Phytochemistry 70 (2009) 1210–1216

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Phytochemistry

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ICChI, a glycosylated chitinase from the latex of Ipomoea carnea

Ashok Kumar Patel a, Vijay Kumar Singh a, Ravi Prakash Yadav a, A.J.G. Moir b, Medicherla V. Jagannadham a,*

a Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, Indiab Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, UK

a r t i c l e i n f o

Article history:Received 27 April 2009Received in revised form 3 July 2009Available online 13 August 2009

Keywords:Ipomoea CarneaConvolvulaceaeProtein purificationMultifunctional enzymeICChIChitinase activityDeglycosylationPolyclonal antibodyN-terminal sequence

0031-9422/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.phytochem.2009.07.005

Abbreviations: BSA, bovine serum albumin; BLASTtool; DMSO, dimethyl sulfoxide; DTNB, 5,5l-dithiobiIpomoea carnea chitinase I; MALDI–TOF, matrix assistetime-of-flight; MES, 2-(N-morpholino)ethanesulfonicgel electrophoresis; SDS, sodium dodecyl sulphate; Tdiamine; TFMS, trifluromethanesulphonic acid.

* Corresponding author. Tel.: +91 542 2367936; faxE-mail addresses: ashokmbu@gmail.com (A.K. Pate

satyam.net.in (M.V. Jagannadham).

a b s t r a c t

A multi-functional enzyme ICChI with chitinase/lysozyme/exochitinase activity from the latex of Ipomoeacarnea subsp. fistulosa was purified to homogeneity using ammonium sulphate precipitation, hydropho-bic interaction and size exclusion chromatography. The enzyme is glycosylated (14–15%), has a molecularmass of 34.94 kDa (MALDI–TOF) and an isoelectric point of pH 5.3. The enzyme is stable in pH range 5.0–9.0, 80 �C and the optimal activity is observed at pH 6.0 and 60 �C. Using p-nitrophenyl-N-acetyl-b-D-glu-cosaminide, the kinetic parameters Km, Vmax, Kcat and specificity constant of the enzyme were calculatedas 0.5 mM, 2.5 � 10�8 mol min�1 lg enzyme�1, 29.0 s�1 and 58.0 mM�1 s�1 respectively. The extinctioncoefficient was estimated as 20.56 M�1 cm�1. The protein contains eight tryptophan, 20 tyrosine andsix cysteine residues forming three disulfide bridges. The polyclonal antibodies raised and immunodiffu-sion suggests that the antigenic determinants of ICChI are unique. The first fifteen N-terminal residues G–E–I–A–I–Y–W–G–Q–N–G–G–E–G–S exhibited considerable similarity to other known chitinases. Owingto these unique properties the reported enzyme would find applications in agricultural, pharmaceutical,biomedical and biotechnological fields.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Chitinases (EC 3.2.1.14) are classified as glycosyl hydrolasesthat catalyze the hydrolysis of chitin, a linear homopolymer of b-1,4-linked N-acetyl-D-glucosamine (GlcNAc) residues. Chitin isthe second most abundant polysaccharide found in fungi, animals,and plants (Brunner et al., 1998). Chitinases are found in a widerange of organisms, including bacteria, fungi, higher plants, insects,crustaceans, and some vertebrates (Gooday, 1990; Flach et al.,1992; Cohen-Kupiec and Chet, 1998; Felse and Panda, 1999).

Plant chitinases are frequently considered as pathogenesis-re-lated (PR) proteins, since their activity can be induced by fungal,bacterial, viral infections, but also by broad sources of stress suchas wounding, salicylic acid, ethylene, auxins, and cytokinins, heavymetal salts or elicitors such as fungal and plant cell wall compo-nents (Graham and Sticklen, 1994). Moreover, chitinases also haveimportant non-defensive functions in growth and development

ll rights reserved.

, basic local alignment searchs(2-nitrobenzoic acid); ICChI,d laser desorption–ionizationacid; PAGE, polyacrylamide

EMED, tetramethylethylene-

: +91 542 2367568.l), jvm@bhu.ac.in, jaganmv@

processes (Kasprzewska, 2003). Chitinases are known to beinvolved in regulating the modulation of signal molecules(Goormachtig et al., 1998), embryogenesis (De Jong et al., 1992;Helleboid et al., 2000), and programmed cell death (Passarinhoet al., 2001).

