-
Abstract. Background: The objective of the present studywas to
evaluate the efficacy of a simple, versatile and cost-effective
immunosuppression protocol, using cyclosporine,ketoconazole and
cyclophosphamide drug regimen to develophuman tumor xenograft in
mice. Materials and Methods:Cyclosporine, ketoconazole and
cyclophosphamide drugregimen was administered to C57BL/6 mice to
induceimmunosuppression. Five million A549, LNCaP and KB cellswere
injected subcutaneously in the immunocompromisedmice for the
development of tumor xenograft. Tumor volumewas calculated every
week. Histopathology of tumor tissuewas analyzed. Results: Prolong
immunosuppression wasachieved by this combination treatment. The
average tumorvolume was found to be greater than 600
mm3.Histopathology of tumor tissue revealed the presence of
largeand irregular nucleus and scanty cytoplasm, which
arecharacteristic of malignant cells. Conclusion: A
versatileimmunosuppression protocol was developed which
wasvalidated for xenograft development using three different
celllines, with a 100% take rate and no mortality.
The International Agency for Research on Cancer (IARC)states
that over 10 million new cases of cancer occur eachyear and over
six million deaths annually occur from cancer.The IARC also
estimates that by 2030, the cancer burdenwill increase to 27
million new cases and 17 million cancer-related deaths globally
(1). In response to these statistics,there is immense research in
the field of medicinal chemistryfor synthesis of novel anticancer
agents (2), isolation andscreening of natural product-based
anticancer compounds
(3), designing of novel drug delivery systems
forchemotherapeutics (4) and biological drugs such as
Smallinterfering RNA/Small hairpin RNA (si/shRNA) (5), to cureor
manage the disease.
In vivo efficacy study of these anticancer agents or
drugdelivery systems majorly employs tumor xenograft model inmice.
Tumor xenograft models are not only useful toestablish the efficacy
of anticancer agents in terms of tumorreduction but are also
valuable in studying the effect ofanticancer agents on key
hallmarks of cancer, such as tumorangiogenesis (6), and metastasis
(5).
All such studies utilize genetically-immunodeficientathymic nude
mice for the development of tumor xenografts.Eventhough the nude
mice xenograft model is widelyemployed, there are serious drawbacks
associated with thismodel. Disadvantages include high cost;
difficultinavailability, especially for developing countries;
difficultyin transportation and aseptic maintenance; tendency for
graftrejection; high mortality rate, etc (7-9). Additionally,
nudemice may not accurately reflect true disease progressionbecause
they lack immune cells which play a critical role intumorigenesis
(10, 11).
In light of these drawbacks of nude mice, many researchershave
proposed different immunosuppression protocols for thedevelopment
of pharmacological immunosuppression with thehelp of appropriate
methods such as total-body irradiation,neonatal thymectomy, and
immunosuppressive drugs. Forinstance, Steel et al. proposed an
immunosuppressive modelby thymectomy and total-body irradiation
combined withsyngeneic bone marrow transplantation or
cytosinearabinoside pre-treatment (12). Floersheim et al. developed
axenograft model of human tumors in mice after
short-termimmunosuppression with procarbazine, cyclophosphamideand
antilymphocyte serum (13). However, the technicalrequirements of
these protocols are expensive, prolonged andmake animals moribund,
which compromise their use forlarge-scale screening procedures. For
instance, Floersheimreported a 33% mortality rate from thymectomy
and a further39% mortality rate within 60 days of irradiation with
9 Gy ofmegavoltage X-rays (14). After the discovery of
cyclosporin
7177
Correspondence to: Manish Nivsarkar, Ph.D., Director,B.V.
PatelPharmaceutical Education and Research Development
(PERD)Centre, Sarkhej-Gandhinagar Highway, Thaltej,
Ahmedabad-380054, Gujarat, India. Tel: +91 7927416409, Fax: +91
7927450449,e-mail: [email protected]
Key Words: Immunosuppression, cyclosporine,
ketoconazole,cyclophosphamide, tumor xenograft model.
ANTICANCER RESEARCH 34: 7177-7184 (2014)
An Improved and Versatile Immunosuppression Protocol for the
Development of Tumor Xenograft in Mice
MEHUL JIVRAJANI1, MUHAMMAD VASEEM SHAIKH1, NEETA SHRIVASTAVA2
and MANISH NIVSARKAR1
Departments of 1Pharmacology and Toxicology, and 2Pharmacognosy
and Phytochemistry,B. V. Patel Pharmaceutical Education and
Research Development Centre, Ahmedabad, India
0250-7005/2014 $2.00+.40
-
A, displaying potent immunosuppressive effects against
theallograft response in animals (15) and Man (16), cyclosporineA
has become a drug of choice for developingpharmacologically
immunocompromised models for thedevelopment of tumor xenograft.
Cyclosporine is a polypeptide derived from the
fungusTolypocaladium inflatum Gams. It has been reported
thatcyclosporine acts mainly by suppressing the release
ofinterleukin-1 from macrophages, required for the activation
ofT-lymphocytes. It also inhibits the release of
interleukin-2,which is essential for the proliferation of activated
T-lymphocytes (17). Floersheim initiated the use of cyclosporinefor
the development of tumor xenografts in C3H mice (14).However,
Floersheim reported minimal tumor developmentafter administration
of cyclosporine for 30 days. After thisinitial success, many
researchers reported development oftumor xenograft by modifying the
dose of cyclosporine, routeof administration, duration of treatment
and combining withother drug regimen in several strains of rat (8,
10, 18-20).Even though these models showed considerable success in
thedevelopment of tumor xenograft, they have several
limitations.These limitations include long duration of
cyclosporinetreatment, requirement for a large number of tumor
cells, andvalidation with only a single cell line. Moreover, all
thesemodels have been developed in rats, which are more difficultto
maintain and handle for the development of tumorxenograft compared
to mice. Due to these limitations, thesemodels are difficult to
scale up and use to screen large numberof anticancer agents. Hence,
an immunosuppressive mousemodel which is easy to develop, causes
little or no mortalityand can be exploited to develop xenograft
from any cancerouscell lines with a 100% take rate is needed.
In the present study, a new, versatile and a
reproducibleimmunosuppression protocol was developed by
usingcyclosporine, ketoconazole and cyclophosphamide drugregimen to
develop human tumor xenograft in mice.
Materials and Methods
Materials. Cell culture mediums, Roswell Park Memorial
Institutemedium (RPMI-1640) and Dulbecco's Modified Eagle
Medium(DMEM) were purchased from Gibco, Grand Island, NY,
USA.Similarly, fetal bovine serum (FBS), sodium bicarbonate,
sodiumpyruvate, trypan blue and trypsin were obtained from
Gibco.Cyclosporine (Sandimmune) and Ketoconazole (Nizral)
werepurchased from, Novartis, Basel, Switzerland and Johnson &
Johnson,New Brunswick, New Jersey, USA respectively.
Cyclophosphamide(Endoxan) was purchased from Baxter, Halle,
Germany. Ampoxinwas purchased from Unichem laboratories Ltd.,
Mumbai, India.Rodent diet was obtained from VRK nutrition, Pune,
India.
Animals. Healthy mice C57 BL/6 were purchased from
MahaveeraEnterprises, Hyderabad, India. All the mice were kept
inindividually ventilated cages, with a relative humidity of 60±5%
anda temperature of 25±2˚C was maintained. A 12:12 h light:dark
cycle
was also regulated for these animals. Balanced rodent food
pelletand water was provided ad libitum. All experimental protocols
werereviewed and accepted (PERD/IAEC/2013/014) by the
InstitutionalAnimal Ethics Committee prior to initiation of the
experiment.
Cell lines. All the human cancer cell lines [A549 (human
lungadenocarcinoma), LNCaP (human prostate adenocarcinoma), andKB
(cervical adenocarcinoma)] were procured from NCCS, Pune,India.
A549 and LNCaP cell lines were maintained in RPMI-1640medium
supplemented with 10% heat-inactivated FBS and 1.0 mMNa pyruvate,
whereas KB cell line was maintained in DMEMsupplemented with 10%
heat-inactivated FBS. The cells were grownin 75 cm2 flasks and
maintained in a standard tissue cultureincubator at 37˚C with 5%
CO2.
Immunosuppression. Healthy male mice (C57 BL/6), 4-6 weeks
old,were divided into seven groups (n=6). Groups 1, 3 and 5
wereadministered 5 mg/kg ketoconazole and groups 2, 4 and 6
wereadministered 10 mg/kg ketoconazole by oral route every day for
7days. Groups 1 and 2 were administered 10 mg/kg, groups 3 and 420
mg/kg, and groups 5 and 6 30 mg/kg cyclosporine byintraperitoneal
route every day for seven days. No treatment wasgiven to the
control group. All the animals were provided autoclavedrodent food
pellet and water ad libitum. Animals were givenampoxin (0.1 μg/ml)
by drinking water during the study. Aftercompletion of the study,
hematology was carried out to determinethe total white blood cells
(WBC) and lymphocyte count to confirmimmunosuppression.
