an improved and versatile immunosuppression protocol for...

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

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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.


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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

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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


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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


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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


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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

Adler, H.I., Fisher, W.D., Cohen, A., Alice, A.H., 1967. Miniature Escherichia coli cellsdeficient in DNA. Proc. Nat. Acad. Sci. U.S.A. 57, 321–326.

Bachmann, B.J., 1983. Linkage map of Escherichia coli K-12. 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. Biol. 33, 635–641.

Davie, E., Sydnor, K., Rothfield, L.I., 1984. Genetic basis of minicell formation inEscherichia coli K-12. J. Bacteriol. 158 (3), 1202–1203.

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 E. coli. Cell 56, 641–649.

Frazer, A.C., Curtiss III, R., 1975. Production, properties and utility of bacterial minicells.Curr. Top. Immunol. Microbiol. 69, 1–84.

Giacalone, M.J., Gentile, A.M., Lovitt, B.T., Xu, T., Surber, M.W., Sabbadini, R.A., 2006. Theuse of bacterial minicells to transfer plasmid DNA to eukaryotic cells. Cell.Microbiol. 8 (10), 1624–1633.

Hall, M.J., Westmacott, D., Wong-Kai-In, P., 1981. Comparative in-vitro activity and modeof action of ceftriaxone (Ro 13–9904), a new highly potent cephalosporin. J.Antimicrob. Chemother. 8, 193–203.

Levy, S.B., 1970. Resistance of minicells to penicillin lysis: a method of obtaining largequantities of purified minicells. J. Bacteriol. 836–839.

MacDiarmid, J.A., Mugridge, N.B., Weiss, J.C., Phillips, L., Bum, A.L., Paulin, P.R., et al.,2007. Bacterially derived 400 nm particles for encapsulation and cancer celltargeting of chemotherapeutics. Cancer Cell 11, 431–445.

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.

Shepherd, N., Dennis, P., Bremer, H., 2001. Cytoplasmic RNA polymerase in Escherichiacoli. J. Bacteriol. 2527–2534.

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 NoticesVolume 2014, Article ID 198251, 10 pageshttp://dx.doi.org/10.1155/2014/198251

http://dx.doi.org/10.1155/2014/198251

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

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


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)).


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


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].


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


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

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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

an improved and versatile immunosuppression protocol for...

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