According to the glycosyl hydrolase classification system basedon amino acid sequence similarity of catalytic domains, chitinaseshave been placed in families 18 and 19 (Henrissat, 1991). Family18 chitinases are found in bacteria, fungi, yeast, viruses, plantsand animals whereas family 19 members are almost exclusivelypresent in plants. In all plants analyzed to date, chitinases of bothfamilies are found (Graham and Sticklen, 1994) and are organizedin five different classes numbered from I to V, according to theirsequences and structure (Neuhaus et al., 1996). Chitinases fromclasses I, II and IV belong to the family 19 whereas classes III andV chitinases are of family 18. Class III includes multi-functionallysozyme/chitinase/exochitinase enzymes (Jekel et al., 1991) withno sequence similarity to plant chitinases from other classes(Meins et al., 1992). Although chitinases from various species ofplants share a high degree of similarity in their amino acid se-quence, but different classes of them show disparate structures.

The biotechnological applications of chitinases are also diverseand widely used in agriculture, industry, environmental protectionand in transgenic plants (Lorito et al., 1998; Felse and Panda, 1999;Patil et al., 2000; Wang and Hwang, 2001; Kishimoto et al., 2004).

A.K. Patel et al. / Phytochemistry 70 (2009) 1210–1216 1211

Chitinases are also used to recycle chitinous waste from arthropodshellfish and for chito-oligosaccharide production (Patil et al.,2000; Wang and Hwang, 2001). The hydrolytic activity of chitinaseis a major consideration in most of these applications.

Ipomoea carnea subsp. fistulosa (morning glory), a member ofconvolvulaceae family is a toxic plant (weed) found in abundancein India, Brazil, USA and other countries. The plant has allelopathiceffect; boiled roots are used as laxative and it provokes menstrua-tion. Other parts of the plant are used by traditional healers for skindiseases treatment, milky juice of plant has been used for the treat-ment of Leucoderma and other related skin diseases (Jain et al.,2009). While screening different parts of I. carnea subsp. fistulosa(morning glory) plant for enzymatic activity, the latex exhibiteda considerable amount of chitinase/lysozyme activity. In this pa-per, the isolation and biochemical characterization of a chitinasefrom the latex of morning glory is reported. Owing to its high pur-ity, high yield, broad stability, morning glory chitinase may haveimmense industrial/biomedical applications.

2. Results and discussion

2.1. Purification and characterization of the chitinase

A novel enzyme (ICChI) has been purified and characterizedfrom the latex of I. carnea. The purification procedure involvedammonium sulphate precipitation; hydrophobic interactionchromatography on Ether–Toypearl 650S column and Phenyl-Toyopearl 650S column; and size exclusion chromatography onSuperdex S-200. The crude protein extract was applied on anEther-Toyopearl column, the unbound showed high chitinaseactivity so it was subjected to Phenyl-Toyopearl column wherethe bound protein was eluted by a linear gradient of ammoniumsulphate from 1.5 M to 0 M (data not shown). After SDS–PAGEgel electrophoretic analysis, the eluted protein peak fractions(15–35) with activity were pooled, desalted, concentrated and ap-plied on Superdex S-200 column and eluted at a flow rate of0.25 ml/minute. The pure and homogeneous fractions in this chro-matographic step show an apparent molecular weight of 35–36 kDa on SDS–PAGE (Fig. 1a and b). The peak fractions (42–63)

Fig. 1. Polyacrylamide gel electrophoresis; (a) SDS–PAGE Coomassie stained gel. Lanepurified chitinase ICChI; lane 3, 20 lg of deglycosylated ICChI. The deglycosylated proteisilver stained gel. Lane M, PageRulerTM Plus Prestained Protein Ladder; lane 1, 2 lg of purifishows reduction in apparent molecular weight. (c) ICChI showing pink band after Schiff’swhen stained with Calcofluor M2R. (e) Isoelectric focusing of chitinase ICChI with pH a300 V for 2 h.

were pooled, concentrated, desalted, stored at 4 �C and used inall biochemical characterizations. MALDI–TOF shows a molecularweight of 34.94 kDa (prominent peak) and two peaks of 17.44and 70.09 kDa due to differential protonation of its basic sites(Fig. 2). The results of purification are summarized in Table 1.The yield and purity of the enzyme was high. Such an economicpurification procedure combined with the easy availability of thelatex makes large-scale preparation of the enzyme possible allow-ing a broad study of its various aspects and hence probableapplications.