Cylophosphamide was injected subcutaneouslyat a dose of 60 mg/kg on
days 3 and 1 before tumor cell injection ingroups of mice showing
the highest immunosuppression.
Total WBC and lymphocyte count. Blood samples were collected
fromall the animals from retro orbital sinus under isoflurane
anesthesia ina heparinized 1.5 ml microcentrifuge tube. Total WBC
and lymphocytecounts were then performed in an automated hematology
analyzer(VetScan HM-5; Abaxis Inc.,Union City, CA, USA).
Preparation of tumor cells. Semi-confluent cells (A549, LNCaP
andKB) were trypsinized by using 0.25% trypsin to detach the
cells.Cells were centrifuged at 200 × g for 7 min at 4˚C,
resuspended andwashed in their respective growth medium i.e.
RPMI-1640 andDMEM. After washing, cells were again resuspended in
theirrespective growth medium. The cells were counted using a
Neubaurchamber and viability was determined by trypan blue
exclusion test.Viable cells were stored on ice and injected
immediately.
Tumor implantation. Immunocompromised male C57 BL/6 mice (4-6
weeks old) were used (n=6 for each cell line). Hairs were removedby
waxing from the shoulder blade of each animal one day
beforeinjection of 0.1ml of cells (approximately 5×106 A549, LNCaP
andKB cells) subcutaneously into the right shoulder blade of
mice.Tumor growth was observed at the site of injection. Tumor
volumewas measured every week externally by digital caliper
usingfollowing formula (21):
Volume (mm3)=(A) × (B2)/2, where A was the largest diameter(mm)
and B the smallest (mm).
At the end of the study, tumors were excised and
histopathologicalanalysis was performed.
ANTICANCER RESEARCH 34: 7177-7184 (2014)
7178
-
Histopathological analysis. At the end of the study, tumors
wereexcised from the animals and maintained in 10% neutral
bufferedformalin. Tumor samples were cut into 5 μm sections and
stainedwith hematoxylin and eosin. The slices were observed
andphotodocumented by optical microscopy (IX 51; Olympus,
Tokyo,Japan) equipped with a digital camera (TL4) in order to
confirm thepresence of malignant cells.
Statistical analysis. All the data are given as the mean±SD.
One-way ANOVA followed by posthoc Bonferroni correction wasapplied
to determine the significance of differences among
groups.Probability values with p≤0.05 were considered to be
significant.
Results
Immunosuppression. Combination of cyclosporine andketoconazole
induced significant immunosuppression in adose-dependent manner
when compared to control animals.Figure 1 shows the mean WBC and
lymphocyte count indifferent groups of mice at the end of
treatment. From thegraph, it can be seen that efficient
immunosuppression wasfound in the animals of group 6, which were
administeredcyclosporine (30 mg/kg) and ketoconazole (10 mg/kg),
whencompared to control animals. Total WBC and lymphocytecounts
were significantly decreased in animals of group 6when compared to
control animals (Figure 1). Hence,animals from group 6 were
selected for the tumor xenograftdevelopment.
Subsequently, cyclophosphamide was injected intoanimals of group
6 subcutaneously at a dose of 60 mg/kg ondays 3 and 1 before tumor
cell injection. Figure 2 shows themean WBC, lymphocyte and
neutrophil count in animals ofgroup 6 after cyclophosphamide
treatment. Administration ofcyclophosphamide significantly reduced
neutrophil andresidual WBC and lymphocyte count in the animals of
group6. Moreover, none of the mice showed any signs of toxicityor
premature death due to drug treatment. Hence,cyclosporine,
ketoconazole and cyclophosphamide inducedsevere immunosuppression
in the treated C57BL/6 mice.
Tumor implantation. Approximately 5×106 cells from each cellline
(A549, LNCaP and KB) were injected subcutaneously intothe shoulder
blade of immunocompromised mice. In allinjected animals, a palpable
tumor was found on the third dayafter tumor injection, with a 100%
take rate. Figure 3 showsthe mean tumor volume each week after
tumor implantation. Itcan be observed from the graph that the mean
tumor volumeincreased radically every week until the fourth week
for eachcell line. Subsequently, the tumor volume was found
toincrease steadily until the eighth week. Thereafter it reached
aplateau and maintained a steady state. The mean tumor volumeof
A549, LNCaP and KB xenograft was found to be 720 mm3,626 mm3 and
668 mm3, respectively. Subsequently, the tumorvolume started to
decrease in some animals. Growth of A549and KB xenografts was found
to be more aggressive than that
of LNCaP xenografts. Figure 4 shows C57BL/6 mice bearingtumor
xenograft eight weeks after tumor implantation (Figure4). Hence,
with this protocol, tumor xenografts successfullydeveloped using
three different cell lines, i.e. A549, LNCaPand KB, and were
maintained for more than two months.
Histopathological analysis. The presence of malignant tumorwas
confirmed by histopathology. Tumors were excised,sectioned and
stained with standard hematoxylin and eosin.Figure 5 shows
hematoxylin and eosin-stained sections ofA549, LNCaP and KB
xenografts. The section shows cellswith large and irregular nuclei
and scant cytoplasm, whichare characteristic of malignant cells
(Figure 5B).Histopathological analysis also revealed the presence
ofangiogenically-activated blood vessels, suggesting theinduction
of angiogenesis (Figure 5Ai). Additionally, it alsoshows malignant
cells invading adjacent stromal tissue(Figure 5Aii and iii).
Discussion
Tumor xenograft models are primarily used to evaluate thein vivo
efficacy of anticancer agents (3-5). These studiesemployed athymic
nude mice for the development of tumorxenograft. However, due to
several limitations of nude mice,many researchers have developed
immunocompromisedmodels which can accept tumor xenograft and
subsequentlyproliferate to produce larger tumor. These models
employtotal body irradiation, neonatal thymectomy,
andimmunosuppressive drugs (12, 13). However, these protocolsare
expensive and cause huge mortality.
Cyclosporine, a potent immunosuppressant, selectivelyinhibits
the activation of T-cells. Cyclosporine binds to thecytosolic
protein cyclophilin of T-cells. This complex inhibitscalcineurin,
which, under normal circumstances, isresponsible for activating the
transcription of interleukin 2,which promotes T-cell activation and
proliferation (22, 23).
Floersheim first reported the use of cyclosporine for
thedevelopment of tumor xenograft (14). However, Floersheimreported
minimal tumor development after dailyadministration of cyclosporine
(100 mg/kg) for 30 days. In1983 Hoogenhout et al. reported a
combination of totallymphoid irradiation, cyclophosphamide and
cyclosporine Afor immunosuppression and achieved a 100% take rate
withmouse osteosarcoma. However, with this protocol theyachieved
only a 63% take rate with human colonicadenocarcinoma (18).
Similarly, Goodman et al. reported thegrowth of human melanoma
section in Lewis rats givencyclosporine at 15-50 mg/kg with a 85%
take rate. However,under the same protocol they were unable to grow
tumorswhen human melanoma cell suspension injectedsubcutaneously
(8). Akhter et al. reported a 100% take rateof the human colonic
adenocarcinoma cells in Sprague
Jivrajani et al: Immunosuppression Protocol for the Development
of Tumor Xenograft
7179
-
Dawley rats when administered 35 mg/kg daily cyclosporineuntil
the end of study. However, the inoculum size injected inthis
protocol was quite high, ranging from 50-100×106 cellsper rat (10).
Recently in 2011, Cunha et al. reportedxenotransplantation of human
glioblastoma cells inimmunosuppressed rats induced by orogastric
cyclosporineat a dose of 5 mg/kg until the end of study (20).
All the above discussed immunosuppression protocols
werevalidated with only single cancer cell line or tumor
xenograft.Moreover, to achieve prolonged
immuno-suppression,cyclosporine was administered daily until the
end of study.Along with its potent immunosuppressive activity,
cyclosporineis also reported to have an anticancer activity (24,
25). Hence,the poor take rate of xenograft with the protocols
discussedabove may be attributed to anticancer activity of
cyclosporine.Prolonged immunosuppression protocol, high inoculum
size,and variable take rate limit the use of such protocols.
In the present study, a simple, versatile and
cost-effectiveimmunosuppression protocol was developed using
acyclosporine, ketoconazole and cyclophosphamide drugregimen to
develop human tumor xenograft in mice.Ketoconazole is an antifungal
agent which interfere withsynthesis of ergosterol, a constitute of
fungal cell membrane.Moreover, it also inhibits cytochrome p450
enzyme whichmetabolizes cyclosporine (26-28). In this way,
ketoconazolehelps in prolonging circulation of cyclosporine and
simultaneously protects from probable fungal infection, whichis
very common with cyclosporine treatment. Cyclo-phosphamide is an
alkylating agent that interferes with DNAreplication. It also
reduces the number of neutrophils, B- and T-cells and natural
killer cells to a significant extent (29-32).
