Department of Botany, B R A Bihar University, India
Submission: September 24, 2018;Published: October 24, 2018
*Corresponding author: Santosh Kumar, Biotechnology Unit, Department of Botany, B R A Bihar University, Muzaffarpur 842001, India.
How to cite this article: Anamika Thakur, Sanju Kumari, Utkarshini Sharma, Rohit Krishna, Kanak Sinha, Santosh Kumar. Molecular Characterization of Protease Producing Stenotrophomonas and Their Cytotoxic Effects on Cancer Cell Lines. Int J cell Sci & mol biol. 2018; 5(2): 555660. DOI: 10.19080/IJCSMB.2018.05.555660.
Proteases are highly required where proteins need some sort of alteration either structurally or functionally. They are widely used in household laundry, paper and leather industries as well as in several pharmaceutical compounds. Thus, the present study was confined to screen more than fifty isolates of microbes from the rhizospheres of fruit orchard for proteolytic activity on skimmed milk agar medium. Isolates SK701 and SK709 were found producing halo regions around the streaked mycelia. However, only isolate SK709 displayed maximum 64.6 U/ml protease activities in starch casein broth after 96 hours of culture at optimum conditions of 35°C and pH 7.6. It was identified as a gram-negative bacterium on the basis of staining, morphological, cultural, biochemical and physiological characteristics. The cytotoxic activity of cell free extracts of isolate SK709 against EAC, SiHa & Hep G2 cell lines in terms of IC50 was found to be 15.1, 26.3 and 35.5μg/ml, respectively. The 16S rRNA gene sequencing, molecular characterization and the BLAST operation as per NCBI directive to construct phylogenetic tree confirmed the putative protease producing isolate SK709 to be the species of Stenotrophomonas. The difference of 2% in the gene sequence with the closest member, Stenotrophomonas sp. MCYF1(KC734883.1) which amounted to 21 out of 1022 nucleotides, made the isolate SK709 qualified for obtaining a separate NCBI accession number KF484911..
Enzymes are biocatalysts obtained from plants, animals & microorganisms. Microbes are known for rich source of natural products with biomedical properties. They are important source of bioactive compounds especially antibiotics  and extracellular enzymes . The ongoing research on microbes has brought a technical platform for industries by way of producing industrial enzymes viz., keratinases, pectinases, xylanases, amylases, lipases, phytases, cellulases and more prominently proteolytic or proteases (caseinases and gelatinases). Microbial exploitation for the production of these protease enzymes is highly attractive for applications in fruit, detergent, pharmaceuticals, paper industries and leather . Microbes producing proteases are preferred for their easy isolation, limited space requirements and optimum temperature near 30°C . The physical factors such as temperature, pH, incubation time, agitation, inoculums density and the availability of carbon and nitrogen source in the media also influence its production.
Proteases almost constitute 25-30% of total global enzyme sales . In recent years many authors have characterized the microbial proteases as an important group of secondary
metabolites for industrial purposes. Actually, the importance of proteases lies in its mode of action: it hydrolyses bonds of amino acids, either at terminal ends (exopeptidases) or in the middle of chain (endopeptidases) . The other group of proteolytic enzymes, proteinases hydrolyses the secondary structure of protein. Thus, proteolytic enzymes are highly required where proteins need some sort of alteration either structurally or functionally , they actually destroy cell components such as lipoprotein membranes and immunoglobulins . The cytotoxic study of the culture filtrate of Stenotrophomonas maltophila caused vigorous endocytosis in cancer cell lines like Vero and HeLa cells . The function of proteases is largely pH dependent, so based on optimum pH, proteases have been recognized as acid proteases, neutral proteases and alkaline proteases [5,6]. Alkalophilic microorganisms such as Streptomyces, Micromonospora, Stenotrophomonas etc., are prone to produce alkaline proteases into extracellular media which are widely used in biotechnological processes.With this background the present study has been aimed to screen the isolated strains of microbes from the rhizosphere of Muzaffarpur (mainly from fruit orchards) for the production and purification of proteases and its characterization with respect to alteration in protein configuration to assess the cytotoxic effect on
cancer cell lines.