2.2. Enzyme activity assay

The enzyme shows multi-functional activities such as chitino-lytic, lysozyme and b-N-acetylglucosaminidase (exochitinase)activity. The chitinolytic activity was performed with several dif-ferent substrates by colorimetric, fluorometric and gel diffusionmethods. The quantitative value for chitinase activity was deter-mined to be 7.9 Units mg�1 using N-acetyl-D-glucosamine as stan-dard. The chitinase activity was further confirmed by in gel activityassay with glycol chitin as substrate and calcofluor as fluorescencebrightener (Fig. 1d). Exochitinase (b-N-Acetylglucosaminidase)activity was assayed by using p-nitrophenyl-b-D-N-acetylglucos-aminide as substrate and found to be 48.6 Units mg�1. In addition,the enzyme also showed lysozyme activity (79.8 Units mg�1) whenassayed using Micrococcus lysodeikticus cells. ICChI migrated as asingle sharp band focused at the apparent isoelectric point (pI) of5.3 (Fig. 1e). The enzyme shows high catalytic efficiency with p-nitrophenyl-b-D-N-acetylglucosaminide. The exochitinase activityin ICChI is approximately 6-fold higher than the chitinase activity.Considering the activity behavior, ICChI is suggested to belong toclass III chitinases of family 18 glycosyl hydrolase which showlysozyme/chitinase/exochitinase activity.

2.3. Carbohydrate content and glycostaining

The carbohydrate content of enzyme (ICChI) was estimated tobe 14–15% using the phenol–sulfuric acid method. The absorbanceof the enzyme (ICChI) at 492 nm was extrapolated from the stan-

M, PageRulerTM Plus Prestained Protein Ladder; lane 1, crude protein; lane 2, 20 lgn shows reduction in apparent molecular weight of around 4000 Da. (b) SDS–PAGEed chitinase ICChI; lane 2, 2 lg of deglycosylated ICChI. The deglycosylated protein

staining indicating it to be glycosylated. (d) Detection of chitinolytic activity of ICChImpholytes 5–8. 25 lg of protein was loaded and electrophoresis was performed at

Fig. 2. Mass spectrometry of chitinase ICChI by MALDI–TOF. Ten picomole ofpurified ICChI was dissolved in 1:1 v/v aqueous formic acid and methanol andsubjected to MALDI–TOF analysis. A calibration was done using horse heartmyoglobin (MW 16,951.5 Da).

Fig. 3. Effects of temperature (s) and pH (d) on stability of enzyme ICChI. Fordetermination of stability against pH, 10 lg of enzyme was subjected to differentpH for 24 h; for temperature stability the same amount of enzyme was incubated atdifferent temperatures for 1 h and assayed. The maximum activity was consideredas 100% activity.

1212 A.K. Patel et al. / Phytochemistry 70 (2009) 1210–1216

dard plot generated from galactose measurement (data notshown). Further, to confirm the glycosylation SDS–PAGE gel wasstained with Schiff’s reagent. The glycosylated enzyme producesa pink stained band on the gel (Fig. 1c) whereas the deglycosylatedenzyme produces no color (data not shown). Deglycosylation of IC-ChI was performed by a chemical method using TFMS and a reduc-tion in molecular weight of around 4000 Da was obtained afterdeglycosylation (Fig. 1a, lane 3; Fig. 1b, lane 2). After deglycosyla-tion ICChI aggregates and loses activity indicating a possible role ofglycans in structural integrity. The glycoprotein nature of the pro-tein could well be the contributing factor for high thermal stability(Rao and Gowda, 2008).

2.4. Effect of temperature and pH

The optimum temperature and pH for chitinase activity was60 �C and pH 6.0 respectively (data not shown). However, the en-zyme was remarkably stable up to 80 �C and pH 5.0–9.0 (Fig. 3).The enzyme is highly stable against temperature and pH and thusmay find application in several industrial and biotechnologicalfields.

2.5. Kinetic parameters of the chitinase enzyme

The results of the kinetic analysis of enzyme ICChI using p-nitro-phenyl-N-acetyl-b-D-glucosaminide as the substrate is shown inFig. 4. The effect of increasing substrate concentration on reactionvelocity follows the typical Michaelis–Menten equation (Fig. 4a).The nature of the kinetics with respect to the substrates is typicallyhyperbolic and at higher concentrations of the substrate, the enzymeactivity attains saturation. The apparent Km value obtained from the

Table 1Summary of purification of chitinase (ICChI) from Ipomoea carnea.

