Chitinase from a Novel Strain of Serratia marcescens JPP1 for

Research ArticleChitinase from a Novel Strain of Serratia marcescensJPP1 for Biocontrol of Aflatoxin Molecular Characterizationand Production Optimization Using ResponseSurface Methodology

Kai Wang Pei-sheng Yan and Li-xin Cao

School of Marine Science and Technology Harbin Institute of Technology Weihai 264209 China

Correspondence should be addressed to Pei-sheng Yan yps6163com and Li-xin Cao caolixin668aliyuncom

Received 20 January 2014 Revised 13 March 2014 Accepted 17 March 2014 Published 9 April 2014

Academic Editor Xiaoling Miao

Copyright copy 2014 Kai Wang et alThis is an open access article distributed under theCreativeCommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Chitinase is one of the most important mycolytic enzymes with industrial significance and produced by a number of organisms Achitinase producing isolate Serratia marcescens JPP1 was obtained from peanut hulls in Jiangsu Province China and exhibitedantagonistic activity against aflatoxins In this study we describe the optimization of medium composition with increasedproduction of chitinase for the selected bacteria using statistical methods Plackett-Burman design was applied to find the keyingredients and central composite design of response surfacemethodologywas used to optimize the levels of key ingredients for thebest yield of chitinaseMaximum chitinase production was predicted to be 2309UmL for a 21-fold increase inmedium containing1270 gL colloidal chitin 734 gL glucose 500 gL peptone 132 gL (NH

4)2SO4 07 gL K

2HPO4 and 05 gL MgSO

4sdot7H2O

Polymerase chain reaction (PCR) amplification of the JPP1 chitinase gene was performed and obtained a 1789 bp nucleotidesequence its open reading frame encoded a protein of 499 amino acids named as ChiBjp

1 Introduction

Chitin is the second most abundant renewable carbohydratepolymer in nature after cellulose and possibly the most abun-dant in marine environments [1] It largely exists in wastesfrom processing of marine food products [2] with an annualrecovery of 1ndash100 billion metric tonnes as chitinous waste[3] Most of the chitinous waste is disposed through oceandumping incineration and land fillingThe lack of cheap andcommercially feasiblemethods for chitinous processing leadsto economic loss wastage of natural resource and problem ofenvironmental pollution Disposal by microbial degradationof chitin offers the best solution to the problem leading torecycling of nutrients in the environment [4 5]

Chitinases (EC 32114) are glycosyl hydrolases whichcatalyze the first step in chitin digestion Recently chitinaseshave received increasing attention because of their wide rangeof biotechnological applications especially in agriculture for

biocontrol of fungal phytopathogens and harmful insectsbecause chitin is the essential component of fungal cell walland cuticle of insects [6ndash9] Moreover the applications ofchitinases offer a potential alternative to the chemical fungi-cides [10 11] A wide range of microorganisms could degradechitin by producing chitinases for nutrition antagonismand combating parasites [12ndash17] However to our knowledgethere is no report performed on chitinase produced byendophytic Serratia marcescens isolated from peanut hullsfor biocontrol of aflatoxins production The ability of Smarcescens to produce chitinases is strain dependent and thequantities depended on the nutritional composition of thegrowth medium and culture condition [18] Meanwhile theapplications of chitinases require large quantities which inturn require optimization of nutritive and physical parameterfor the selected isolate

Several statistical and nonstatisticalmethods are availablefor optimization of medium constituents Plackett-Burman

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 482623 8 pageshttpdxdoiorg1011552014482623

2 BioMed Research International

and response surface methodology are the most widely usedstatistical approaches for reducing the time and expenseCentral composite design (CCD) was used to determine lev-els of various parameters with the interrelation between eachparameter evolved simultaneously [19] Studies on mediumoptimization for chitinases production are the worthwhiletechnique for multifactor experiments because they coulddetect the true optimum of the factor [20ndash22] In additionmedium composition greatly influenced the microbial pro-duction of extracellular chitinase and its interaction plays animportant role in the synthesis of the chitinases [23 24] Theobjective of the present work was to characterize the mediumof S marcescens JPP1 for maximum antagonistic effect onaflatoxin in favor of the chitinase production using statisticaldesigns of Plackett-Burman and central composite design ofresponse surface methodology The molecular properties ofthe extracellular chitinase were also determined

2 Materials and Methods

21 Bacterial Strain Thestrain used in this study was isolatedfrom the peanut hulls collected from the sampling site inHuaian city Jiangsu Province China Itwas identified asSerratia marcescens JPP1 based on its morphological andphysiological characteristics and 16S rRNA gene sequenceanalysis The nucleotide sequence of 16S rRNA gene wassubmitted to NCBI GenBank database under the accessionnumber JQ308601 [25]

22 Media Composition PGY medium peanut hulls weredried at 40∘C and then ground The ground peanut hullswere boiled with water for 1 h at the final concentration of25 and then centrifuged at 6600 g at room temperature for5min The supernatant was supplemented with 2 glucoseand 05 yeast extract and then autoclaved for 20min at121∘C pH in nature S marcescens JPP1 was maintained onsolid PGY medium The stocks were kept in the refrigeratorand subcultured at monthly intervals

23 Preparation of Colloidal Chitin Colloidal chitin wasprepared from pure chitin (Sangon Biotech China) by themethod of Roberts and Selitrennikoff [26] Commercialchitin (40 g) was weighed and taken in a beaker 500mL ofconcentrated hydrochloric acid was added followed by con-tinuous stirring at 4∘C After stirring for 1 h the hydrolyzedchitin in the beaker was washed several times with distilledwater to remove the acid completely and hence bring the pHinto the range of 6-7 Once this pHwas obtained the colloidalchitin was filtered using Whatman filter paper The filteredcolloidal chitin was then collected and stored in the form of apaste at 4∘C