The soil samples were collected from rhizosphere of different
locations of Muzaffarpur (26°7’N &85°25’E) district in Bihar,
India. The soil samples were taken from 2-5 inches below the
surface using sterilized spatula and zipped bags. The samples
were kept first at 70°C for 15 minutes to evaporate moisture. It
was then mixed thoroughly and sieved through a domestic 1.5mm
pore size sieve to get rid of large debris.One gram of thus obtained
sieved soil sample was suspended in conical flask having 10ml
sterile distilled water. The flask was thoroughly shaken. It was
then filtered through a two layered muslin cloth. Thereafter the
sample was diluted to 10-5 dilutions and stored in sterilized test
tubes at 4°C for 24h.
Aliquots (ml) of each dilution were spread on the surface of
starch agar medium (Starch-9g, L-asparagine-9g , Ammonium
sulphate -2g,Tris -2g, Sodium chloride –1g, Dipotassium sulphate
-0.5g, Magnesium sulphate -0.2g, Calcium chloride -0.1g, Trace
solution - 1ml, Potassium Dihydrogen Phosphate -0.5g, agar
-15g, dissolved in one litre distilled water; pH -7.0) plates,
supplemented with antifungal nystatin (50μg ml-1) and incubated
at 35±2ºC for seven days. Plates with around 200 colonies were
selected. Streaking of single colony was carried out to purify the
selected colonies on the same medium. Gram staining smears
were prepared and examined under the microscope to ascertain
the nature of isolates.
More than fifty isolates, purified from the soil samples were
screened by streaking the mycelium on skim milk agar medium
(Skimmed milk powder-100g, peptone -5g, agar-15g, dissolved in
one litre Distilled water, pH 7.0) andinoculated at 35±2°C for 5-7
days. The culture plates showing transparent halo zone against an
opaque non-hydrolysed medium indicated the protease producing
strains. Proteolytic activities of the isolates were measured by
diameter of clear zone around colony on skim milk agar plates and
finally two isolates, SK701 and SK709 were selected for further
The colour of colony, aerial mycelium and soluble pigment
of the selected isolates when grown on starch agar medium
were recorded after 96h of incubation. Smears of isolates
were prepared for gram staining and spore chain morphology
studied under a compound light microscope (Nikon, Japan)
using 1000× magnification power. Colour of the substrate
mycelia (reverse of the plate) was also observed along with
diffusible pigments. Various biochemical and physiological
tests performed for the identification of the potential isolates
were indole production, methyl red, Voges Proskauer,
citrate utilization, casein hydrolysis, starch hydrolysis, urea
hydrolysis, gelatin hydrolysis, H2S production, temperature
tolerance, utilization of carbon and nitrogen sources and
Sodium chloride resistance.
Optimum conditions for growth (optical densities at
540nm) and proteinase production of the selected isolates,
SK701 and SK709 were carried out in starch casein broth
(soluble starch: 10g, K2HPO4: 2g, KNO3: 2g, casein: 0.3g,
MgSO4.7H2O: 0.05g, CaCO3: 0.02g, FeSO4.7H2O: 0.01g, Distilled
water: 1litre, pH:7.0) by incubating on shaker at 35°C for 7 days.
Effects of different incubation temperatures (25, 30, 35, 40,
45, 50, 55 and 60°C) and initial pH (4, 5, 6, 7, 8 and 9) of the
medium on protease estimation just after 96 h were screened
in broth. Effect of casein degradation on pH was also noticed
on daily basis. Different carbon sources such as 1.0% glucose,
sucrose, fructose, lactose, and mixture of glucose + sucrose,
both at 0.5% were also tested. Nitrogen sources were 0.5%
yeast extract, ammonium sulphate, sodium nitrate, urea and
ammonium acetate. All the experiments were carried out in
triplicate and averages were reproduced.