Purification step Volume (ml) Total protein(mg)

Crude extract 325 87.80Ammonium sulphate precipitation (85%

saturation)100 64.50

HIC on ETP unbound 200 30.70HIC on PTP eluted (fractions 15–35) 100 11.20SEC on Superdex S-200 (fractions 42–63) 21 6.64

a Activity was measured under standard assay conditions with colloidal chitin as sub

Lineweaver–Burk plot was 0.5 mM with p-nitrophenyl-N-acetyl-b-D-glucosaminide as substrate (Fig. 4b). Values of Vmax and Kcat weredetermined to be 2.5 � 10�8 Moles min�1 lg enzyme�1 and29.0 s�1. From this, the specificity constant, Kcat/Km, was calculatedto be 58.0 mM�1 s�1.

2.6. Specific amino acid residues

Tryptophan and tyrosine contents of ICChI were 8 (measuredvalues 8.22) and 20 (measured values 19.6), respectively. Theextinction coefficient was estimated as 20.56 M�1 cm�1. The totalsulfhydryl content of the protein was found to be 6 (measured val-ues 6.28) forming three disulfides bridges, a characteristic featureof most of class III chitinases (Jekel et al., 1991; Terwisscha vanScheltinga et al., 1994; Cavada et al., 2006). Under similar experi-mental conditions ribonuclease, papain, proteinase K, and lyso-zyme gave the reported values.

2.7. Polyclonal anti-ICChI antibodies and immunoassays

The presence of polyclonal antibodies raised in rabbit waschecked by immunodiffusion. A precipitin line was not observedwith the pre-immunoserum indicating that there is no prior anti-body against ICChI in the pre-immune serum. However with raisedpolyclonal anti-ICChI antibodies, precipitin lines start appearingafter 10–12 h of incubation at room temperature and are distinctlyvisible by 24–30 h. The precipitin line is formed only against ICChIas a result of precipitation of antigen–antibody complexes near theequivalence zone. Antisera to ICChI did not cross-react with

Total activitya

(units)Specific activity (units/mg)

Purification(fold)

Yield (%)

176.40 2.00 1.00 100.00146.10 2.26 1.13 82.80

104.38 3.40 1.70 59.1764.96 5.80 2.90 36.8352.46 7.9 3.95 29.74

strate as described in Materials and methods.

Fig. 4. Determination of kinetic parameters for chitinase ICChI using p-nitrophenylN-acetyl-b-D-glucosaminide as substrate. (a) Activity of ICChI undergoes saturationat the higher substrate concentration in accordance with Michaelis–Mentenequation. Different concentration of substrates (0.0–8.0 mM) was taken with equalamount of enzyme (20 lg) and activity was measured as described in materials andmethods section. (b) A Lineweaver–Burk plot, 1/(S) vs. 1/(V) has been used for thedetermination of kinetic parameters. The calculated value of Km, Vmax, Kcat andspecificity constant (Kcat/Km) of the enzyme was found to be 0.5 mM, 2.5 �10�8 Moles min�1 lg enzyme�1, 29.0 s�1 and 58.0 mM�1 s�1 respectively.

A.K. Patel et al. / Phytochemistry 70 (2009) 1210–1216 1213

carnein (Patel et al., 2007), Chitinase II and MGP (Patel et al., 2008)purified from the same plant suggesting that the antigenic deter-minants of ICChI are unique (Fig. 5). The expression of chitinasesis altered in response to biotic and abiotic stress (Takenaka et al.,2009) in this context the polyclonal antibodies raised would beof immense importance as a probe to detect the localization and

Fig. 5. Ouchterlony’s double immunodiffusion was carried out in 1% agarosedissolved in phosphate buffered saline. 100 ll of rabbit serum was added in thecentral well and 40 lg of four purified proteins chtinase ICChI, carnein, chitinase II,and MGP were added in peripheral wells. The precipitin band shown by arrow wasobserved only against the chitinase ICChI after 24 h of incubation suggestingantigenic determinants of ICChI to be unique.

study the expression of ICChI in response to various stressessignals.

2.8. Amino-terminal sequence

The N-terminal sequence of the ICChI was generated by Edmannautomated degradation method. The first 15 amino acid residues,G–E–I–A–I–Y–W–G–Q–N–G–G–E–G–S showed high% identity withseveral plant class III chitinases (Table 2), which are extracellularacidic proteins important for plant defense against pathogenic bac-teria and fungi (Jekel et al., 1991). Further sequence and gene anal-ysis is required to classify ICChI.

