172 J SCI IND RES VOL 72 MARCH 2013Journal of Scientific & Industrial ResearchVol. 72, March 2013, pp. 172-178

*Author for correspondenceE-mail: sk2410@yahoo.co.uk

Optimization of mycolytic enzymes (Chitinase, β1,3-Glucanase andCellulase) production by Bacillus subtilis, a potential biocontrol agent

using one-factor approach

Ashwini Narasimhan, Deepak Bist, Samantha Suresh & Srividya Shivakumar*

Department of Microbiology, Centre for PG Studies, Jain University, 9th Main, Jayanagar 3rd Block, Bangalore, India.

Received : 27 June2012; revised:28 October 2012 ; accepted:02 January 2013

This study presents one-factor approach for optimization of growth and physical parameters for increasing production ofthree mycolytic enzymes viz. chitinase, β-1,3-glucanase and β1,4-glucanase (cellulase) by B.subtilis, a potent biocontrol agent.Maximum enzyme activity of all three enzymes was observed in the following physical parameters: pH 7.0, temperature of 30°C,and incubation period of 72h. Optimum nutritional parameters were found to be as follows: Carboxymethyl cellulose (CMC, 10g/L);Corn steep liquor (CSL, 1g/L) or KNO3 (1 g/L), CaCl2 (5mM) and Triton X 100 (0.1% v/v). Using one factor approach, productionof all three enzymes significantly increased for chitinase (5.49 folds); â-1,3-glucanase (7.39 folds) and cellulase (1.91 folds)respectively as compared to the un-optimized media containing Nutrient broth. The crude mycolytic enzymes had activity inacidic pH (4 - 6) and temperature (50 - 60°C) range with optima at pH 6.0 and 55°C, respectively. However, chitinase showed twopH optima at pH 6 and 11 indicating the possibility of having two isoforms. The enzymes showed varied response to metal ions.Identification of essential nutrients affecting co-production of three enzymes by a single strain would help to formulate a suitablemedium for their concerted production. Moreover, low cost cellulosic materials can be used as carbon source for these enzymesproduction by the isolate for industrial and agricultural applications.

Keywords: mycolytic enzymes, biocontrol agent, B subtilis, one factor approach.

IntroductionChitin and β-1,3-glucan are the main structural

components of fungal cells walls, except those frommembers of the class Oomycetes, which contain β -1,3-glucan and cellulose1 β -Glucanases, produced byseveral fungi and bacteria are one of the most potentenzymes for degrading fungal cell walls2. Lytic enzymes(chitinase, β-1,3-glucanase, protease) are responsible forthe lysis and hyperparasitism of antagonists againstdeleterious fungal pathogens. In these mechanisms, chitin,β-1,3-glucan and protein components of the fungal cellwall are digested by these extracellular enzymes3. Theability of B. subtilis to suppress a wide range of plantpathogens and also its reputation as a safe and friendlymicroorganism is attractive. Many isolates of B. subtilisdisplay antagonistic activity against a number of pre-harvest and post-harvest pathogens on several differentcrops6,7. They offer a potential additive or alternative touse of chemical fungicides8,9. However, such applicationsrequire these enzymes to be produced in large quantities

which in turn require optimization of nutritive and physicalparameters like pH and temperature for its productionby selected isolate.

Several statistical and non statistical methods areavailable for optimization of medium constituents10 .Plackett-Burman and Response Surface Methodology(RSM) are the most widely used statistical approaches.Although optimizing the parameters by statistical methodreduces the time and expense, selection of mediacomponents for use in Plackett Burman design is eitherdecided by borrowing or by random selection. Borrowinginvolves literature survey to select components used byother workers for growth and production of desiredproduct from same genus being analysed. The problemwith this method is that there are not too many options ifthe organism under analysis has not been previouslystudied for production of desired product11. Thus, beforestatistical optimization of medium for production ofdesired product from a new source bacterium it isessential to screen large number of possible mediumconstituents. Component replacing is the most commonlyused method for screening number of carbon, nitrogenand phosphorous sources12 while the effect of surfactants,

173SHIVAKUMAR et al: OPTIMIZATION OF MYCOLYTIC ENZYMES BY BACILLUS SUBTILIS

metal ion, antibiotics etc is checked by one factor at atime approach13. This approach can generate informationon medium constituents for desired product fromorganism under study and can also identify newcomponents affecting its production. It is assumed thatno single study has attempted to optimize production ofmore than one mycolytic enzyme from a single strain.Previous study has identified B.subtilis JN032305, isolatedfrom chilli rhizosphere soils sampled from Hesaraghattaand other regions in the outskirts of Bangalore, to havemycolytic enzyme mediated antagonism towards chilliphytopathogens14,15. The present study focuses onimprovement of growth and cultivation conditions ofB.subtilis, a potential broad spectrum biocontrol agentfor increasing mycolytic enzyme production by this strain.

Experimental sectionOrganism and culture conditions

The mycolytic enzymes producing bacteria wasisolated from the Chilli rhizosphere. It was identified asBacillus subtilis by morphological, biochemical and16S rDNA sequence analysis. The Gene-Bank accessionno. for the nucleotide sequence is JN032305.

Enzyme assayThe culture was grown at 30oC with 120 rpm for 24 h.