24 Chitinase Activity Assay Chitinase activity was testedaccording to the method of Monreal and Reese detectingN-acetylglucosamine (NAG) as the final product [27] Thereaction mixture for the chitinase assay contained 1mL of5 acid swollen chitin 1mL of 50mM acetate buffer (pH50) and 1mL of enzyme solution The reaction mixture

was incubated at 50∘C for 1 h and then the reaction wasstopped after boiling for 15minThemixture was centrifugedat 5000 rpm for 20min and the concentration of releasedNAG was assayed at 530 nm spectrophotometrically withcolloidal chitin as substrate One unit of chitinase activity wasdefined as the amount of enzyme that catalyzed the release of1 120583mol of NAG per hour at 50∘C Data is expressed as mean plusmnSD of three experiments

25 Design of Experiment The optimization of medium con-stituents to improve chitinase activity of S marcescens JPP1was carried out in two stages Plackett-Burman and responsesurface methodology Firstly eight variables including threebest carbon sources three best nitrogen sources magnesiumsulfate and potassium hydrogen phosphate anhydrous wereselected on the basis of their role in chitinase secretionenhancementThe variables having themost significant effecton chitinase activity were identified using a 2-level Plackett-Burman design

Secondly response surface methodology was used tooptimize the screened components to enhance chitinaseactivity using central composite design A 24 full factorialCCD of RSM was employed to optimize the four mostsignificant factors (glucose peptone ammonium sulfate andchitin) for enhancing chitinase activity The concentrationsof 4 variables were previously investigated for chitinaseactivity using single-factor experiments (data not shown)In this study the experimental plan consisted of 31 trialsand the independent variables were studied (Table 1) All theexperiments were done in triplicate and the average chitinaseactivity was taken as the dependent variables or responsesThe data obtained from CCD on chitinase production weresubjected to analysis of variance (ANOVA) Then a second-order polynomial equation was fitted to the data by multipleregression procedure This resulted in an empirical modelthat related the response measured in the independentvariables to the experiment The behavior of the system wasexplained by the following quadratic equation

119884 = 120573

0+sum120573

119894119909

119894+sum120573

119894119895119909

119894119909

119895+sum120573

119894119894119909

119894

2 (1)

where 119884 is predicted response 119909119894and 119909

119895are the input

variables 1205730is the intercept term 120573

119894is the linear effects 120573

119894119894is

the squared effects and 120573119894119895is the interaction term

The statistical software package Minitab 160 (MinitabInc State College PA) was used to analyze the experimentaldesign The response obtained was statistically evaluated andthe model was built based on the variables with confidencelevels more than 95

26 Nucleotide Sequence of Chitinase Gene Genomic DNAfrom strain S marcescens JPP1 was extracted using a bacterialgenomic DNA FastPrep Extraction Kit (Sangon BiotechChina) The oligonucleotides designed using Primer Premier50 on the basis of the published sequence of chitinase (ChiB)gene [28] were synthesized and used for determination ofthe chitinase gene sequence The upstream primer (51015840-CC-AAGACAGGCGGCAGTAAATAAAA-31015840) and downstream

BioMed Research International 3

Table 1 Experimental design and results of CCD of 4 variables in coded and actual units

Runs 119860 glucose (gL) 119861 peptone (gL) 119862 chitin (gL) 119863 (NH4)2SO4 (gL) Chitinase activity (UmL)Coded Conc Coded Conc Coded Conc Coded Conc Observed Predicted

1 minus1 4 minus1 2 minus1 8 minus1 2 747 plusmn 009 7562 1 8 minus1 2 minus1 8 minus1 2 929 plusmn 015 9243 minus1 4 1 4 minus1 8 minus1 2 1616 plusmn 014 16114 1 8 1 4 minus1 8 minus1 2 1818 plusmn 007 18085 minus1 4 minus1 2 minus1 8 1 4 879 plusmn 008 8756 1 8 minus1 2 minus1 8 1 4 949 plusmn 010 9877 minus1 4 1 4 minus1 8 1 4 1768 plusmn 012 17458 1 8 1 4 minus1 8 1 4 1838 plusmn 003 18879 minus1 4 minus1 2 1 12 minus1 2 505 plusmn 006 50510 1 8 minus1 2 1 12 minus1 2 636 plusmn 008 70311 minus1 4 1 4 1 12 minus1 2 1283 plusmn 019 128912 1 8 1 4 1 12 minus1 2 1465 plusmn 021 151713 minus1 4 minus1 2 1 12 1 4 504 plusmn 007 55714 1 8 minus1 2 1 12 1 4 646 plusmn 005 70015 minus1 4 1 4 1 12 1 4 1303 plusmn 017 135716 1 8 1 4 1 12 1 4 1495 plusmn 014 152917 minus2 2 0 3 0 10 0 3 95 plusmn 004 95118 2 10 0 3 0 10 0 3 1384 plusmn 008 129119 0 6 minus2 1 0 10 0 3 465 plusmn 006 40520 0 6 2 5 0 10 0 3 2121 plusmn 011 208921 0 6 0 3 0 10 minus2 1 1182 plusmn 008 117222 0 6 0 3 0 10 2 5 1384 plusmn 003 130323 0 6 0 3 minus2 6 0 3 1364 plusmn 009 138624 0 6 0 3 2 14 0 3 889 plusmn 012 77625 0 6 0 3 0 10 0 3 1343 plusmn 005 132326 0 6 0 3 0 10 0 3 1313 plusmn 009 132327 0 6 0 3 0 10 0 3 1374 plusmn 017 132328 0 6 0 3 0 10 0 3 1364 plusmn 014 132329 0 6 0 3 0 10 0 3 1303 plusmn 011 132330 0 6 0 3 0 10 0 3 1304 plusmn 005 132331 0 6 0 3 0 10 0 3 1263 plusmn 006 1323

primer (51015840-AAAAGCGATGTCTACAGCCTGATGG-31015840)were used to amplify and sequence the CHI gene