A recent report on the role of latex chitinase in plant defense(Wasano et al., 2009) indicates a possible role of ICChI in protectionfrom the insects. Various molecular and biotechnological aspectssuch as regulation strategies, gene cloning of chitinase from micro-organism and plants, and various industrial and agricultural appli-cations of chitinases have been described by Patil et al. (2000). Thispractical aspect may result in the induction of some agronomicallyimportant crops resistant to various diseases.

2.9. Conclusion

ICChI is highly stable enzyme and has potential to be utilized inindustrial and biotechnological applications. In addition, it may beemployed in agriculture, environmental protection, recycling chi-tinous waste, and chito-oligosaccharide production. Further studywould reveal the physiological role, tissue localization, and struc-ture–function relationship.

3. Experimental

3.1. Material and methods

Latex (milky sap) was collected by superficial incisions of youngstems of I. carnea subsp. fistulosa (morning glory) found abundantlyin Banaras Hindu University campus, Varanasi, India. Glycol chito-san, chitin, p-nitrophenyl N-acetyl-b-D-glucosaminide, calcofluorM2R fluorescent brightener 28, allosamidine, MES buffer, sodiumazide, BSA, ribonuclease A, hen egg white lysozyme, DTNB, glyc-erol, urea, b-mercaptoethanol, N,N0-methylene-bis-acrylamide,TEMED, Coomassie brilliant blue R-250 as well as G-250, Freund’scomplete, incomplete adjuvants, potassium sodium tartrate; 3,5-dinitrosalicylic acid, and agarose were obtained from SigmaChemical Co. (USA); ampholine carriers in all pH ranges, SDS–PAGEstandard from Fermentas Life Sciences (Canada); SDS from ServaFeinbiochemica, Heidelberg, electrophoretic markers from GEHealthcare Limited (UK); orthophosphoric acid, periodic acid,Schiff’s reagent was from Loba Chemie Pvt Ltd., India, acrylamide,Tris buffer, ammonium sulphate, TFMS, sodium metabisulphite,anisole, pyridine, Triton-X, were purchased from SpectrochemPvt Ltd. (India).

3.2. Isolation and purification of chitinase

All steps for purification of the enzyme were carried out at 4 �Cand the solutions were prepared using Milli Q water (Millipore).Fresh latex secretions by superficial incisions made on youngstems and apical buds of I. carnea plants were collected into0.05 M MES buffer, pH 6.0 and stored at �20 �C for 24 h. The latexwas thawed at room temperature and centrifuged at 12,000g(Sorvall RC-5C plus) for 20 min to remove all insoluble cell debris.Clear supernatant referred as crude proteins was used for furtherinvestigations.

The crude protein was subjected to 85% ammonium sulphateprecipitation and the resultant precipitate was collected by centri-

Table 2N-Terminal sequence alignment of chitinase ICChI with amino acid sequences of other known chitinases.

Accession Source Sequences Identity (%)

>gi|chitinase ICChI [Ipomoea carnea] GEIAIYWGQNGGEGS 100>gi|998516|gb|AAB34670.1| [Phytolacca americana] GGIAIYWGQNGGEGT 87>gi|3451147|emb|CAA09110.1| [Hevea brasiliensis] GGIAIYWGQNGNEGT 80>gi|106647236|gb|ABF82271.1| [Panax ginseng] GGISIYWGQNGGEGT 80>gi|119393870|gb|ABL74451.1| [Casuarina glauca] GGIAIYWGQNGNEGT 80>gi|33323055|gb|AAQ07267.1| [Ficus awkeotsang] GGIAIYWGQNGNEGT 80>gi|13560118|emb|CAB43737.2| [Trifolium repens] AGIAVYWGQNGGEGS 80>gi|28971736|dbj|BAC65326.1| [Vitis vinifera] GGIAIYWGQNGNEGT 80>gi|871764|emb|CAA61279.1| [Vigna unguiculata] GGIAIYWGQNGNEGT 80>gi|10954033|gb|AAG25709.1| AF309514 [Malus x domestica] GGIAIYWGQNGNEGT 80>gi|4530607|gb|AAD22114.1| [Fragaria x ananassa] GGIAIYWGQNGNEGT 80>gi|762879|dbj|BAA08708.1| [Psophocarpus tetragonolobus] AGIAVYWGQNGGEGS 80>gi|75708015|gb|ABA26457.1| [Citrullus lanatus] AGIAIYWGQNGNEGS 80>gi|7595839|gb|AAF64474.1| AF241266 [Cucumis melo] AGIAIYWGQNGNEGS 80>gi|5823010|gb|AAD53006.1| AF082284 [Cucurbita moschata] AGIAIYWGQNGNEGS 80>gi|4835584|dbj|BAA77676.1| [Glycine max] AGIAIYWGQNGGEGT 80>gi|28848952|gb|AAO47731.1| [Rehmannia glutinosa] GKISIYWGQNGNEGT 73>gi|124294787|gb|ABN03967.1| [Gossypium hirsutum] GDIAIYWGQNGNEGT 73ClustalW multiple sequence alignments . *:: * * * * * *. * *:

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fuged at 12,000g for 30 min. The precipitate was suspended in0.05 M MES buffer pH 6.0 (buffer A) followed by dialysis againstthe same buffer. The dialysate was adjusted to 1.5 M ammoniumsulphate concentration and subjected to a hydrophobic interactionchromatography (HIC) on Ether-Toyopearl 650S (ETP)(2.0 � 7.0 cm) column pre-equilibrated with buffer A containing1.5 M ammonium sulphate. The unbound proteins from HICshowed high chitinase activity so they were subjected to HIC ona Phenyl-Toyopearl 650S (PTP) (2.0 � 10.0 cm) column pre-equili-brated as above. The column was washed with an excess of thesame buffer containing 1.5 M ammonium sulphate to removeloosely bound proteins followed by elution with a linear gradientof ammonium sulphate from 1.5 M–0 M in buffer A.

The eluted active fractions were pooled, desalted, concentratedusing Viva-spin concentrator (MWCO 10,000 Da) and subjected tosize exclusion chromatography on Superdex S-200 (Pharmacia)column (1.2 � 120 cm) pre-equilibrated with buffer A containing0.5 M NaCl. The proteins were eluted isocratically with the samebuffer at a flow rate of 0.25 ml/minute. The gel filtration elutionprofile consisted of an initial symmetrical peak with high chitinaseactivity followed by small peak of other protein lacking in chitinaseactivity. The homogenous fraction from SDS–PAGE analysis werepooled, concentrated, desalted and stored in buffer A at 4 �C forall biochemical characterization. The enzyme was named as ICChI(I. carnea chitinase I). The chitinase activity and protein contentwas analyzed at each purification step.

3.3. Polyacrylamide gel electrophoresis

Protein samples were analyzed by SDS–PAGE (Laemmli, 1970)under reducing conditions to check the homogeneity of the en-zyme preparations as described using a mini electrophoresis sys-tem (Bio-Rad). After electrophoresis, gel was stained with 0.1%(w/v) Coomassie brilliant blue R-250.

3.4. Enzyme assay

The enzymatic activity was assayed with several substratesusing spectrophotometric, fluorometric methods and in gel diffu-sion assay.

(a) Colloidal chitin chitinase assayChitinase activity was measured by reduction of 3,5-dinitrosal-

icylic acid, in presence of N-acetyl-D-glucosamine released by

enzymatic hydrolysis of chitin (Monreal and Reese, 1969). Oneml of 10% wet colloidal chitin; suspended in 0.2 M phosphate buf-fer pH 6.5 and 1 ml of enzyme were mixed and incubated for60 min at 50 �C. Enzymatic reaction was stopped by addition of1 ml NaOH (1% w/v). The undigested chitin was centrifuged at5000g for 10 min and the pellet was discarded. One ml of superna-tant was added to 1 ml of 1% 3,5-dinitrosalicylic acid dissolved in30% sodium potassium tartrate in 2 M NaOH in a test tube andincubated for 5 min in boiling water. Soluble reducing sugar asN-acetyl-D-glucosamine produced by chitinase activity was deter-mined by absorbance measurement at 535 nm. One unit of chiti-nase activity was defined as the amount of enzyme required toproduce 1 lg of N-acetyl-D-glucosamine in 1 h (Rojas-Avelizapaet al., 1999).