Immunosuppression of animals receiving cyclosporine
andketoconazole was evident by significant reduction in total
WBCand lymphocyte count. Administration of cyclophosphamide tothese
animals suppressed neutrophils and residual B- and
ANTICANCER RESEARCH 34: 7177-7184 (2014)
7180
Figure 1. Graph showing the mean white blood cells (WBC)
andlymphocyte count of different groups of mice at the end of
cyclosporineand ketoconazole treatment (n=6) (*p
-
T-cells, which prolonged immunosuppression, as well asachieving
a 100% take rate of tumor xenograft. Ketoconazoleand ampoxin
protected immunosuppressed animals frombacterial and fungal
infection, which is a major cause of deathin immunosuppression
protocols.
Thus, using a combination treatment with
cyclosporine,ketoconazole and cyclophosphamide, a 100% take rate
wasachieved with human lung adenocarcinoma, prostateadenocarcinoma
and cervical adenocarcinoma in C57/BL6mice. Increasing tumor volume
was maintained for eight
Jivrajani et al: Immunosuppression Protocol for the Development
of Tumor Xenograft
7181
Figure 4. Immunocompromised C57BL/6 mice bearing tumor xenograft
of A549(a), LNCaP(b), and KB(c) as indicated by arrows.
Figure 5. Light microscopy observation of HE-stained section of
A549(i), LNCaP(ii), and KB(iii) tumor xenograft. A: Light
microscopy at ×100magnification. Angiogenesis and tumor cell
invasion are indicated by arrows. B: Light microscopy at ×400
magnification. Tumor cells having largenucleus and scanty cytoplasm
are indicated by arrows.
-
weeks after tumor implantation with all three xenograft
types.Histopathological analysis of tumor xenograft confirmed
thepresence of malignant tumor cells. It also showed invasion
oftumor cells into adjacent stromal tissue; however, themetastatic
potential of xenografts was not evaluated in thisstudy. It also
revealed the presence of angiogenic bloodvessels, which is a
prerequisite for tumor formation. Thus, itwas confirmed that the
xenografts that developed were not theresult of simple hyperplasia
but was malignant and invasivetumor. However, there is a
possibility for neoplastictransformation of host (mouse) stromal
cells by the injectedhuman tumor cells (33, 34). Hences further
characterizationof the tumors is required depending upon the
specific use.
In conclusion, a new, versatile, and relatively
shortimmunosuppression protocol was developed using a combinationof
cyclosporine, ketoconazole and cyclophosphamide drugregimen. The
protocol was validated with three different
humanadenocarcinomatypes, namely lung, prostate and
cervicalcarcinoma for induction of tumor xenograft in C57BL/6
mice.A 100% take rate was achieved by this protocol, with
nomortality until the end of study. Moreover, in this protocol,
allthe immunosuppressive drugs were administered before
tumorimplantation hence interaction of cyclosporine
andcyclophosphamide with any anticancer drug to be evaluated canbe
avoided. The developed model is cost effective and relativelysimple
to establish as compared to previously reported modelsand can be
used in place of athymic nude mice to evaluateefficacy of novel
anticancer drugs, targeted drug delivery systemsand even to study
pathophysiology of human tumors. This modelwill be a boon for
developing countries where nude mice areoften unavailable for
cancer research.
Acknowledgements
The Authors are thankful to B. V. Patel Pharmaceutical
Educationand Research Development (PERD) Centre, Ahmedabad
forproviding all the facility for the successful completion of the
work.Additionally, the Authors would like to thank CSIR, India
forproviding financial assistance to Mehul Jivrajani in terms of
SeniorResearch Fellowship #113353/2K12/1 for this work.
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Received July 16, 2014Revised September 5, 2014
Accepted September 9, 2014
Jivrajani et al: Immunosuppression Protocol for the Development
of Tumor Xenograft
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Journal of Microbiological Methods 92 (2013) 340–343
Contents lists available at SciVerse ScienceDirect
Journal of Microbiological Methods
j ourna l homepage: www.e lsev ie r .com/ locate / jmicmeth
Note
A combination approach for rapid and high yielding purification
ofbacterial minicells
Mehul Jivrajani a, Neeta Shrivastava b, Manish Nivsarkar a,⁎a
Department of Pharmacology and Toxicology, B. V. Patel
Pharmaceutical Education and Research Development (PERD) Centre,
Sarkhej-Gandhinagar Highway, Thaltej, Ahmedabad,380 054 Gujarat,
Indiab Department of Pharmacognosy and Phytochemistry, B. V. Patel
Pharmaceutical Education and Research Development (PERD) Centre,
Sarkhej-Gandhinagar Highway, Thaltej, Ahmedabad,380 054 Gujarat,
India
⁎ Corresponding author at: B.V. Patel Pharmaceuticavelopment
(PERD) Centre, Sarkhej-Gandhinagar High054 Gujarat, India. Tel.:
+91 7927416409; fax: +91 7
E-mail address: [email protected] (M. Ni
0167-7012/$ – see front matter © 2012 Elsevier B.V.
Allhttp://dx.doi.org/10.1016/j.mimet.2012.12.002
a b s t r a c t
a r t i c l e i n f o
Article history:Received 19 November 2012Received in revised
form 4 December 2012Accepted 4 December 2012Available online 9
December 2012
Keywords:Minicell purificationDifferential
centrifugationCeftriaxoneFiltration
A method for bacterial minicell purification was developed by
combining antibiotic (ceftriaxone) lysis andfiltration. This method
is fast, cost effective and facilitates high yield of purified
minicells, with no parentstrain contamination as confirmed by
fluorescent microscopy, average particle size and polydispersity
index.
© 2012 Elsevier B.V. All rights reserved.
Bacterial minicells are nano sized anucleated cells produced
dur-ing the abnormal cell division in several mutant strains of
Escherichiacoli as well as other gram positive bacteria (Adler et
al., 1967; Frazerand Curtiss, 1975; MacDiarmid et al., 2007;
MacDiarmid et al., 2009).Minicells are produced by depressing
cryptic polar sites of cell fissionthrough inactivating bacterial
genes, minCDE chromosomal deletionwhich controls the normal
bacterial cell division (De Boer et al.,1989). The resultant
minicells contain all of the molecular compo-nents of the parent
cell, except the chromosome. Minicells do notpossess the capacity
to divide and their production does not interferewith normal cell
division which occurs simultaneously. Severalminicells producing
bacterial strains are summarized in the Table. 1.Minicells can be
used in various areas of biology. Conventionally,minicells were
useful in deducing cellular functions in the environ-ment devoid of
nucleus (Cohen et al., 1968), while the currentapproach is focussed
on its use for targeted delivery of chemothera-peutics as well as
si/shRNA (MacDiarmid et al., 2007; MacDiarmid etal., 2009). In case
of every bacterial-derived biologic development, it
l Education and Research De-way, Thaltej,
Ahmedabad-380927450449.vsarkar).
rights reserved.
is indispensable to eliminate living parent bacteria and
endotoxinbefore in vivo administration to reduce the risk of
infection and anyadverse reaction.
Several methods have been developed for the purification
ofminicells such as differential centrifugation, multiple density
gradientcentrifugation, multistep filtration, etc. (Adler et al.,
1967; Shepherdet al., 2001). These methods of purification have
certain limitationslike applicable to limited sample size, low
recovery from a densitygradient resulting in reduced yield and
increased cost of production.Consequently, to overcome these
limitations, antibiotic treatment(penicillin) method (Levy, 1970)
was developed. This method isbased on the principle of non dividing
nature of minicells. Although,this method has advantage of high
yield, it is not suitable for allbacterial strains. Hence, in such
cases potent antibiotic that can killlarge variety of bacterial
strain and at the same time do not harmminicells, can be replaced
with penicillin. Moreover, combination ofthis method with other
suitable methods may yield large amount ofpure minicells.
We developed a new method for the purification of
minicellswherein a combination of antibiotic treatment
(ceftriaxone) andfiltration was used, resulting in high yield of
purified minicells. Themethod developed is simple and
cost-effective and is a modificationof method as reported by S.B.
Levy (1970).
Ceftriaxone is a semisynthetic, broad-spectrum cephalosporin
an-tibiotic. The bactericidal activity of ceftriaxone results from
inhibition
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Table 1List of minicell producing strains.
Strain Genotype Source
E. coli P678-54 F-, thr-1, leuB6(Am), secA208, fhuA2, lacY1,
glnV44(AS)?, gal-6,λ−, minB-2,hns-1?, rfbC1, galP63, rpsL135(strR),
fic-1, malT1(λR),xyl-7, mtlA2, thi-1
Adler et. al.