PCR was performed under the following conditions94∘C for 5min followed by 94∘C for 1min 58∘C for 1minand 72∘C for 15min for 35 cycles and with a final 10minextension at 72∘C PCR sequencing was performed at SangonBiotech Shanghai In order to confirm the fidelity of thesequence two independent PCR products were sequencedin both directions Sequence comparison was performedin the GenBank database using BLAST through the NCBIserverThe protein sequence alignment was performed usingCLUSTAL X program of MEGA 5 package

3 Results and Discussion

Achitinase producing isolate JPP1 was obtained from peanuthulls in Jiangsu Province China The JPP1 bacterium wasa rod-shaped organism that was gram negative casein

hydrolysis catalase reactivity and citrate degradation posi-tive It was identified as Serratia marcescens based on bio-chemical and genetic characteristics In preliminary studiesthe strain exhibited antagonistic activity against myceliagrowth and subsequent aflatoxin production The mainmechanism of strain JPP1 for biocontrol against the growthof A parasiticus and aflatoxin production was also deter-mined The strain JPP1 could produce chitinase to degradephytopathogenic fungal cell walls [25]

Since the ability of S marcescens to produce chitinasesis strain dependent and the medium composition greatlyinfluenced the chitinase synthesis optimization of culturemedia for chitinase production was performedThe Plackett-Burman design could provide an efficient way of a largenumber of variables to identify the most important onesA total of 8 variables were analyzed with regard to theireffects on chitinase production using the Plackett-Burmandesign Three best carbon sources (glucose fructose andbeef extract) three best nitrogen sources (ammonium sulfate


peptone and ammonium chloride) magnesium sulfate anddipotassiumhydrogen phosphate (K

2HPO4) were selected on

the basis of their role in chitinase secretion enhancementTheresults revealed that the most significant three factors whichwere more effective in chitinase production were peptoneglucose and ammonium sulfate (119875 lt 001) [29]

Central composite design is a very useful tool for deter-mining the optimal level of significant constituents andtheir interaction In this study CCD was used to determinethe optimum level of the four selected significant variables(peptone glucose chitin and ammonium sulfate) for thechitinase production A total of 31 experiments with differentcombinations of the four selected variables were performedThe design matrix with the corresponding results of CCDexperiments as well as the experimental results is presentedin Table 1 The 119875 values for the model (lt00001) and forldquolack of fitrdquo (0066) suggested that the obtained experi-mental data were a good fit with the model By applyingmultiple regression analysis on the experimental data theexperimental results of the CCD design were fitted witha second-order polynomial equation for chitinase activityThe response of chitinase production (119884) by S marcescensJPP1 can be expressed in terms of the following regressionequation

119884 = 1323 + 085119860 + 421119861 + 033119862 minus 152119863

+ 008119860 lowast 119861 minus 014119860 lowast 119862 + 008119860 lowast 119863

+ 004119861 lowast 119862 minus 018119861 lowast 119863 minus 017119862 lowast 119863

minus 051119860

2minus 019119861

2minus 022119862

2minus 061119863

2

(2)

where 119884 is chitinase production (response) 119860 glucose 119861peptone 119862 chitin and119863 (NH

4)2SO4

The second-order response surface model of (2) waschecked by an 119865-test and the results by the analysis ofvariance (ANOVA) were given in Table 2The ANOVA of thequadratic regressionmodel demonstrated that themodel washighly significant due to its high 119865 value (119865 = 9319) and avery low probability value (0)The119865 value shows howwell thefactors describe the variation in the data about their meanThe greater the 119865 value was from unity the more certain itwas that the factors explain adequately the variation in thedata about their mean and the estimated factor effects werereal The 119875 values were employed to confirm the significanceof each coefficient Regression coefficients and significancewere determined by 119875 values which were summarized inTable 3 The fit goodness of the model can be checked by thedetermination coefficient 1198772 (09879) indicating that 9879of the variability in the response could be explained by themodel The value of the adjusted 1198772 (09773) was also veryhigh to advocate for a high significance of the model

The 3D response surface was the graphical repre-sentation of the regression equation using Minitab 160software The response surface curves were shown inFigure 1 to help visualize the effects of peptone glucosechitin and ammonium sulfate on chitinase activity whileeach figure demonstrated the effect of two factors FromFigure 1 it is evident that an increase in glucose chitin

Table 2 ANOVA of chitinase activity for the RSM parameters fittedto second-order polynomial equation

Source ofvariation DF SS MS 119865 value 119875 value

Model 14 51902 37073 9319 0119860

1 17323 17323 4354 0119861

1 425294 425294 106905 0119862

1 2581 2581 649 0022119863

1 55724 55724 14007 0119860 lowast 119860

1 5105 7303 1836 0001119861 lowast 119861

1 0362 1036 26 0126119862 lowast 119862

1 0658 1326 333 0087119863 lowast 119863

1 10522 10522 2645 0119860 lowast 119861

1 0092 0092 023 0638119860 lowast 119862

1 0311 0311 078 039119860 lowast 119863

1 0095 0095 024 0632119861 lowast 119862

1 0023 0023 006 0812119861 lowast 119863

1 0494 0494 124 0282119862 lowast 119863

1 0439 0439 11 0309Residual 16 6365 0398Lack of fit 10 5451 0545 358 0066Pure error 6 0914 0152Total 30 5253851198772= 09879 DF degrees of freedom SS sum of squares MS mean square