(b) A gel diffusion chitinase assayChitinase was visualized by SDS–PAGE containing 0.01% glycol

chitin synthesized from glycol chitosan (Trudel and Asselin,1989). Parallel SDS–PAGE was performed for visualization of chiti-nase activity with a fluorescence brightener and Coomassie stain-ing. After electrophoresis, gels were incubated for 13 to 20 h at37 �C in 100 mM sodium-acetate pH 5.0, 1% Triton–X 100 (v/v),to remove SDS and promote chitinase activity against glycol chitin.First gel was stained with 0.01% Calcofluor White M2R (Sigma) in0.5 M Tris–HCl pH 8.9 and the chitinase activity was visualized un-der UV transilluminator. The other gel stained with Coomassie bril-liant blue G-250 was used for comparison of position of the proteinband.

(c) Exochitinase activity assayb-N-Acetylglucosaminidase activity of enzyme was assayed by

using p-nitrophenyl-b-D-N-acetylglucosaminide as substrate. Oneml of reaction mixture containing 2 mM substrate, 50 mM acetatebuffer, pH 6.0 and 20 lg enzyme was incubated at 30 �C for 30 min.The reaction was terminated by the addition of 1 ml of 0.2 MNa2CO3 and amount of p-nitrophenol liberated was measured byabsorbance at 405 nm. One unit of the enzyme activity was definedas the amount of enzyme that liberates 1 lmol of p-nitrophenolper min under the conditions (Kyoung-Ja et al., 2003).

(d) Lysozyme activityLysozyme activity of the purified enzyme was measured by the

rate of lysis of M. lysodeikticus cell walls as described by Shugar(1952). The reaction was monitored continuously as decrease in

A.K. Patel et al. / Phytochemistry 70 (2009) 1210–1216 1215

light scattering at 450 nm using a Beckman DU Spectrophotometerat 37 �C. The reaction contained 0.3 mg of M. lysodeikticus cell wallsin a volume of 2.9 mL in 0.1 M phosphate buffer, pH 7.0 and 0.1 mlenzyme. One unit is defined as the amount of enzyme that pro-duces a decrease in absorbance of 0.001/min under the specifiedcondition.

3.5. Protein assay

Protein concentration was determined by the method describedby Bradford (1976). Bovine serum albumin was used as standard.

3.6. Carbohydrate content and deglycosylation

The carbohydrate content of the enzyme was determined usingphenol–sulfuric acid method (Hounsell et al., 1997) using galactoseas a standard. The deglycosylation of the enzyme was done bychemical method using TFMS (Edge, 2003). The electrophoresiswas carried with glycosylated and deglycosylated protein sampleby the method as described above. The gel was stained with Schiff’sreagent specific to glycoprotein (Wilson et al., 2006).

3.7. Isoelectric focusing

The isoelectric point of the purified enzyme was determinedby isoelectric focusing on polyacrylamide disc gels as described(Patel et al., 2007). Electrophoretic runs were carried out withampholine carrier ampholytes pH range 5.0–8.0 at 300 V for 2 husing 0.1 M NaOH as catholyte and 0.1 M orthophosphoric acidas anolyte. Enzyme in IEF–PAGE gel was visualized by Coomassiestaining G-250.

3.8. Mass spectrometry

Molecular weight of the purified enzyme was determined byMicromass TofSpec MALDI–TOF. Samples were dissolved at a con-centration of 10 pmol/ll in 1:1 (v/v) 1% aqueous formic acid andmethanol. Positive ionization was used for the sample analyseswith an electro spray voltage of 1.0 kV. A sampling cone voltageof 40 V and MCP detector of 2700 V. Nitrogen was employed asthe API gas and data were acquired over the appropriate m/z rangeat a scan speed of 3.0 s in continuum mode. An external calibrationwas made using horse heart myoglobin (MW 16,951.5 Da) anddata were processed using the MassLynx suite of software pro-grams supplied with the mass spectrometer.

3.9. pH and temperature optima

The effect of pH on the enzymatic activity of the purified en-zyme was determined in the range of pH 0.5–12.0. Buffers usedwere KCl–HCl (pH 0.5–1.5), glycine–HCl (pH 2.0–3.5), sodium-ace-tate (pH 4.0–5.5), sodium-phosphate (pH 6.0–7.5), Tris–HCl (pH8.0–10.0) and glycine–NaOH (pH 10.5–12.0). A buffer of 50 mMwas used throughout the experiment.

Similarly, the effect of temperature on the enzymatic activity ofthe purified enzyme was determined in the temperature range 20–95 �C. The colloidal chitin was used as substrate for the activity as-say as described above.