E. coli χ984 F-, purE41, glnV42(AS)?, λ−, serC53, minB-2,
his-53, metC56,rpsL97(strR),xyl-14, cycA1, T3R, ilv-277, cycB2
R. Curtiss
E. coli PB114 minB::kan dadR1 trpA62 tna-5 purB+ λ− minΔ minC
mind minE P.de Boer et. al.E. coli χ1411 F-, λ−, minB-2, glnV44(AS)
or glnV42(AS), T3R R. CurtissE. coli χ1488 F-, purE41, glnV42(AS),
λ−, serC53, minB-2, his-53, xyl-14, metB65,cycA1,
hsdR2, cycB2, tte-1, ilv-277R. Curtiss
E. coli χ2224 F-, thr-1, leuB6(Am), secA208, fhuA2, lacY1,
glnV44(AS),galK2(Oc), minB-2,tdk-2, udk-30, upp-30,
rpsL109(strR),malT1(λR), xyl-7, mtlA2, thi-1
R. Curtiss
E. coli χ2338 F-, ΔaraC766, fhuA53, dapD8, gltA16,
Δ(gal-modC)690, minB-2,Δ(trpB-trpC)513,rfbC1, gyrA25(NalR),
ΔthyA57, endA1, aroB15?,cycA1, hsdR2, oms-1, cycB2
R. Curtiss
E. coli χ1849 F-, fhuA53, dapD8, purE41, glnV42(AS),
Δ(galK-uvrB)40, λ−, minB-2, his-53,gyrA25(NalR), Δ(bioH-asd)29,
metB65, cycA1, hsdR2,cycB2, ilv-277, tte-1, oms-1
R. Curtiss
E. coli RS3242 Zcf-117::Tn10 Min+ Bachmann, 1983E. coli KL99 Hfr
Min+ Bachmann, 1983E. coli KL208T Hfr trp::Tn10 Min+ Davie et al.,
1984E. coli χ1081T1 minB minA (?) zcf-117::Tn10 Davie et al.,
1984E. coli MG1655 F–,λ∠,ilvG–,rfb-50,rph-1 Coli Genetic stock
center, YaleE. coli MPX1B9 F–,λ∠,ilvG–,rfb-50,rph-1,zac::aph,
laclQ, Ptac ftsZ20 ΔphoA Mpex PharmaceuticalsS. typhimurium
ΔminCDE::CmlR MacDiarmid J.A. et al.S. flexneri ΔminCDE::CmlR
MacDiarmid J.A. et al.L. monocytogenes ΔminCD::CmlR MacDiarmid J.A.
et al.
Table 2Average size and polydispersity index of different
fractions of minicell purification.
Sr. No. Fraction Size(nm)
PDI (polydispersityindex)
1. Starting culture of GFP transformedE. coli PB114
929.4 1.0
2. Culture of E. coli PB114 afterdifferential centrifugation
639.9 0.156
3. Cells after antibiotic treatment 584.9 0.0814. Cells after
successive filtration with
0.45 μ and 0.22 μ filter522.3 0.042
341M. Jivrajani et al. / Journal of Microbiological Methods 92
(2013) 340–343
of cell wall synthesis. Ceftriaxone has a high degree of
stability in thepresence of betalactamase, both penicillinases and
cephalosporinases,of gram-negative and gram-positive bacteria (Hall
et al., 1981). Be-cause of these advantages, ceftriaxone is
selected for the minicell pu-rification from parent strain.
E. coli minicell producing strain PB114 was obtained from
Dr.Lawrence Rothfield, University of Connecticut, USA. Strain was
grownin 200 ml of LB broth supplemented with 50 μg/ml kanamycin.
Thestrain was transformed with plasmid pEZ43G-D which posses GFPas
a reporter gene to track the purification under fluorescent
micro-scope. Transformed strain, E. coli PB114 GFP was grown in LB
brothsupplemented with 50 μg/ml kanamycin and 100 μg/ml
ampicillin.Approximately, 90% of parent strain was separated from
the minicellsby initial differential centrifugation for 10 min at
2000×g at room tem-perature which was confirmed by fluorescent
microscopy at 1000×magnification (Fig. 1). The supernatant was
centrifuged for 10 min at10,000×g to pellet the minicells. Minicell
pellet was resuspended in50 ml of fresh LB broth and broth was
incubated at 37 °C, 180 rpm for45 min to facilitate reinitiation of
cell growth. In the next step, ceftriax-onewas added at a dose of
100 μg/ml and brothwas again incubated at37 °C, 180 rpm for 45 min.
The dose of ceftriaxone was adequate tocause cell lysis. Most
importantly, ceftriaxone has no detrimental effectonminicells at
this concentration as confirmed byfluorescentmicrosco-py.
Ceftriaxone is very potent in cell lysis,where only single
treatment issufficient to kill majority of parent strain. The broth
was centrifuged at400 ×g for 5 min to remove cell debris and dead
cells, after ceftriaxonetreatment. Supernatant was again
centrifuged at 10,000 ×g to pelletminicells. Harvestedminicells
werewashed in fresh broth and observedunder fluorescent microscope
at 1000× magnification to check themorphology of minicells as well
as their purity. Further, to removeany residual parent
strain,minicells were filtered through 0.45 μm filter(Millipore).
Finally, minicells were filtered through 0.22 μm filter to re-move
any cell debris and free endotoxins. At every step of
purification,purity and morphology of minicells were observed under
fluorescentmicroscope. Simultaneously, sample was platted on LB
agar plate tocheck the presence of viable parent strain (Fig. 1).
Additionally, to fur-ther confirm the purity of minicells, the size
of cells at every step ofpurification was determined by using
dynamic light scattering method(Zeta sizer ZS 90, Malvern) (Fig.
2). Eventually, purified minicells weregrown for 14 days in
thioglycolate broth to confirm the absence of anyslow-growing
organisms in the final fraction.Minicell number obtained
after final step of purification was calculated
spectrophotometrically bytaking optical density at 600 nm and
employing following equation(Giacalone et al., 2006).
A600 � 5:0� 1010=ml
Total number of minicells after final step of purification
wasfound to be 3.81×1010 minicells from the 200 ml of starting
cul-ture. Obtained minicell yield was remarkably high in
comparisonto the other reported methods. For instance, the yield of
minicellsusing the method reported by S.B. Levy was found to be
approx-imately 1.8×108 minicells from same starting culture
(Levy,1970).
Previous reports suggest that sucrose gradients are toxic to E.
colicells and show a lag phase of 2 to 4 h in growth. This
alteration ingrowth shows that it may affect the biosynthetic
activity of minicellstoo (Levy, 1970). Minicells are resistant to
ceftriaxone treatmentbecause of their non dividing nature.
Fluorescent microscopy of cellsand corresponding colony forming
unit on the plate at every stepof purification clearly demonstrated
that subsequent to ceftriaxonetreatment, parent strain
contamination is occasional. Finally, purifiedminicells were
obtained after filtration with 0.45 μm and 0.22 μmfilters (Fig. 1).
Average size of cells decreased significantly from929.4 nm
(starting culture) to 522.3 nm (final fraction), suggestingthe
purity of minicells (Fig. 2) which was also confirmed
frompolydispersity index (PDI) value that reduced from 1.0 to
0.042(Table. 2).
-
Fig. 1. Fluorescent microscopy (1000×) and growth of cells at
different stages of minicell purification: A) Starting culture of
GFP transformed E. coli PB114 and its growth on LB agarplate, B)
culture of E. coli PB114 after differential centrifugation and its
growth on LB agar plate, C) cells after antibiotic treatment and
its growth on LB agar plate, and D) minicellsafter successive
filtration with 0.45 μ and 0.22 μ filter and its purity check on LB
agar plate.
342 M. Jivrajani et al. / Journal of Microbiological Methods 92
(2013) 340–343
-
Fig. 2. Graphs showing average size of cells at different stages
of minicells purification A) starting culture of GFP transformed E.
coli PB114, B) culture of E. coli PB114 after differ-ential
centrifugation, C) cells after antibiotic treatment, and D) cells
after successive filtration with 0.45 μ and 0.22 μ filter.
343M. Jivrajani et al. / Journal of Microbiological Methods 92
(2013) 340–343
Acknowledgments
The authors are thankful to B. V. Patel Pharmaceutical
Educationand Research Development (PERD) Centre, Ahmedabad for
providingall the facility for the successful completion of the
work. We wouldlike to thank Dr. Lawrence Rothfield (University of
Connecticut, Con-necticut, USA) for kindly providing E. coli
PB114.
References
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Microbiol. Rev. 47, 180–230(edition 7).
Cohen, A., et al., 1968. The properties of DNA transferred to
minicells during conjuga-tion. Cold spring harbour Symp. Quant.
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Davie, E., Sydnor, K., Rothfield, L.I., 1984. Genetic basis of
minicell formation inEscherichia coli K-12. J. Bacteriol. 158 (3),
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De Boer, P.A., Crossley, R.E., Rothfield, L.I., 1989. A division
inhibitor and a topologicalspecificity factor coded for by the
minicell locus determine proper placement ofthe division septum in
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Bum, A.L., Paulin, P.R., et al.,2007. Bacterially derived 400 nm
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MacDiarmid, J.A., Amaro-Mugridge, N.B., Weiss, J.M., Sedliarou,
I., Wetzel, S., Kochar, K., etal., 2009. Sequential treatment of
drug resistant tumors with targeted minicellscontaining siRNA or a
cytotoxic drug. Nat. Biotechnol. 27, 643–651.