Adj 1198772 = 09773

Table 3 Test of significance for regression coefficient

Modelterm

Coefficientestimate

Standarderror 119905 value 119875 value

Intercept 132343 02384 55514 0119860 08496 01287 6599 0119861 42096 01287 32696 0119862 03279 01287 2547 0022119863 minus15237 01287 minus11835 0119860 lowast 119860 minus05053 01179 minus4284 0001119861 lowast 119861 minus01903 01179 minus1614 0126119862 lowast 119862 minus02153 01179 minus1826 0087119863 lowast 119863 minus06066 01179 minus5143 0119860 lowast 119861 00756 01577 048 0638119860 lowast 119862 minus01394 01577 minus0884 0390119860 lowast 119863 00769 01577 0488 0632119861 lowast 119862 00381 01577 0242 0812119861 lowast 119863 minus01756 01577 minus1114 0282119862 lowast 119863 minus01656 01577 minus105 0309

and ammonium sulfate concentrations caused enhancementin chitinase secretion followed by a decrease in its secretionFigure 1 also shows that maximum chitinase activity wasobtained at high concentrations of peptone In our studychitinase production was perhaps related to the high con-centrations of peptone The high peptone content would


lt50

50ndash7575ndash100100ndash125125ndash150

150ndash175175ndash200

Peptone (gL)

Glu

cose

(gL

)

10

8

6

4

2

1 2 3 4 5

gt200

(a)

lt50



Chitin (gL)

Pept

one (

gL)

5

4

3

2

1

6 8 10 12 14

gt200

(b)

lt50



Ammonium sulfate (gL)

Pept

one (

gL)

5

5

4

4

3

3

2

2

1

1

gt200

(c)

Figure 1 Response surface plot of chitinase production showing the interactive effects of the glucose and peptone concentrations (a) peptoneand chitin concentrations (b) and ammonium sulfate and peptone concentrations (c) keeping all other parameters constant

assure the availability of amino acids required for the syn-thesis of chitinase in general [18] Figure 1(c) shows theinteractive effect of peptone and (NH

4)2SO4concentrations

on chitinase production The chitinase activity increasedas (NH

4)2SO4increased from 1 to 132 gL but further

(NH4)2SO4content showed a declining trend for chitinase

production The medium containing peptone as organicnitrogen source led to the highest chitinase activity while(NH4)2SO4was inorganic nitrogen source for chitinase

production Because inorganic nitrogen sources were easilyutilized in early bacterial fermentation and organic nitro-gen sources could be used in the formation of metabolic


98

9494

9851

100

0005

Serratia marcescens (1E15 A)Serratia marcescens (1OGB A)Serratia marcescens (AFU067791)

Serratia marcescens (P117971)Serratia liquefaciens (AAN035971)

Serratia proteamaculans (YP 0014797021)Serratia odorifera (ZP 061912521)

Serratia plymuthica (ZP 101101031)

ChiBjp

Figure 2 Phylogenetic tree based on the amino acid sequence of ChiB from strain JPP1 and some other related taxa The numbers on thetree indicate the percentage of bootstrap based on 1000 replications

enzymes it preferably includes both organic nitrogen sourceand inorganic nitrogen source in the medium compo-nents

Because Serratia is a member of the Enterobacteriaceaeits direct use as a biocontrol agent would be limited due toits ability to cause disease in humans as an opportunisticpathogen For this reason it is important to find cheapand effective culture media that will permit the optimalproduction of chitinase on a medium scale The optimizedfactors for obtaining the highest level of chitinase productionwere 734 gL glucose 500 gL peptone 1270 gL chitin and132 gL ammonium sulfate In these conditions the chitinaseproduction was predicted to be 2309UmL for a 21-foldincrease

Polymerase chain reaction (PCR) amplification of thechitinase gene was performed and obtained a 1789 bpnucleotide sequence The sequence was submitted to NCBIGenBank database under the accession number KJ562867Similarity search of the nucleotide sequence was performedin the GenBank database using BLASTn The results showedthat the most similar 10 sequences all belonged to Smarcescens ChiB gene with the similarities in range of95ndash99 Nucleotide sequencing analysis revealed an openreading frame (ORF) consisting of 1500 nucleotides withATG as a start codon and TAA as a stop codon This ORFencoded a protein of 499 amino acids named as ChiBjpThe deduced protein sequence of ChiBjp was compared withentries in the GenBank database using BLASTp The N-terminal moiety of ChiBjp showed sequence homology withenzymes classified into family 18 of glycosyl hydrolases suchas S marcescens ChiB (WP 0169267611 sequence identity998 3WD0 A 998) and complex of S marcescens ChiA(1H0G A 988) The phylogenetic relationship of ChiBjpwith those of representative chitinase from the genus Serratiawas shown in Figure 2 After similarity alignment and phy-logenetic analysis the chitinase produced by strain JPP1 wasdetermined as ChiB ChiB is an exochitinase that degradeschitin chains from their nonreducing ends In addition tothe catalytic domain this enzyme has a small chitin-bindingdomain that extends the substrate-binding cleft towards thereducing end of the polysaccharide chain [30]