The isolates, SK701 and SK709 with protease activities
were allowed to grow starch casein broth medium at 35°C for
2 days on a rotary shaker at 150rpm. Then 1ml of such grown
bacterial suspension was again cultivated in starch casein
broth medium (40mL) in 250mL Erlenmeyer flasks for 7 days
at 37°C at 200rpm. After due period, centrifugation of broth
suspension was carried out at 10,000rpm for 15min and cell
free supernatant was separated for protease activity assay.
The method of Tsuchida et al.  for protease estimation
was followed. 250ml of starch casein broth was prepared and
inoculated with SK701 and SK709 separately and incubated at
37°C for 7 days in incubator shaker at 180rpm. After that the whole
broth was centrifuged at 10000rpm at 4°C for 20min and the clear
supernatant was taken as crude enzyme and was stored at 4°C.
A mixture of 500μl of 1% (w/v) of casein in 50mM phosphate
buffer (pH 7) and 200μl crude enzyme extract were incubated in a
waterbath at 40°C for 20min. After that the enzyme reaction was
terminated by adding 1ml of 10% (w/v) TCA and was kept at room
temperature for 15min. The reaction mixture was centrifuged
to separate the unreacted casein at 10,000rpm for 5min. The
supernatant was then mixed with 2.5ml of 0.4M Na2CO3. 1ml of
3-fold diluted Folin and Ciocalteus Phenol reagent was added. The
resulting solution was incubated at room temperature in dark for
30min and the absorbance of the blue colour, thus developed, was
measured at 660nm against a reagent blank using tyrosine.Since
SK709 had shown maximum proteolytic activity and thus selected
for further investigation.
10ml of overnight culture of the isolate SK709 was transferred
into conical flask containing 500ml of Bennet medium (sterilized
with steam explosion) individually and incubated for 7 days at
24°C at 200rpm and pH (7.0±0.2). Further the mass cultured broth
was filtered through cheese cloth to get clear filtrate. Further,
equal volume (1:1) of ethanol was added & mixed by vortexing
& kept without disturbance. The organic phase was collected &
evaporated at 70°C in an incubator. The obtained residue was
stored at-20°C for cytotoxicity assay.
EAC (Ehrlich Ascites Carcinoma), SiHa (Human cervical
cancer) and HepG2 (Hepatic carcinoma) cell lines were procured
from NCCS Pune, India. Cells were cultured and maintained in
Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% Fetal
Bovine Serum and antibiotics in a humidified atmosphere of 5%
CO2at 37°C in culture dishes/flasks. Stock culture was maintained
in the exponential growth phase by passaging as monolayer
culture using in 0.02% EDTA. The dislodged cells were suspended
in complete medium and reseeded routinely.
The cytotoxic effects were assessed against EAC, SiHa and
HepG2 cells at the varying concentrations of plant extracts by the
MTT assay. The 3-(4,5-dimethyl-2-yl)-2,5-diphynyl tetrazolium
bromide (MTT) is metabolic substrate which is reduced by the
mitochondrial succinate dehydrogenase enzyme and forms
formazan crystal. In brief cells were seeded overnight and then
incubated with various concentration of extracts for 72h. At
the end of the treatment, medium was removed, and cells were
incubated with 20μl of MTT (5mg/ml in PBS) in fresh medium
(50μl) for 4 hrs in CO2 incubator. After the treatment formazan
crystal, formed by mitochondrial reduction of MTT were
solubilised in DMSO (150μl /well) and the absorbance was read at 570nm after 10min incubation on the iMark Microplate Reader
(Bio-Rad, USA). Percent cytotoxicity (IC50) values were determined
by extrapolating the graph.