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Research ArticlePurification and Characterization of
Haloalkaline,Organic Solvent Stable Xylanase from Newly
IsolatedHalophilic Bacterium-OKH
Gaurav Sanghvi,1,2 Mehul Jivrajani,2,3 Nirav Patel,2,4 Heta
Jivrajani,5
Govinal Badiger Bhaskara,2 and Shivani Patel2
1 Department of Pharmaceutical Sciences, Saurashtra University,
Rajkot, Gujarat 360 005, India2Department of Biotechnology, M.
& N. Virani Science College, Rajkot, Gujarat 360 005, India3
Department of Pharmacology and Toxicology, B. V. Patel
Pharmaceutical Education andResearch Development (PERD) Centre,
Sarkhej-Gandhinagar Highway, Thaltej, Ahmedabad, Gujarat 380 054,
India
4Department of Biochemistry and Molecular Biology, Miller School
of Medicine, University of Miami, Miami, FL 33136, USA5Department
of Biochemistry, Saurashtra University, Rajkot, Gujarat 360 005,
India
Correspondence should be addressed to Mehul Jivrajani;
[email protected]
Received 5 March 2014; Revised 21 June 2014; Accepted 23 June
2014; Published 8 September 2014
Academic Editor: Anwar Sunna
Copyright © 2014 Gaurav Sanghvi et al.This is an open access
article distributed under the Creative CommonsAttribution
License,which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly
cited.
A novel, alkali-tolerant halophilic bacterium-OKH with an
ability to produce extracellular halophilic, alkali-tolerant,
organicsolvent stable, and moderately thermostable xylanase was
isolated from salt salterns of Mithapur region, Gujarat,
India.Identification of the bacteriumwas done based upon
biochemical tests and 16S rRNA sequence.Maximumxylanase
productionwasachieved at pH 9.0 and 37∘C temperature in themedium
containing 15%NaCl and 1% (w/v) corn cobs. Sugarcane bagasse
andwheatstraw also induce xylanase production when used as carbon
source.The enzyme was active over a range of 0–25% sodium
chlorideexamined in culture broth.The optimum xylanase activity was
observed at 5% sodium chloride. Xylanase was purified with
25.81%-fold purification and 17.1% yield. Kinetic properties such
as Km and Vmax were 4.2mg/mL and 0.31 𝜇mol/min/mL, respectively.The
enzyme was stable at pH 6.0 and 50∘C with 60% activity after 8
hours of incubation. Enzyme activity was enhanced by Ca2+,Mn2+, and
Mg2+ but strongly inhibited by heavy metals such as Hg2+, Fe3+,
Ni2+, and Zn2+. Xylanase was found to be stable inorganic solvents
like glutaraldehyde and isopropanol.The purified enzyme hydrolysed
lignocellulosic substrates. Xylanase, purifiedfrom the halophilic
bacterium-OKH, has potential biotechnological applications.
1. Introduction
Biomass has been recognized as one of the major worldrenewable
energy sources in which cellulose and hemicellu-lose are considered
as its major fraction [1]. Hemicelluloserepresents a group of plant
polysaccharides with differentstructures and different
monosaccharide composition, whichcan be present in various amounts
or traces depending onthe natural source [2]. Xylan is the
principal hemicellulosesand major plant cell wall polysaccharide
component, com-posed mainly of D-xylose. It is a
heteropolysaccharide witha homopolymeric chain of 1,4,𝛽-d-xylosidic
linkages withthe backbone comprising of O-acetyl,
𝛼-L-arabinofuranosyl,
and 1, 2-linked glucuronic or 4-O-methylglucuronic acid[3].
Xylanases (EC 3.2.1.8) randomly hydrolyze the 𝛽-1,4-glycosidic
bonds of xylan to produce xylooligomers ofdifferent lengths [4].
Many kinds of xylanases have beenisolated from various
microorganisms like fungi, bacteria,actinomycetes, and yeasts
[5].
In the recent years, microorganisms from extreme condi-tions
have been the focus of researchers attention as enzymesfrom
extremophilic microorganisms can withstand harshconditions like
extreme temperature, salt, alkaline condition,and so forth.
Extremophiles can be classified into ther-mophiles, psychrophiles,
acidophiles, alkaliphiles, halophiles,and others [6]. Halophiles
have gained attention due to their
Hindawi Publishing CorporationInternational Scholarly Research
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pageshttp://dx.doi.org/10.1155/2014/198251
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2 International Scholarly Research Notices
extensive mechanism of adaptation to extreme
hypersalineenvironments and are differentiated based on salinity
intononhalophile (15% NaCl)[7]. Halophiles are the most likely
source of such enzymes,because not only their enzymes are
salt-tolerant, but manyare also thermotolerant [8]. Furthermore,
exoenzymes fromhalophiles are not only interesting from the basic
scientificviewpoint but they may also be of potential interest in
manyindustrial applications, owing to their stability and activity
atlow water activities [9, 10].
Currently, major application of xylanase is in pulp andpaper
industries where xylanases replace chemical bleachingagents, which
results in greater brightness in pulp. Mostindustrial pulping is
done at high temperature and underalkaline conditions, hence
requiring xylanases to be opera-tionally stable under such
conditions. To meet the specificindustry’s needs, an ideal xylanase
should equipped withspecific properties, such as good pH and
thermal stability,high specific activity, and strong resistance to
metal cationsand chemicals, are also pivotal factors to the
applications.However, the great majority of xylanases reported so
far areneither active nor stable at both high temperature and
highpH [11]. Thus, much research interest has been generated inthe
production of xylanase under halophilic conditions [12].
In the present study, production and characterizationof
haloalkaline thermostable xylanase by a newly isolated,halophilic
bacterium-OKH is reported.
2. Material and Methods
2.1. Isolation and Maintenance of Microorganism. The halo-philic
bacterium-OKHwas isolated from sediments collectedfrom salt
salterns around Mithapur. Culture was grown onagar plates
containing 0.5% (w/v) Birchwood xylan, 0.5%yeast extract in mineral
salt medium containing (g/L) NaCl150, MgCl
25.0, K
2SO40.2, and agar with pH adjusted to
9.0. After 96 hrs, plates were flooded with 0.1% Congo
redsolution for 15–20mins and then destained with 1MNaCl
for10–15mins [13].The colonies showing clear zone of hydrolysiswere
picked and used for xylanase production. Based on thezone of
clearance, xylanase from halophilic bacterium-OKHwas selected for
further studies.
2.2. Bacterial Identification and Phylogenetic Analysis.
Themorphological, cultural, and biochemical characteristic of
theisolated strain was studied according to Bergey’s Manual
ofDeterminative Bacteriology [14]. GenomicDNAof
halophilicbacterium-OKH was isolated by SDS lysozyme method
[15]with slight modification in method by adding extra P:C:Iand C:I
step to remove high amount of protein impuritiesobtained. PCR
amplification of 16srRNA was performedusing the forward
5-AGAGTTTGATCCTGGCTCAG-3and reverse primer 5-CAACCTTGTTACGACT-3,
respec-tively. The obtained PCR product was sequenced and16srRNA
gene sequence was compared with GenBank sub-missions using BLASTn
programme. The phylogenetic anal-ysis was done by RDP PHYLIP
software.
2.3. Enzyme Production. 2mL of 96 hr old culture was inoc-ulated
to 250mL Erlenmeyer flasks containing the followingmedia (g/L):
yeast extract 3.0, NaCl 150, MgCl
25.0, K
2SO4
0.2, and CaCl20.02 gm, respectively. Media were supple-
mented by 0.5 gm of Birchwood xylan and 1 gm of corn cobsas
substrates. Production media were autoclaved at 121∘C for15mins at
15 lbs pressure. Flasks were incubated in rotaryshaker at 120 rpm.
After every 24 hrs of interval, flasks wereremoved and the content
was centrifuged at 10,000 rpm for20mins. The crude supernatant was
used for xylanase assay.
2.4. Study of Physicochemical Factors on Xylanase Production
2.4.1. Effect of Carbon and Nitrogen Sources on Enzyme
Pro-duction. Carbon sources such as glucose, maltose,
lactose,arabinose, glucose, galactose, sucrose, fructose,
mannose,and xylose were used in 1% (w/v) to check the effect of
thesesupplements on enzyme production. Additionally,
variousconcentrations of rice straw, wheat straw, sugarcane
bagasse,corn cobs, rice husks, groundnut shells, and saw dust
werealso used to enhance the production of xylanase. In caseof
nitrogen sources, effect of both organic and inorganicnitrogen
sources on enzyme production was studied. Pep-tone, malt extract,
beef extract, and yeast extract were usedas organic nitrogen
sources, whereas for inorganic nitrogensources, urea, ammonium
sulphate, and sodium nitrate wereused.