50 150 200 250 300 350 400100

Figure 3 Predicted secondary structure for ChiB from strain JPP1by GOR4 program (blue alpha helix red extended strand purplerandom coil)

Using Protparam program the predicted molecularweight and the isoelectric point of ChiBjp were 554803Daand 593 respectively It was a fat-soluble protein relativelystable and hydrophilic with formula C

2508H3766

N662

O738

S15

The predicted secondary structure of ChiBjp was 3166alpha helix 2064 extended strand and 4770 random coilusing GOR4 program as shown in Figure 3 The catalyticdomains of family 18 chitinases have a (120573120572)

8barrel fold The

120573-strand four of the barrel contains a characteristic DXDXEsequence motif that includes the glutamate residue whichprotonates that oxygen in the scissile glycosidic bond [31] Itspredicted tertiary structure exhibited similarity of 986withthe template by Geno3d program [32] as shown in Figure 4The template was complex of ChiB (1H0G) from wild typeS marcescens with the natural product cyclopentapeptideargadin from Clonostachys [33]

4 Conclusion

The optimization of medium composition with increasedproduction of chitinase from Serratia marcescens JPP1 wascarried out using two statistical experimental methodsincluding Plackett-Burman design and central compositedesign Maximum chitinase production was predicted tobe 2309UmL for a 21-fold increase in medium con-taining 1270 gL colloidal chitin 734 gL glucose 500 gLpeptone 132 gL (NH

4)2SO4 07 gL K

2HPO4 and 05 gL

MgSO4sdot7H2O The results suggested that statistical experi-

mental designs provided an efficient and economical methodin optimizing chitinase production for biocontrol of afla-toxins Similarity alignment and phylogenetic analysis ofthe nucleotide sequence and deduced amino acid sequencedetermined that the chitinase produced by strain JPP1 was


(a) (b)

Figure 4 Predicted tertiary structure for ChiB from strain JPP1 by Geno3d program ((a) predicted tertiary structure (b) template)

ChiB The molecular properties of the chitinase includingpredicted secondary and tertiary structure were also deter-mined

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (Grant nos 30870003 and 21273056)and COMRA Project (no DY125-15-R-01) and Project (HIT-NSRIF200806) supported by Natural Scientific ResearchInnovation Foundation in Harbin Institute of Technology

References

[1] V B Bansode and S S Bajekal ldquoCharacterization of chitinasesfrommicroorganisms isolated from Lonar lakerdquo Indian Journalof Biotechnology vol 5 no 3 pp 357ndash363 2006

[2] M N V Ravi Kumar ldquoA review of chitin and chitosan applica-tionsrdquo Reactive and Functional Polymers vol 46 no 1 pp 1ndash272000

[3] N Rattanakit A Plikomol S Yano M Wakayama and TTachiki ldquoUtilization of shrimp shellfish waste as a substratefor solid-state cultivation of Aspergillus sp S1-13 evaluation ofa culture based on chitinase formation which is necessary forchitin-assimilationrdquo Journal of Bioscience and Bioengineeringvol 93 no 6 pp 550ndash556 2002

[4] V Gohel V Maisuria and H S Chhatpar ldquoUtilization of vari-ous chitinous sources for production of mycolytic enzymesby Pantoea dispersa in bench-top fermenterrdquo Enzyme andMicrobial Technology vol 40 no 6 pp 1608ndash1614 2007

[5] R Pichyangkura S Kudan K Kuttiyawong M Sukwattanasi-nitt and S-I Aiba ldquoQuantitative production of 2-acetamido-2-deoxy-D-glucose from crystalline chitin by bacterial chitinaserdquoCarbohydrate Research vol 337 no 6 pp 557ndash559 2002

[6] V B Maisuria V Gohel A N Mehta R R Patel and H SChhatpar ldquoBiological control of Fusarium wilt of pigeonpea byPantoea dispersa a field assessmentrdquo Annals of Microbiologyvol 58 no 3 pp 411ndash419 2008

[7] N Mathivanan V Kabilan and K Murugesan ldquoPurificationcharacterization and antifungal activity of chitinase fromFusarium chlamydosporum a mycoparasite to groundnut rustPuccinia arachidisrdquo Canadian Journal of Microbiology vol 44no 7 pp 646ndash651 1998

[8] A S De Pinto C C Barreto A Schrank and H V MarileneldquoPurification and characterization of an extracellular chitinasefrom the entomopathogen Metarhizium anisopliaerdquo CanadianJournal of Microbiology vol 43 no 4 pp 322ndash327 1997

[9] E S Mendonsa P H Vartak J V Rao and M V DeshpandeldquoAn enzyme fromMyrothecium verrucaria that degrades insectcuticles for biocontrol ofAedes aegyptimosquitordquoBiotechnologyLetters vol 18 no 4 pp 373ndash376 1996

[10] C-J Huang and C-Y Chen ldquoSynergistic interactions betweenchitinase ChiCW and fungicides against plant fungal patho-gensrdquo Journal of Microbiology and Biotechnology vol 18 no 4pp 784ndash787 2008

[11] B Bhushan andG SHoondal ldquoEffect of fungicides insecticidesand allosamidin on a thermostable chitinase from Bacillus spBG-11rdquo World Journal of Microbiology and Biotechnology vol15 no 3 pp 403ndash404 1999

[12] K M Ghanem S M Al-Garni and N H Al-Makishah ldquoSta-tistical optimization of cultural conditions for chitinase pro-duction from fish scales waste by Aspergillus terreusrdquo AfricanJournal of Biotechnology vol 9 no 32 pp 5135ndash5146 2010