16S rRNA gene of isolated genomic DNA of SK709 was
amplified  in a PCR thermal cycler (Genetix) by universal
primers, 9F (5’GAGTTTGATCCTGGCTCAG3’) & 1541R
(5’AAGGAGGTGATCCAACC3’). The reaction mixture contained
2μg of dNTPs mixture (1.25mM each), 1μg of each of the primers,
2.5μg of DNA, 1μl of Taq polymerase and sterile deionised water to
make final volume 100μl. PCR consists of an initial denaturation
at 94°C for 1min, annealing at 63°C for 1min and 72°C for 1min
& final 5min extension at 72°C. The PCR amplified product were
stained with ethydium bromide and run on agarose (1.2%) gel
electrophoresis and examined under gel documentation system.
The complete 16S rRNA gene of SK709 was sequenced by using
PCR products at Samved Biotech, Ahmedabad.
The 16S rRNA gene sequence of SK709 were
compared with other bacterial sequences by using NCBI BLAST
programme. Multiple sequence alignment & phylogenetic tree
were constructed using software programme MEGA 4.0 . The
16S rRNA sequence of strain SK709 was submitted to GenBank
(Nucleotide database of NCBI). The sequence was accepted and
the accession number (KF849411) for SK709 was obtained.
Bacterial strains, isolated from the rhizosphere of Muzaffarpur
were grown on starch agar medium, supplemented with
nystatin and were screened colony colour, spore morphology,
[14,15] mycelia colour, etc. (Table 1). Two bacterial strains,
SK701 and Sk709 of litchi orchard of Muzaffarpur were
confirmed with proteolytic activities as they developed halo
regions on Skimmed milk agar plates.But isolate SK709 was
more prominent having approximately 28 mm clear halo
zones around colonies or streaked mycelia on fourth day of
incubation (Figure 1). Purified strains were examined under
microscope for gram staining and Isolate SK709 was found
to be gram negative whereas isolate SK701 with many others
were found to be gram positive. Further both isolated reacted
positively in different biochemical and physiological tests
(Table 2). Sucrose was found to be the optimum source among
carbon compounds and beef extract for protease production. The
optimum growth of bacterial isolates was found at pH 7.8 and
temperature at 35±2°C.
Growth of the bacteria and protease production were found
to be maximum on 4th day of incubation. The casein-degradation
activities by releasing proteinase was found maximum (64.6 U/
ml) on 4th day (Figure 2).The pH above 7.0 was required in case
of gram negative isolate SK709 whereas pH with less than 7.0
favoured the growth of other bacterial strains including SK 701.
The pH of the broth started to increase with the growth of isolate
SK709, the value reached to 7.8 on 4th day after 96h of incubation
(Figure 3). The effective temperature for maximum protease
activities was noticed at 35±2°C. At high temperature (>45°C), the
protease activities dropped to the level of 20% (Figure 4).
The cytotoxicity effect of crude extract of isolate SK709
was assayed against EAC, SiHa and HepG2 cells at the varying
concentration by MTT assay. The data of MTT assay were used
to extrapolate IC50 of the extracts of these cell lines, which was
found dose (200 μg/ml) and time dependent (72h). Among the
three cancer cell lines (Figure 5), EAC showed the minimum
concentration IC50value (17.2μg/ml) followed by SiHa (26.5μg/
ml) and Hep G2(35.5μg/ml).
The genomic DNA of the proteolytic isolate SK709 was
isolated by standard protocol  and the samples were run on
0.8% agarose gel electrophoresis. Thereafter PCR amplification
using universal primers resulted into a single discrete amplicon
band of 1.5kb of 16S rRNA gene (Figure 6).
The NCBI BLAST homology analysis of 16S rRNA gene of SK709
showed maximum similarity (≥98%) with Stenotrophomonas sp.