2.4.2. Effect of NaCl, pH, and Temperature on Xylanase
Pro-duction. To study the effect of NaCl on enzyme
production,organism was cultivated at different NaCl
concentrationsranging from 0 to 25%. Effect of pH and temperature
onenzyme production was evaluated by varying pH (2.0–10.0) and
temperature (10–70∘C) of the production medium.Extracellular
xylanase activitywasmeasured in culture super-natant.
2.5. Xylanase Assay. Xylanase activity was determined at37∘C for
30min in 0.05M Tris-HCl buffer (pH 9.0) by
DNSA(3,5-dinitrosalicylic acid) [16]. In blank, enzyme was
addedafter adding DNSA reagent. The absorbance was measuredat 540
nm. One unit of xylanase activity was defined as theamount of
enzyme produced 1𝜇mol of xylose equivalentperminute under specified
conditions. Protein concentrationwas estimated by Lowry’s method
[17] using BSA (bovineserum albumin) as the standard.
2.6. Purification of Xylanase. All the purification steps
werecarried out at 4∘C unless stated otherwise. The crude enzymewas
subjected to 0–80% ammonium sulphate precipitation.The precipitated
protein was collected by centrifugation(10,000 rpm) and dissolved
in 0.05M Tris-HCl buffer (pH9.0). Collected fraction was dialysed
and concentrated usingrotary vacuum evaporator. Dialyzed sample was
loaded onDEAE cellulose column (10 cm × 10 cm) and fractions
wereeluted at flow rate of 10mL/hr. Fractions were eluted by
lineargradient of 0-1M NaCl. Fraction with maximum activityin ion
exchange chromatography was further purified by
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International Scholarly Research Notices 3
size exclusion chromatography. Preequilibrated column ofSephadex
G-100 was used for size exclusion chromatography.1mL fraction was
collected at a flow rate of 10mL/hr. Proteinconcentration of each
fraction was determined by measuringOD at 280 nm.The purified
fractions were checked for purityon SDS PAGE.
2.7. SDS PAGE and Zymogram Analysis. Homogeneity
andmolecularweight of the purified xylanasewere determined byusing
12% SDS PAGE as described by Laemmli [18]. Proteinbands were
visualised by staining with silver stain. Themolecular weight
standard used was the medium molecularweight marker for SDS
electrophoresis procured from Genei(India). Zymogram analysis for
xylanase was carried out asdescribed by Hung et al. [19].
2.8. Influence of pH, Temperature, and Salinity on
XylanaseActivity and Stability. The optimal temperature of the
puri-fied xylanase was determined in 0.05M Tris-HCl buffer (pH9.0)
at a temperature range of 10–70∘C. To evaluate stability,the enzyme
solution was incubated at temperature the rangeof 10–70∘C for 24
hours. Percentage relative enzyme activitywas recorded at 4-hour
intervals during 24-hour incubation.
The optimal pH of the purified xylanase was determinedby
measuring the activity between the pH 3.0 and 11.0. Threebuffers
(0.05M)were utilized. Sodiumacetate bufferwas usedfor pH 3–5,
sodium phosphate buffer for pH 4–7, and Tris-HCl buffer for pH
8–11. To test stability of purified xylanase,enzyme solution was
incubated in 0.05M Tris-HCl buffer(pH 9.0) for 24 hours. Aliquots
were withdrawn at an intervalof 4 hours. The xylanase activity was
measured according tothe standard assay method.
The optimal salt concentration for purified xylanase
wasdetermined in 0.05M Tris-HCl buffer (pH 9.0) containingvarious
concentrations of NaCl (0–30% w/v) concentrations.For stability,
purified xylanasewas incubatedwith 0.05MTrisbuffer (pH 9.0) with
salinity in the range of 0–30% for 24hours at 37∘C. Each assay was
presented as the average ofthree trials.
2.9. Effect of Metal Ions and Organic Solvents on
XylanaseActivity. Effect of various metal ions such as HgCl
2, MnCl
2,
CuCl2, CoCl
2, AgNO
3, ZnCl
2, FeCl
2, NiCl
2, and NH
4Cl was
studied by adding each metal ion at two different
concen-trations (2mM and 5mM) in reaction mixture. Thereafter,the
residual enzyme activities were determined under thestandard assay
conditions. Activity in the absence of metalions was considered as
100%. To evaluate enzyme stability inorganic solvent, different
organic solvents like methanol, ace-tone, acetic anhydride,
isopropanol, and glutaraldehyde wereused. The enzyme activity was
determined under standardassay conditions.
2.10. Storage Stability. To determine storage stability,
enzymewas kept under different conditions with different
timeintervals; that is, it has been kept at room temperature for
3-4days; it has been kept at storage temperature for one monthand
enzyme activity was checked. The kinetic constants, Km
and Vmax, were estimated using linear regression plots
ofLineweaver and Burk [20].
2.11. Application of Xylanase. Various lignocellulosics
sub-strates like wheat straw, rice straw, and the commercial
paperpulp samples sugarcane bagasse were saccharified by
crudexylanase [21]. Each substrate (100 g/L of 0.05M Tris-Cl,
pH9.0) was mixed with 50mL of crude enzyme
preparation.Saccharification was performed in shake flasks (120
rpm) at37∘C for 24 and 48 hours. The supernatants were assayed
forestimation of reducing sugar.
2.12. Statistical Analysis. All the data were represented
asaverage of least three independent experiments. Data havebeen
represented as mean ± standard deviation.
TheGenBank accession number of the sequence reportedin this
paper is EF063150.
3. Results and Discussion
3.1. Characterisation of Bacterial Strain. Halophilic
bacteriaare metabolically more versatile than the Archaea and
theirenzymatic activities are more diverse. To suit the
industrialrequirement halophilic bacteria are perfect resource to
beused as it produces salt tolerant enzymes which are resistantto
low pH. The halophilic bacterium-OKH used in thepresent study was
isolated from soil sample collected nearMithapur, Gujarat, India.
It is Gram-positive, rod shaped,translucent, and nonmotile bacteria
which is catalase positiveandhydrolysed gelatin and casein. It is
sensitive to teicoplaninand chloramphenicol antibiotic. However, it
is resistant tobacitracin, metronidazole, cefpodoxime,
levofloxacin, tetra-cycline, and streptomycin (Table 1). It was
able to fermentsugars like glucose, sucrose, xylose, and lactose
without gasproduction.
The result of the RDP Seqmatch and BLAST clearlyshowed that 16s
rRNA gene sequence of isolate was distinctfrom the data available
in the database. The 16s rRNAsequence showed ∼90% identity to the
Bacillus sp. BHO502.Henceforth, isolate belongs to class
unclassified Bacillus sp.and designated as halophilic bacterium-OKH
in currentstudy as sample was collected from Okha region
(nearMithapur) in Gujarat (Figure 1).
3.2. Growth Characteristics and Xylanase Production
fromHalophilic Bacterium-OKH. Most of the halophiles are
slowgrowing. Production of xylanase started in early log phaseand
increased till late stationary phase but after that
itdeclined.These results indicate that xylanase production
wasindependent of growth phase which is in harmony withearlier
reports on xylanase production by Chromohalobactersp. [22].
3.2.1. Effect of NaCl, pH, and Temperature on Enzyme
Pro-duction. Facilitated growth of OKH was observed in widerange of
salinity from 5 to 20%. Highest growth and xylanaseactivity were
obtained at pH 9.0 after 72 hours of incubationat 15% NaCl. The
strain-OKH was found to be moderately
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4 International Scholarly Research Notices
Halophilic bacterium OKHBacillus sp. BH052
Bacillus sp. 17-5Bacillus sp. BH164
Bacillus sp. BH069Bacterium SL2.26
Bacillales bacterium MSU3010Bacillus sp. SL5-2
Halophilic bacterium MBIC3303Bacillus sp. CM1
Scale:0.01
Figure 1: Dendrogram showing phylogenetic position of halophilic
bacterium-OKH.
Resid
ual a
ctiv
ity (%
)
NaCl (%)
00
0
20
40
60
80
100
120
2
4
6
10 20 30
Biom
ass (
A660
nm)
(a)
Resid
ual a
ctiv
ity (%
)
pH
00
0
20
40
60
80
100
120
6 8 10 1242
2
3
1
4
5
Biom
ass (
A660
nm)
(b)
Resid
ual a
ctiv
ity (%
)
00
120
30
60
90
20 40 60 80 1000
1
2
3
Temperature (∘C)
Biom
ass (
A660
nm)
(c)
Figure 2: Effect of NaCl (a), pH (b), and temperature (c) on the
growth and xylanase production by halophilic bacterium-OKH. Growth
isrepresented by squares whereas xylanase activity is represented
by circles.
halophilic in nature as no growth was observed in absenceof
NaCl. There was marked to be increased in activity withan increase
in concentration of NaCl up to 15% and a furtherincrease in NaCl
concentration decline growth as well as pro-duction of xylanase
(Figures 2(a) and 2(b)). This observationis in agreementwith other
halophilic organisms, namely,Gra-cilibacillus sp. TSCPVG[12] and
Chromohalobacter sp.TPSV101[22], where increase in salinity above
optimum decreasesenzyme production.