[13] K M Ghanem F A Al-Fassi and R M Farsi ldquoStatistical opti-mization of cultural conditions for chitinase production fromshrimp shellfish waste by Alternaria alternatardquo African Journalof Microbiology Research vol 5 no 13 pp 1649ndash1659 2011


[14] M A Faramarzi M Fazeli M T Yazdi et al ldquoOptimization ofcultural conditions for production of chitinase by a soil isolateofMassilia timonaerdquoBiotechnology vol 8 no 1 pp 93ndash99 2009

[15] M F Kern S D F Maraschin D Vom Endt A SchrankM H Vainstein and G Pasquali ldquoExpression of a chitinasegene from Metarhizium anisopliae in tobacco plants confersresistance against Rhizoctonia solanirdquo Applied Biochemistry andBiotechnology vol 160 no 7 pp 1933ndash1946 2010

[16] P Patidar D Agrawal T Banerjee and S Patil ldquoOptimisationof process parameters for chitinase production by soil isolatesof Penicillium chrysogenum under solid substrate fermentationrdquoProcess Biochemistry vol 40 no 9 pp 2962ndash2967 2005

[17] H Yamazaki A Tanaka J-I Kaneko A Ohta andHHoriuchildquoAspergillus nidulans ChiA is a glycosylphosphatidylinositol(GPI)-anchored chitinase specifically localized at polarizedgrowth sitesrdquo Fungal Genetics and Biology vol 45 no 6 pp963ndash972 2008

[18] M I Gutierrez-Roman F Holguın-Melendez R Bello-Mendo-za K Guillen-Navarro M F Dunn and G Huerta-PalaciosldquoProduction of prodigiosin and chitinases by tropical Serratiamarcescens strains with potential to control plant pathogensrdquoWorld Journal of Microbiology and Biotechnology vol 28 pp145ndash153 2012

[19] S-L Lee and W-C Chen ldquoOptimization of medium compo-sition for the production of glucosyltransferase by Aspergillusniger with response surface methodologyrdquo Enzyme and Micro-bial Technology vol 21 no 6 pp 436ndash440 1997

[20] Y R Abdel-Fattah E R El-Helow K M Ghanem and WA Lotfy ldquoApplication of factorial designs for optimization ofavicelase production by a thermophilic Geobacillus isolaterdquoResearch Journal of Microbiology vol 2 no 1 pp 13ndash23 2007

[21] A Q M Al-Sarrani and M Y M El-Naggar ldquoApplication ofPlackett-Burman factorial design to improve citrinin produc-tion in Monascus ruber batch culturesrdquo Botanical Studies vol47 no 2 pp 167ndash174 2006

[22] M Y El-Naggar S A El-Aassar A S Youssef N A El-Sersyand E A Beltagy ldquoExtracellular 120573-mannanase production bythe immobilization of the locally isolated Aspergillus nigerrdquoInternational Journal of Agriculture and Biology vol 8 pp 57ndash62 2006

[23] K J Al Ahmadi M T Yazdi M F Najafi et al ldquoOptimizationof medium and cultivation conditions for chitinase productionby the newly isolated Aeromonas sprdquo Biotechnology vol 7 no2 pp 266ndash272 2008

[24] N N Nawani and B P Kapadnis ldquoOptimization of chitinaseproduction using statistics based experimental designsrdquo ProcessBiochemistry vol 40 no 2 pp 651ndash660 2005

[25] K Wang P S Yan L X Cao Q L Ding C Shao and T FZhao ldquoPotential of chitinolytic Serratia marcescens strain JPP1for biological control of Aspergillus parasiticus and aflatoxinrdquoBioMed Research International vol 2013 Article ID 397142 7pages 2013

[26] W K Roberts and C P Selitrennikoff ldquoPlant and bacterialchitinases differ in antifungal activityrdquo Journal of GeneralMicrobiology vol 61 pp 1720ndash1726 1988

[27] J Monreal and E T Reese ldquoThe chitinase of Serratia marces-censrdquo Canadian Journal of Microbiology vol 15 no 7 pp 689ndash696 1969

[28] S J Horn M Soslashrlie G Vaaje-Kolstad et al ldquoComparativestudies of chitinases A B and C from Serratia marcescensrdquoBiocatalysis and Biotransformation vol 24 no 1-2 pp 39ndash532006

[29] KWang P S Yan and L X Cao ldquoOptimization of nutrients forchitinase production by Serratia marcescens JPP1 against afla-toxin using statistical experimental designrdquo Journal of Chemicaland Pharmaceutical Research vol 5 no 12 pp 447ndash449 2013

[30] DM F Van Aalten B SynstadM B Brurberg et al ldquoStructureof a two-domain chitotriosidase from Serratiamarcescens at 19-A resolutionrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 97 no 11 pp 5842ndash5847 2000

[31] D M F Van Aalten D Komander B Synstad S GaseidnesM G Peter and V G H Eijsink ldquoStructural insights into thecatalyticmechanism of a family 18 exo-chitinaserdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 98 no 16 pp 8979ndash8984 2001

[32] A Sahay andM Shakya ldquoIn silico analysis and homologymod-elling of antioxidant proteins of spinachrdquo Journal of Proteomicsand Bioinformatics vol 3 no 5 pp 148ndash154 2010

[33] D R Houston K Shiomi N Arai et al ldquoHigh-resolutionstructures of a chitinase complexed with natural productcyclopentapeptide inhibitors mimicry of carbohydrate sub-straterdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 99 no 14 pp 9127ndash9132 2002

