al., 2012). in molecular wieght, we assume that these proteases
The extracellular proteases L. plantarum S31 may belong to serine-type or metal-type protease.
showed thermal stability property as these enzymes The dependency of protease activity on metal ions
could retain proteolytic activity even the temperature such as Ca2+ and Mg2+ as cofactors more strengthen
was raised above 65oC. Proteolytic activity was our previous prediction of which one of extracellular
also highest at pH 5 and its enzyme activity totally proteases L. plantarum S31 would be metal-type
disappeared when pH increased above 7. The protease. For example, proteolytic activity of neutral
characteristic of thermostabilty of proteases in our metalloprotease (34.7 kDa) isolated from L. brevis
study was closely similar to that of extracellular was stimulated under metal ion co-treatment (5
proteases, named Lmm-protease-Lh (29 kDa) and mM) such as Ca2+, Mg2+, Na+ and K+ (Amund et al.,
Hmm-protease-Lh (62 kDa), isolated from Kefir- 1990). To clarify which of these proteases belong to
originated Lactobacillus helveticus which showed either class, next step of purification using cation/
higest actvity at 60oC to 80oC (Valasaki et al., 2008). anion exchanger chromatography will be done in the
Other thermo-tolerance proteases also have been further study.
identified in other positive-gram bacteria such as Types of surfactant and DTT have quite
Bacillus cereus KCTC 3674 (36 kDa and 38 kDa different effect on enzymes activity. SDS treatment
protease) and Bacillus stearothermophilus strain tremendously increased enzymes activity while
TLS33 (36 kDa proteases S, 53 kDa protease N, and tween-20 completely abolished it. The discrepancy
71 kDa protease B) (Sookkheo et al., 2000; Kim et effect of these surfactants on enzymes activity
al., 2001). Additionally, proteases activity exhibited were probably due to characteristic of surfactant’s
by extracelular proteases L. plantarum S31 in broad ionic charge that would greatly impact on stability
acidic pH (at pH 4 to 7 the enzymes retained mostly and solubilty of enzymes in medium. For instance,
80% protease activity) seemed to be an adaptive denaturated proteases occured during purification
mechanism to compensate medium acidification due steps could be restored its proteolytic activity
to accumulation of lactic acid during growth. This by SDS treatment (2 mM or equally to 0.05%)
result was in accordance with Takehana et al. (1999) using concentraion that belowed Critical Micelle
who succeeded purifying two extracellular acid Concentration (CMC) (Prieto et al., 2014). Altough
proteases CPMB8 (61 kDa) and CPMB11 (36 kDa) SDS concentration (aboved its CMC value) used in
from acid-tolerant bacteria. our experiment was 50 to 100 time higher than that
Reduction in proteases activity due to proteases of previously reported, the effect of SDS on enzymes
inhibitor treatment is a good indicator to classify property indeed enhanced its activity. This result was
proteolytic enzymes (Rao et al., 1998). Extracellular also observed in antiviral serine protease silkworm
proteases L. plantarum S31 was sensitive to both and in serine protease Bacillus cereus SIU1 whose
proteases inhibitors, PMSF (serine-type protease its respective protease activity was induced under
inhibitor) and EDTA (metal-type protease inhibitor), 0.2% or 0.1% up to 1% SDS treatment (Matti et al.,
347 Budiarto et al./IFRJ 23(1): 340-349

2010; Singh et al., 2012). Moreover, several bacterial obtained from 20-day culture of L. plantraum
surfacant-resistant proteases have been reported S31 and used as proteolytic enzymes to generate
recently such as 20 kDa serine protease B. mojavensis bioactive peptides from skimmed-milk. The purifed
A21 (0.1% SDS treatment), (Haddar et al., 2009), 21 enzymes contained mainly two proteases with
kDa alkaline metalloprotease B. firmus CAS 7 (1% molecular mass of 18 kDa and 37 kDa and this mixed
to 10% SDS treatment) (Annamalai et al., 2014), enzymes showed thermotolerance characteristic and
and alkalithermophilic protease Alkalibacillus sp. mostly active at pH 5. The activity of the enzymes
NM-Fa4 (1% to 5% SDS treatment) (Mesbah and was also enhanced by present of, SDS, cofactors
Wiegel, 2014). DTT enhanced caseinolytic activity (Mg2+ and Ca2+) and DTT. Moreover, skimmed-milk
of extracellular proteases L. plantarum S31 (1.34 hydrolysate generated by these enzymes showed
fold-increment) due to reduction of disulphide bonds promising antibacterial activity towards E. coli and
within casein substrate that make easier for enzymes L. monocytogenes tested.
to act. This observation was close similar with the
enhancing effect of DTT on keratinolytic activity Acknowledgment
of protease Aspergillus parasiticus (Anitha and
Palanivelu, 2013). This work was financially supported by
Discovery of bioactive peptides derived from competitive LIPI program 2014.
enzymatically-proteolyzed milk products has gained
much attention recently (Fitzgerald and Meisel, References
2003). Many of which have been clinically proven
to show broad biological activity against the most Aguilar, G., Morlon-Guyot, J., Trejo-Aguilar, B. and
deadly illness such as cancer and infecious deseases Guyot, J.P. 2000. Purification and characterization of
(Kilara and Panyam, 2003; Muro et al., 2011). an extracellular α-amylase produced by Lactobacillus
Here, we have applied the extracellular proteases L. manihotivorans LMG 18010T, an amylolytic lactic
acid bacterium. Enzyme Microbiology Technology
plantarum S31 to generate bioactive peptides using
27(6): 406-413.
skimmed-milk as substrate and tested its potency Amund, O.O., Omidji, O. and Ilori, O. 1990. Purification
as natural antibacterial resource. The hydrolysis and properties of a neutral protease produced by
proccess of skimmed-milk has been done at maximal Lactobacillus brevis. Journal of Biotechnology 13:
proteolytic activity of enzyme which is 37oC, pH 7 361-365.
and 18-hrs hydrolysis. The pH 7 was chosen instead Anitha, T.S. and Palanivelu, P. 2013. Purification and
of pH 5 due to solubility of skimmed-milk was much characterization of an extracellular karatinolytic
better at this condition. Skimmed-milk hydrolysate protease from a new isolate of Aspergillus parasiticus.
in our study exhibited antibacterial activity towards Protein Expression and Purification 88: 214-220.
L. monocytogenes and E. coli (two kinds of bacteria Annamalai, N., Kumar, A., Saravanakumar, Vijaylakshmi ,
A. S. and Balasubramanian, T. 2011. Characterization
which are common found in spoilage foods) with
of protease from Alcaligens faecalis and its
different degree of sensitivity. IC50 for E. coli is antibacterial activity on fish patogens. Journal of
at around 0.3 mg/mL while L. monocytogenes Environmental Biology 32: 781-786.
seemed to be slight resistance to the treatment at Annamalai, N., Rajeswari, M.V., Sahu, S.K. and
this concentration. Further increasing the treatment Balasubramanian, T. 2014. Purification and
doses reduced bacterial killing ability of skimmed- characterization of solvent stable, alkaline protease
milk hydrolysate. This could be happen probably due from Bacillus firmus CAS 7 by microbial conversion
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