Based on the result, phylogenetic tree was constructed using
neighour joining method (Figure 7).Thesequence (Table3) was
published after trimming and assembling to NCBI(http//: www.
ncbi.nlm.nih.gov). It was revealed that the strain SK709 showed
1001 base similarity out of 1022 (98%) with Stenotrophomonas
sp. MCYF1(NCBI Accession no. KC734883.1). Thus, the difference
of 21 nucleotides in the 16S rRNA gene qualified the strain SK 709
to earn a separate NCBI Accession no. KF484911.
Microorganisms are usefulness in food and dairy industries,
pharmaceutical sectors, leather industries etc. They produce a
variety of bioactive substances and secondary metabolites. Nowa-
days, they are an important producers of industrial significant
enzymes viz. protease, amylase, phytase, cellulase, lipase, etc.
[17,18]. Proteases are one of such novel enzyme that is useful in
day-to-day activities. Most of the bacterial strains were isolated
on starch agar medium. In order to obtain a specific byproduct,
the medium requires some modifications. Thus, a specific
protease screening medium (skim milk agar medium) was used
for screening casein degrading isolates. Cultural, morphological,
physiological, biochemical properties revealed the strains SK701 &
SK709, isolated from rhizosphere of litchi orchard of Muzaffarpur
to be the species of genera Streptomyces (actinomycetes group)
and Stenotrophomonas (Pseudomanad group), respectively. The
generic status of these strains SK701 and SK709 was not only
confirmed by physiological and cultural characterization but also
by the Burgey’s manual of Determinative Bacteriology [16,17].
However, the final support of the recognition Stenotrophomonas
was established on the basis of phylogenetic tree constructed
using 16S rRNA gene sequencing [19,20].
The proteolytic activity in broth was observed after 96h of
incubation during the stationary phase. The protease activity
was optimum at 35°Cand showing maximum activity pH 7.8,
confirming alkaline nature of protease enzyme . Different
alkaline protease producing alkalophilic strains have been
reported .Alkaline proteases showed better resistance to alkali
and other denaturing chemicals in the reaction mixture & have
higher affinity towards proteinaceous substrates thus it could
be proved as a very good detergent supplement. The organism
showed optimum growth near 35±2°C indicating its mesophilic
nature. In present investigation optimum protease production
was obtained by combination of sucrose (carbon source) & beef
extract (nitrogen source).
In present study an attempt was made to find cytotoxic activity
from the extract of Stenotrophomonas sp. Earlier Figueiredo
et al.  have reported cytotoxic activity in Stenotrophomonas
maltophila. The bacterial isolate showed IC50 value 17.2μg/ml,
26.5μg/ml and 35.5μg/ml in EAC, SiHa and Hep G2 cancer cell
lines, respectively. Suffness et al., have reported that the IC50
values less than 30μg/ml in cancer cell lines can be considered as
potent for anticancer drug development. This report augurs
that the extract of SK 709 may prove a beneficial chemical to fight
against cancer and the protease may have the ability to make
alteration in protein configuration in cancer cell lines leading to
apoptosis and thus hindering the growth of tumour cells.
The isolate SK709 with proteolytic activity on skimmed
milk agar medium was identified as Stenotrophomonas on the
basis of morphological, cultural, biochemical and physiological
characteristics as well as 16 S rRNA gene sequencing. On the basis
of 98% blast homology data base similarity with Stenotrophomonas
sp.MCYF1(KC734883.1), the isolate SK709 was christened as
Stenotrophomonas sp strain SK 34L and because of the difference
of 21 nucleotides which amounts to 2%, the strain earned a fresh
accession no. KF849411.
Authors are thankful to the Head of the Department of Botany
for providing proper facilities to work. SKas Project Fellows and
US as as P I, DST WOS-A,are thankful to the University Grants
Commission, New Delhi for granting financial support under
UGC SAP (DRS-Phase 1) scheme to the University Department of
Botany, B R A Bihar University Muzaffarpur (File No. F.3-13/2009-
SAP-II) and to the Department of Science and Technology, New
Delhi (File No. SR/WOS-A/LS-289/2011), respectively.