Since enzymes are very sensitive to pH, determinationof the
optimal pH is essential for xylanase production.In the present
study, the effect of pH on production ofenzyme was thus studied by
carrying out fermentation over
a wide range of pH (2.0–10.0). The production of xylanasewas
found to be highest at pH 9.0. There are reports ofmaximum xylanase
production by halophiles from pH 7.5to 9.0. It is evident from the
data that xylanase fromOKH is alkali-tolerant and offers use in
pulp and paperindustries. Maximal activity (28.14U/mL) was observed
at atemperature of 37∘C. The optimal temperature for
xylanaseproduction by various halophiles has been previously
studied;they have a wide range of temperature preferences
depend-ing upon nature of adaptation and salt requirements [23].In
the present study, OKH, being a mesophilic species,showed an
optimal temperature at 37∘C for maximal enzymeproduction (Figure
2(c)).
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International Scholarly Research Notices 5
Table 1: Morphological, physiological, and biochemical
character-istics of halobacterium-OKH.
Character Halobacterium-OKHColony characteristics Translucent,
slimyPigmentation Cream pigmentationMorphology Gram-positive
rodsAnaerobic growth −Motility −NaCl range 7–15%NaCl optimum
12.5%Temperature range 25–40∘CTemperature optimum 37∘CpH range
5.0–10.0pH optimum 8.0Catalase +Gelatinase test −Lipase test
−Amylase test +Indole production −H2S production −Nitrate
production −Sugar fermentation
Glucose +Maltose +Sucrose −Mannitol −Xylose +Lactose +
Antibiotic resistanceBacitracin +Metronidazole +Cefpodoxime
+Teicoplanin −Streptomycin and pencillin G +Chloramphenicol
−Tetracycline +
3.2.2. Effect of Carbon and Nitrogen Sources on Enzyme
Pro-duction. In the present study, oat spelt xylan was provento be
the best carbon sources for xylanase production fol-lowed by
Birchwood xylan. Among all other carbon sources,supplementation of
xylose increases the yield of xylanasewhereas sucrose and lactose
did not support growth aswell as xylanase production. Increased
yield of xylanaseproduction by xylose supplementation was reported
in thestrain Bacillus pumilus GESF-1 [24]. To attain
cost-effectiveproduction, different agro residues were used as
substratefor xylanase production. The effective utilization of
suchagricultural wastes not only solves environmental problemsbut
also promotes the economic value of the agriculturalproducts.
Appreciable xylanase activity was observed using2% corn cobs as a
carbon source (Table 2). Increased levelof xylanase production
using corn cobs may be due to itslow lignin content and higher
sugar content as compared
Table 2: Effect of different sugars and agro residues as
carbonsources for xylanase production.
Carbon sources % Residual activityGlucose 65Maltose 45Fructose
50Mannose 52Lactose 13Arabinose 52Galactose 43Sucrose 12Xylose
85Oat spelt xylan 100Birchwood xylan 94Agro residues
Rice Straw 29Wheat Straw 72Sugarcane bagasse 64Corn cobs 87Rice
husks 42Groundnut shells 25Saw dust 21
to other substrates. Sugarcane bagasse and wheat straw
alsoincrease xylanase production. Similar reports have
shownxylanase induction using lignocellulosic substrate in
strainsof Cellulomonas flavigena [25], Staphylococcus sp. [26],
andBacillus pumilus GESFI [24].
Xylanase with minimal cellulases can be produced usinglow
nitrogen to carbon ratio. Therefore, effect of concentra-tion of
nitrogen on production of enzyme is very important.Effect of
nitrogen source on xylanase production is shownin Figure 3 (Figure
3). Very less activity was observed onsupplementation of inorganic
nitrogen sources compared toother organic nitrogen sources.
Xylanase production was alsosupported by urea. Among organic
nitrogen sources, yeastextract, peptone, tryptone, and beef extract
resulted in bettergrowth and xylanase production. Similar behavior
has beenreported in Chromohalobacter sp. TPSV 101 where
organicnitrogen sources gave maximum xylanase production [12].
3.3. Purification of Xylanase. The crude enzyme was
pre-cipitated using ammonium sulphate to 80% saturation.
Theproteinwas purified by ion exchangeDEAECellulose columnand
SephadexG-100 gel filtration chromatography (Figure 4).The active
fractions of purified ion exchange column wereinjected into
Sephadex G-100 column. The purification hasbeen summarized in Table
3 (Table 3). The purified enzymexylanase exhibited 28.14U/mg
specific activities. Overallrecovery of 17.1%- and 25.8-fold purity
was observed. In caseofGracilibacillus strain acetone precipitated
xylanase showedspecific activity of 46.1 U/mg with 7% yield.
Similar findingswere reported in Bacillus pumilus sp. where 21-fold
puritywas observed with 2% yield. The purified enzyme showed
asingle band on SDS PAGE with a molecular mass of 55 KDa
-
6 International Scholarly Research Notices
Table 3: Purification of xylanase isolated from
halobacterium-OKH.
Total protein Total activity Specific activity Fold purity %
yieldCrude enzyme 525.6 574.8 1.09 1 100Ammonium sulphate
precipitation 155.8 319.2 2.04 1.87 55.5Ion exchange chromatography
42.3 185.9 4.39 4.02 32.3Size exclusion chromatography 3.5 98.5
28.14 25.81 17.1
Resid
ual a
ctiv
ity (%
)
Nitrogen sources
Peptone
Beef extractYeast extract
Tryptone
Urea
Ammonium sulphate
Sodium nitrate
0
50
100
150
Figure 3: Effect of nitrogen sources on enzyme production.
(Figure 5). The zymogram of xylanase exhibited a
significantactivity band that corresponds to result of SDS PAGE.
Highmolecular weight xylanase (62KDa) has been reported instrain
Cl8 [23].
3.4. Effect of NaCl, pH, and Temperature on Enzyme Activityand
Stability. The results in Figure 6(a) demonstrate that theoptimal
temperature of purified xylanase was 37∘C and it wasstable in
temperature range of 10–50∘C. The enzyme activitydeclined rapidly
as the temperature increased above 50∘C and15% of the activity was
retained at 60∘C after 4 hours of incu-bation (Figure 6(b)). In
comparison, Bacillus pumilus GESF1xylanase showed maximum activity
at 40∘C and retainedabout 80% at 60∘C [24]. The xylanase of
Gracilibacillus sp.TSCPVG, a moderate halophile, had the highest
activityretained at 60∘C whereas 83% of activity retained at
55∘Cand 61% of activity retained at 50∘C, respectively [12].
Twoextremely halophilic strains SX15 and CL8 also showedmaximum
activity at 60∘C and 30∘C [23, 27].
StrainOKHxylanase exhibitedmaximal activity at pH9.0(Figure
7(a)). It was stable over a wide range of pH rangesfrom pH 6.0 to
10.0. About 35% of activity was observedat pH 6.0 after incubation
of 8 hours. However, xylanaseretained 80%of activity at pH 10.0
after 12 hours of incubation(Figure 7(b)). Bacillus sp. NG 27
showed maximum xylanase
Table 4: Effect of metal ions and reducing agents on xylanase
activ-ity from halobacterium-OKH.
Metal ion/chemical Relative activity (%)2mM 5mM
Control 100 100Ca2+ 146 151Ag2+ 0 0Hg2+ 0 0Co2+ 65 63Fe3+ 0
0Mg2+ 119 125Mn2+ 138 142Ni2+ 121 107Cu2+ 0 0Cd2+ 0 0Zn2+ 0
0Mercaptoethanol 126 117EDTA 35 21
activity at pH 8.4 [28]. Similar to our finding, strain
Bacillushalodurans showedmaximumactivity at pH9.0 [11]. Xylanasewas
active over a broad range of NaCl concentration of 0–25%with
optimal concentration (5%). At NaCl concentrationof 25%, the enzyme
retained 22% of its activity (Figure 8).Similar description has
also been reported from Bacilluspumilus [24].
3.5. Effect of Additives on Enzyme Activity. Effect of metalions
and effectors are summarized in Table 4 (Table 4).Xylanase was not
affected by addition of metal ions suchas Ca2+, Mg2+, and Mn2+, but
it was inhibited by othermetal ions such as Ag2+, Hg2+, Fe3+, Ni2+,
and Zn2+. Similarfindings on inhibitory effect ofmetal ions on
xylanase activityhave also been reported fromhalophilic
bacteriumCL8 strain[23]. On the contrary, Zn2+ has stimulatory
effect on xylanasefrom TSPVS strain while Mn2+ has been reported to
inhibitxylanase activity of Bacillus sp. K-1 and Bacillus
haloduransS7, respectively [11, 29].