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


and response surface methodology are the most widely usedstatistical approaches for reducing the time and expenseCentral composite design (CCD) was used to determine lev-els of various parameters with the interrelation between eachparameter evolved simultaneously [19] Studies on mediumoptimization for chitinases production are the worthwhiletechnique for multifactor experiments because they coulddetect the true optimum of the factor [20ndash22] In additionmedium composition greatly influenced the microbial pro-duction of extracellular chitinase and its interaction plays animportant role in the synthesis of the chitinases [23 24] Theobjective of the present work was to characterize the mediumof S marcescens JPP1 for maximum antagonistic effect onaflatoxin in favor of the chitinase production using statisticaldesigns of Plackett-Burman and central composite design ofresponse surface methodology The molecular properties ofthe extracellular chitinase were also determined

2 Materials and Methods

21 Bacterial Strain Thestrain used in this study was isolatedfrom the peanut hulls collected from the sampling site inHuaian city Jiangsu Province China Itwas identified asSerratia marcescens JPP1 based on its morphological andphysiological characteristics and 16S rRNA gene sequenceanalysis The nucleotide sequence of 16S rRNA gene wassubmitted to NCBI GenBank database under the accessionnumber JQ308601 [25]

22 Media Composition PGY medium peanut hulls weredried at 40∘C and then ground The ground peanut hullswere boiled with water for 1 h at the final concentration of25 and then centrifuged at 6600 g at room temperature for5min The supernatant was supplemented with 2 glucoseand 05 yeast extract and then autoclaved for 20min at121∘C pH in nature S marcescens JPP1 was maintained onsolid PGY medium The stocks were kept in the refrigeratorand subcultured at monthly intervals

23 Preparation of Colloidal Chitin Colloidal chitin wasprepared from pure chitin (Sangon Biotech China) by themethod of Roberts and Selitrennikoff [26] Commercialchitin (40 g) was weighed and taken in a beaker 500mL ofconcentrated hydrochloric acid was added followed by con-tinuous stirring at 4∘C After stirring for 1 h the hydrolyzedchitin in the beaker was washed several times with distilledwater to remove the acid completely and hence bring the pHinto the range of 6-7 Once this pHwas obtained the colloidalchitin was filtered using Whatman filter paper The filteredcolloidal chitin was then collected and stored in the form of apaste at 4∘C

24 Chitinase Activity Assay Chitinase activity was testedaccording to the method of Monreal and Reese detectingN-acetylglucosamine (NAG) as the final product [27] Thereaction mixture for the chitinase assay contained 1mL of5 acid swollen chitin 1mL of 50mM acetate buffer (pH50) and 1mL of enzyme solution The reaction mixture

was incubated at 50∘C for 1 h and then the reaction wasstopped after boiling for 15minThemixture was centrifugedat 5000 rpm for 20min and the concentration of releasedNAG was assayed at 530 nm spectrophotometrically withcolloidal chitin as substrate One unit of chitinase activity wasdefined as the amount of enzyme that catalyzed the release of1 120583mol of NAG per hour at 50∘C Data is expressed as mean plusmnSD of three experiments

25 Design of Experiment The optimization of medium con-stituents to improve chitinase activity of S marcescens JPP1was carried out in two stages Plackett-Burman and responsesurface methodology Firstly eight variables including threebest carbon sources three best nitrogen sources magnesiumsulfate and potassium hydrogen phosphate anhydrous wereselected on the basis of their role in chitinase secretionenhancementThe variables having themost significant effecton chitinase activity were identified using a 2-level Plackett-Burman design

Secondly response surface methodology was used tooptimize the screened components to enhance chitinaseactivity using central composite design A 24 full factorialCCD of RSM was employed to optimize the four mostsignificant factors (glucose peptone ammonium sulfate andchitin) for enhancing chitinase activity The concentrationsof 4 variables were previously investigated for chitinaseactivity using single-factor experiments (data not shown)In this study the experimental plan consisted of 31 trialsand the independent variables were studied (Table 1) All theexperiments were done in triplicate and the average chitinaseactivity was taken as the dependent variables or responsesThe data obtained from CCD on chitinase production weresubjected to analysis of variance (ANOVA) Then a second-order polynomial equation was fitted to the data by multipleregression procedure This resulted in an empirical modelthat related the response measured in the independentvariables to the experiment The behavior of the system wasexplained by the following quadratic equation

119884 = 120573

0+sum120573

119894119909

119894+sum120573

119894119895119909

119894119909

119895+sum120573

119894119894119909

119894

2 (1)

where 119884 is predicted response 119909119894and 119909

119895are the input

variables 1205730is the intercept term 120573

119894is the linear effects 120573

119894119894is

the squared effects and 120573119894119895is the interaction term

The statistical software package Minitab 160 (MinitabInc State College PA) was used to analyze the experimentaldesign The response obtained was statistically evaluated andthe model was built based on the variables with confidencelevels more than 95

26 Nucleotide Sequence of Chitinase Gene Genomic DNAfrom strain S marcescens JPP1 was extracted using a bacterialgenomic DNA FastPrep Extraction Kit (Sangon BiotechChina) The oligonucleotides designed using Primer Premier50 on the basis of the published sequence of chitinase (ChiB)gene [28] were synthesized and used for determination ofthe chitinase gene sequence The upstream primer (51015840-CC-AAGACAGGCGGCAGTAAATAAAA-31015840) and downstream
















119884 = 1323 + 085119860 + 421119861 + 033119862 minus 152119863



minus 051119860

2minus 019119861

2minus 022119862

2minus 061119863

2

(2)