3.10. Temperature and pH stability of the chitinase

The thermal stability of the purified enzyme was estimated bydetermining the residual activity of the enzyme solution after incu-bation for 60 min at various temperatures from 20 to 95 �C.

Similarly, the stability of the enzyme was estimated by deter-mining the residual activity of the enzyme solution after exposure

to different pH from 0.5 to 12.0 for 24 h at room temperature. Theenzyme was assayed as described above with colloidal chitin assubstrate.

3.11. Kinetic parameter of the chitinase enzyme

A kinetic property of the enzyme was determined using p-nitro-phenyl-N-acetyl-b-D-glucosaminide as the substrate (Tronsmo andHarman, 1993). Reactions mixture containing increasing substrateconcentrations from 0 to 5 mM in 50 mM acetate buffer pH 6.0 and20 lg of purified enzyme was incubated for 30 min at 50 �C. Reac-tions were terminated by adding 500 ll of 0.4 M sodium carbonate,which enhanced the color of p-nitrophenol formed by the enzy-matic cleavage of the substrate. Absorbance at 405 nm was mea-sured by a Beckman DU spectrophotometer.

3.12. Specific amino acid residue

The tyrosine and tryptophan contents of the enzyme were mea-sured using the method of Goodwin and Morton (1946). The absor-bance spectrum of the enzyme in 0.1 M NaOH was recordedbetween 220 and 320 nm using a Beckman DU 640 B spectropho-tometer and the absorbance values at 280 and 294.4 nm were de-duced from the spectra.

The exposed and total cysteine residues of ICChI were estimatedby Ellman’s (1959) method. The molar extinction coefficient of TNBanion at 412 nm is 14,150 M�1 cm�1 (Creighton, 1989) was usedfor the calculation. For exposed sulfhydryl group estimation, thepurified enzyme was reduced with 0.01 M b-mercaptoethanol for15 min, in 0.05 M Tris–HCl buffer, pH 8.0, and dialyzed against0.1 M acetic acid at 4 �C for 24 h with frequent changes. For theestimation of total sulfhydryl content, the enzyme was reducedwith 0.01 M b-mercaptoethanol in the presence of 6 M GuHCl for15 min at 37 �C and dialyzed against 0.1 M acetic acid. The numberof disulfide bonds, in the protein, was deduced by comparison ofthe number of free and total cysteine residues.

To validate these measurements, tyrosine, tryptophan and cys-teine content of some standard proteins like papain, ribonucleaseA, BSA and lysozyme were determined under similar conditions.

3.13. Extinction coefficient

The extinction coefficient of the protein (ICChI) at 280 nm wasdetermined by a spectrophotometric method (Aitken andLearmonth, 1997) using Beer–Lambert’s law.

3.14. Amino-terminal sequence analysis of chitinase

The purified enzyme (ICChI) dialyzed against distilled waterwas concentrated by freeze drying and ten picomole sample wasapplied on a protein sequencer (Applied Biosystems Procise Se-quencer). The N-terminal was sequenced by automated Edman’sdegradation. The N-terminal sequence obtained was compared toother known plant chitinases using NCBI–BLAST. The multiplealignments were performed using CLUSTALW.

3.15. Antigenic properties of the chitinase

Antibodies against the purified enzyme ICChI were raised in amale albino rabbit (1 kg body weight) as described (Patel et al.,2007). The presence of antibodies was confirmed by immunodiffu-sion studies by Ouchterlony’s double immunodiffusion as de-scribed by Ouchterlony and Nilsson (1986). Enzyme (ICChI) andthe other three proteins purified from the latex of I. carnea wereloaded in the peripheral well and anti-ICChI serum was loaded inthe central well to check the specificity of raised antibody.

1216 A.K. Patel et al. / Phytochemistry 70 (2009) 1210–1216

Acknowledgements

Funding to AKP from CSIR (Council of Scientific & Industrial Re-search, India), VKS from UGC (University Grants Commission, In-dia), RPY from DBT (Department of Biotechnology, India) isgratefully acknowledged. We appreciate the funding aid of BIF(Boehringer Ingelheim Fonds, Germany) in form of travel Grantfor the short three months to furnish the experiments and data col-lection in abroad laboratory. We are thankful to Prof. D.W. Rice andS.E. Sedelnikova of University of Sheffield for their experimentalsupport and fruitful discussions.

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