Among the effectors tested, 𝛽-mercaptoethanol increasedthe
activity considerably, indicating that reduced cystine res-idues
are not involved, similar to xylanase of Bacillus sp. SPS-0 [30].
EDTA inhibited the activity suggesting that xylanas-e was metal ion
dependent. Similar finding was reported inBacillus pumilus sp.
[24].
-
International Scholarly Research Notices 7
Prot
ein
conc
entr
atio
n (m
g/m
L)
Fraction number
00
0
50
100
150
200
20 40 60 80 100
20
40
60Xy
lana
se ac
tivity
(U/m
L)
(a)
00
020 40 60
40
80
120
160 4
2
Prot
ein
conc
entr
atio
n (m
g/m
L)
Fraction number
Xyla
nase
activ
ity (U
/mL)
(b)
Figure 4: (a) Ion exchange chromatography. (b) Sephadex G-100
gel filtration chromatography of pooled, active fraction from ion
exchangechromatography. Protein concentration is represented by
circles whereas xylanase activity is represented by square.
97.4
66
43
29
18.4
6.5
(KD
a)
(KD
a)
55
1 2 3
Figure 5: SDS-PAGE and zymogram analysis of purified
xylanasefrom halophilic bacterium-OKH. Lane 1: marker, lane 2:
xylanase in12% SDS-PAGE, and lane 3: zymogram analysis of xylanase
activity.
3.6. Effect of Organic Solvents on Xylanase Activity. To
date,the use of halophilic extremozymes in organic solvents hasbeen
limited to very few enzymes [31]. The influences ofdifferent
organic solvents on xylanase activity are shown inTable 5 (Table
5). Organic solvents like methanol, acetone,acetic anhydride,
isopropanol, and glutaraldehyde were usedto evaluate xylanase
activity. Significant decrease in enzymeactivity was found in the
presence of 10% (v/v) solvents.Maximum stability was observed in
presence of glutaralde-hyde followed by isopropanol. Xylanase
activity in presenceof isopropanol has been reported using
halophilic bacteriumCL8 strain [23]. However, to the best of our
knowledge, anactivating effect of glutaraldehyde has not been
observed forxylanases to date. Factors affecting the enzymes
stability inorganic solvents are changes in solvent-exposed surface
areasand increase in the extent of secondary structure formationand
truncated amino and carboxyl termini. Consequently, insurroundings
with lower salt concentrations, the solubility
Table 5: Effect of organic solvents on xylanase activity.
Solventswereused in 5% and 10%, respectively, and residual activity
was recorded.
Solvents Relative activity (%)5% 10%
Methanol 11 0Isopropanol 42 21Acetone 15 0Acetic anhydride 19
0Glutaraldehyde 51 39
of halophilic enzymes is often very poor which could limittheir
applicability [32]. However, this propertymakes enzymestable in
nonaqueous media [33, 34].
3.7. Storage Stability. Xylanases used in industrial
applica-tions are stored at different temperatures, that is, at
roomtemperature, cooled, or frozen [34]. Enzyme retained 95%
ofactivity at 4-5∘C after storage for 1 month. Enzyme retained85%
of activity when stored at room temperature for 3 days.A 2-3mL
aliquot of xylanase was frozen for 3 weeks andresidues of semisolid
lyophilized enzyme retained nearly 60%of activity.
3.8. Application of Xylanase. All the lignocellulosic
sub-strates, used for saccharification, were found to be
susceptiblefor enzymatic hydrolysis as shown by a significant
increasein the production of reducing sugars (Table 6).
Reducingsugars were released from all agro residues following
theirtreatment with the purified enzyme preparation. Corncobwas
saccharified more efficiently in comparison with wheatstraw and
rice straw after 24 hours, and the release ofreducing sugars was
increasedwith increase in the incubationperiod. The effect of
xylanase treatment was more intensiveon sugarcane bagasse pulp.
Currently, industrial applicationof xylanases is in prebleaching of
Kraft pulp in order tominimize the use of toxic chlorine-containing
chemicals inthe subsequent bleaching step [35, 36]. Since this
xylanasecould also saccharify natural lignocellulosic substrate,
it
-
8 International Scholarly Research Notices
Temperature
00
10
20
30
40
20 40 60 80
Xyla
nase
activ
ity (U
/mL)
(a)Re
sidua
l act
ivity
(%)
Time (hours)0
0
20
40
60
80
100
4 8 12 16 20 24
10203040
506070
(b)
Figure 6: Graph showing (a) effect of temperature on enzyme
activity. (b)Thermal stability of xylanase activity of halophilic
bacterium-OKH.The values represent averages from triplicate
experiments.
pH
00
5
10
15
20
25
30
10 122 4 6 8
Xyla
nase
activ
ity (U
/mL)
(a)
Resid
ual a
ctiv
ity (%
)
Time (hours)0
0
20
40
60
80
100
4 8 12 16 20 24
678
910
(b)
Figure 7: Graph showing (a) effect of pH on the activity of
xylanase of halophilic bacterium-OKH. (b) pH stability of xylanase
activity ofhalophilic bacterium-OKH.The values shown represent
averages from triplicate experiments.
seems to be a good candidate for use in the paper pulpindustry
to produce quality pulps. Optimization of xylanaseby various
statistical approaches is currently in progress.
4. Conclusion
The present work reports the characterization of
haloalkali-moderately thermostable xylanase from newly
isolatedhalophilic bacterium-OKH. It also addresses the
property
of xylanase such as stability in broad pH range, tempera-ture,
and NaCl concentration. Moreover, the ability of thestrain
halophilic bacterium-OKH to produce xylanase withagro residues
supplements has been explored for economicxylanase production
process. Application of purified xylanasein saccharification of
agro residues was checked and efficientsaccharification was found
in sugarcane pulp after 24 hoursof incubation. Thus, this strain
could be good contenderfor different biotechnological applications
under extreme
-
International Scholarly Research Notices 9Re
sidua
l act
ivity
(%)
Time (hours)
00
20
40
60
80
100
4 8 12 16 20 24
0%5%10%
15%20%25%
Figure 8: Graph showing NaCl stability of xylanase residual
activityof halophilic bacterium-OKH at various time intervals.
Table 6: Treatment of lignocellulosic substrate with purified
xylan-ase
Substrate Reducing sugar (mg/mL)Lignocellulosic substrate 24 (h)
48 (h)Corn cobs 2.45 ± 0.23 4.42 ± 0.36Wheat straw 2.15 ± 0.76 3.87
± 0.21Rice Straw 1.89 ± 0.13 3.01 ± 0.91Sugarcane bagasse 7.12 ±
0.63 11.69 ± 0.59
conditions. Further, improvements in enzyme productionusing
optimization parameters by statistical approach and usein
biobleaching are in progress.
Conflict of Interests
The authors declare that there is no conflict of
interestsregarding the publication of this paper.
Authors’ Contribution
Gaurav Sanghvi, Mehul Jivrajani, and Nirav Patel
contributedequally to this paper.
Acknowledgments
Authors are thankful to Department of Biotechnology, M.& N.
Virani Science college, Rajkot, as well as Departmentof
Pharmaceutical Sciences, Saurashtra University, Rajkot,Gujarat,
India, for providing all the facility for the completionof this
work.
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Research ArticleAntiestrogenic and Anti-Inflammatory Potential
ofn-Hexane Fraction of Vitex negundo Linn Leaf Extract:A Probable
Mechanism for Blastocyst ImplantationFailure in Mus musculus
Mehul Jivrajani,1 Nirav Ravat,1 Sheetal Anandjiwala,2 and Manish
Nivsarkar1
1 Department of Pharmacology and Toxicology, B. V. Patel
Pharmaceutical Education and Research Development (PERD)
Centre,Sarkhej-Gandhinagar Highway, Thaltej, Ahmedabad, Gujarat 380
054, India
2Department of Natural Products, National Institute of
Pharmaceutical Education and Research (NIPER), Ahmedabad,Gujarat
380054, India
Correspondence should be addressed to Manish Nivsarkar;
[email protected]
Received 20 June 2014; Revised 7 August 2014; Accepted 13 August
2014; Published 29 October 2014
Academic Editor: Marie Aleth Lacaille-Dubois
Copyright © 2014 Mehul Jivrajani et al.This is an open access
article distributed under the Creative Commons Attribution
License,which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly
cited.
The anti-implantation potential of different fractions of Vitex
negundo Linn leaf extract was evaluated in female Swiss
Albinomice.Animals from different groups were dosed orally either
with 0.2% agar (vehicle) or with fractions of V. negundo leaf
extract (n-hexane, chloroform, n-butanol, and remnant fractions) at
10:00 a.m., from day 1 to day 6 of pregnancy. The pregnant
femalesfrom each group were sacrificed on different days of
pregnancy (𝑛 = 6), and uterus was excised and used for estimation
of lipidperoxidation and assay of superoxide dismutase activity as
a marker for blastocyst implantation. Animals treated with n-h