4)2SO4





Model 14 51902 37073 9319 0119860

1 17323 17323 4354 0119861

1 425294 425294 106905 0119862

1 2581 2581 649 0022119863

1 55724 55724 14007 0119860 lowast 119860

1 5105 7303 1836 0001119861 lowast 119861

1 0362 1036 26 0126119862 lowast 119862

1 0658 1326 333 0087119863 lowast 119863

1 10522 10522 2645 0119860 lowast 119861

1 0092 0092 023 0638119860 lowast 119862

1 0311 0311 078 039119860 lowast 119863

1 0095 0095 024 0632119861 lowast 119862

1 0023 0023 006 0812119861 lowast 119863

1 0494 0494 124 0282119862 lowast 119863


Adj 1198772 = 09773


Modelterm

Coefficientestimate





lt50



Peptone (gL)

Glu

cose

(gL

)

10

8

6

4

2

1 2 3 4 5

gt200

(a)

lt50



Chitin (gL)

Pept

one (

gL)

5

4

3

2

1

6 8 10 12 14

gt200

(b)

lt50




Pept

one (

gL)

5

5

4

4

3

3

2

2

1

1

gt200

(c)










98

9494

9851

100

0005





ChiBjp





50 150 200 250 300 350 400100



2508H3766

N662

O738

S15


8barrel fold The


4 Conclusion


4)2SO4 07 gL K

2HPO4 and 05 gL




(a) (b)





Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















119884 = 1323 + 085119860 + 421119861 + 033119862 minus 152119863



minus 051119860

2minus 019119861

2minus 022119862

2minus 061119863

2

(2)


4)2SO4





Model 14 51902 37073 9319 0119860

1 17323 17323 4354 0119861

1 425294 425294 106905 0119862

1 2581 2581 649 0022119863

1 55724 55724 14007 0119860 lowast 119860

1 5105 7303 1836 0001119861 lowast 119861

1 0362 1036 26 0126119862 lowast 119862

1 0658 1326 333 0087119863 lowast 119863

1 10522 10522 2645 0119860 lowast 119861

1 0092 0092 023 0638119860 lowast 119862

1 0311 0311 078 039119860 lowast 119863

1 0095 0095 024 0632119861 lowast 119862

1 0023 0023 006 0812119861 lowast 119863

1 0494 0494 124 0282119862 lowast 119863


Adj 1198772 = 09773


Modelterm

Coefficientestimate





lt50



Peptone (gL)

Glu

cose

(gL

)

10

8

6

4

2

1 2 3 4 5

gt200

(a)

lt50



Chitin (gL)

Pept

one (

gL)

5

4

3

2

1

6 8 10 12 14

gt200

(b)

lt50




Pept

one (

gL)

5

5

4

4

3

3

2

2

1

1

gt200

(c)










98

9494

9851

100

0005





ChiBjp





50 150 200 250 300 350 400100



2508H3766

N662

O738

S15


8barrel fold The


4 Conclusion


4)2SO4 07 gL K

2HPO4 and 05 gL




(a) (b)





Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






119884 = 1323 + 085119860 + 421119861 + 033119862 minus 152119863



minus 051119860

2minus 019119861

2minus 022119862

2minus 061119863

2

(2)


4)2SO4





Model 14 51902 37073 9319 0119860

1 17323 17323 4354 0119861

1 425294 425294 106905 0119862

1 2581 2581 649 0022119863

1 55724 55724 14007 0119860 lowast 119860

1 5105 7303 1836 0001119861 lowast 119861

1 0362 1036 26 0126119862 lowast 119862

1 0658 1326 333 0087119863 lowast 119863

1 10522 10522 2645 0119860 lowast 119861

1 0092 0092 023 0638119860 lowast 119862

1 0311 0311 078 039119860 lowast 119863

1 0095 0095 024 0632119861 lowast 119862

1 0023 0023 006 0812119861 lowast 119863

1 0494 0494 124 0282119862 lowast 119863


Adj 1198772 = 09773


Modelterm

Coefficientestimate





lt50



Peptone (gL)

Glu

cose

(gL

)

10

8

6

4

2

1 2 3 4 5

gt200

(a)

lt50



Chitin (gL)

Pept

one (

gL)

5

4

3

2

1

6 8 10 12 14

gt200

(b)

lt50




Pept

one (

gL)

5

5

4

4

3

3

2

2

1

1

gt200

(c)










98

9494

9851

100

0005





ChiBjp





50 150 200 250 300 350 400100



2508H3766

N662

O738

S15


8barrel fold The


4 Conclusion


4)2SO4 07 gL K

2HPO4 and 05 gL




(a) (b)





Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


lt50



Peptone (gL)

Glu

cose

(gL

)

10

8

6

4

2

1 2 3 4 5

gt200

(a)

lt50



Chitin (gL)

Pept

one (

gL)

5

4

3

2

1

6 8 10 12 14

gt200

(b)

lt50




Pept

one (

gL)

5

5

4

4

3

3

2

2

1

1

gt200

(c)










98

9494

9851

100

0005





ChiBjp





50 150 200 250 300 350 400100



2508H3766

N662

O738

S15


8barrel fold The


4 Conclusion


4)2SO4 07 gL K

2HPO4 and 05 gL




(a) (b)





Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


98

9494

9851

100

0005





ChiBjp





50 150 200 250 300 350 400100



2508H3766

N662

O738

S15


8barrel fold The


4 Conclusion


4)2SO4 07 gL K

2HPO4 and 05 gL




(a) (b)





Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b)





Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Chitinase from a Novel Strain of Serratia marcescens JPP1 for