200µL of the culture inoculum was transferred to 250mLErlenmeyer flask containing 100mL of broth medium with(g/L): CMC, 10; peptone, 5; Beef extract, 3 and NaCl, 5.The pH of the medium was adjusted to 7.0 using 1NNaOH before autoclaving. All the experiments wereperformed in duplicate. The culture broth after 72 h ofincubation was centrifuged at 10,000 g for 10min at 4° Cto separate the cells. The cell-free supernatant wasanalyzed for enzyme activity16.

Chitinase assayChitinase activity was assayed following the release

of glucose according to the method17. 3 ml of reactionmixture containing 1 ml of 1 % colloidal chitin in 0.1 Mphosphate buffer (pH 6.5) was incubated with 1.0 ml ofculture filtrate at 37 °C for 45 minutes. One unit ofenzyme activity is defined as the amount of enzymerequired to produce 1 µg of glucose per min. Specificactivity was expressed as units/mg protein.

β -1, 3 and β -1,4 Glucanase assayThe specific activity of β-1, 3 and β-1,4 Glucanase

was determined by measuring the amount of reducing

sugars liberated using dinitrosalicylic acid solution(DNS)16. The culture broth was centrifuged and enzymesolution 1 ml was added to, 1.0 ml of substrate solutionwhich contained Yeast Cell Wall Extract(YCW, 1%, v/v)18 for β-1,3 Glucanase and 1 ml ofCarboxy Methyl Cellulose solution (CMC, 1%, v/v) forβ-1,4 Glucanase. The mixture was incubated in a waterbath at 50°C for 30 min and the reaction was terminatedby adding 1ml of DNS solution and incubated in boilingwater bath for 10 – 15 min till the development of thecolour of the end product. Reducing sugar concentrationwas determined by optical density at 540 nm19.

Screening of essential medium componentsNutrient broth media including 12 carbon sources, 8

nitrogen sources, 5 surfactants and 9 different metal ionswere screened for their effect on chitinase, β-1,3glucanase and cellulase production by Bacillus subtilis.Effect of different carbon sources was checked bysupplementing basal media (NB) with different carbonsources (equivalent weight) or 5 g/l of different testsugars or 1 g/l of pure sugars (with/without puresubstrates-CMC, chitin & YCW, respectively). Nitrogenwas screened by replacing the corresponding source inthe basal medium in equimolar concentrations(1.0 g/l for peptone) with respect to N. Surfactants andmetal ions were screened by supplementing the mediawith the test component. The concentration of surfactantsused while screening was 0.1% while the concentrationof metal ions used were 5mM for macronutrients and1mM for micronutrients.

ReproducibilityAll the experiments were done in triplicates and the

values represented statistically are in ANOVA form.

Results and discussionIsolation and screening of chitinase producing bacteria

15 chilli rhizosphere soils samples were collectedfrom in and around Bangalore, heat treated and platedon chitin amended plates. Out of 15 samples plated, 9chitinolytic colonies were isolated based on the clearancezones that they formed. The isolate 2 which showedinhibition of all the nine pathogens tested was chosen forfurther work. The selected isolate showed broadspectrum antagonism against Alternaria (3) spp. (55%),Colletotrichum gloeosporioides (57%), Phytophthoracapsici (55%), Rhizoctonia solani (42%), Fusariumsolani (42%), Fusarium oxysporum (40%) and

174 J SCI IND RES VOL 72 MARCH 2013

Verticillium sp. (36%), the range of percentage inhibitionvaried from 40-62. The isolate was identified as Bacillussubtilis by 16S rDNA sequence analysis the NCBIGeneBank accession no. for which was given asJN032305. The isolate produced 3 mycolytic enzymes-chitinase, ß-1,3- glucanase and cellulase.

Effect of carbon sources on mycolytic enzyme productionIt was reported that medium composition is one of

the main factors that enhance chitinase and cellulaseproduction by microorganisms20. Glucose supplementedwith chitin as well as CMC alone was identified as thebest carbon source among all the carbon sources checked(Fig. 1) yielding increase in chitinase by 4.15 folds;glucanase by 6.28 folds and cellulase by 1.95 folds.Addition of simple sugars like glucose, lactose, maltose,and sucrose in the medium as sole carbon sourcesuppressed the activity of all 3 enzymes drastically21-23.Similar observation has been reported for Thermococcuschitonophagus for chitinase. However, incorporationof glucose along with chitin enhanced chitinase andcellulase enzyme production as compared to control asreported by Bhushan & Hoondal (1998)8. Colloidal chitinwas found to induce chitinase production byThermococcus chitonophagus24. It has been reportedthat CMC shows inducing effect on cellulase production.These results are in agreement with other reports25-28.

Effect of Nitrogen sources on mycolytic enzyme productionAmong various organic and inorganic sources tested

for mycolytic enzyme production by Bacillus subtilis,CSL and KNO3 were identified as the best nitrogensource (Fig.2). Supplementation of CSL and KNO3(1.0 g/l) in the media increased chitinase production by4.87 and 6.09 folds, respectively; glucanase production

by 3.51 folds with CSL; cellulase production by 1.85 and1.26 folds respectively. Ray et al. (2007)29 reported thatorganic nitrogen sources were found to be more suitablefor optimizing cellulase production by Bacillus subtilisand Bacillus circulans than inorganic sources. Urea wasfound to be the suitable nitrogen source for chitinaseproduction by Paenibacillus sp. D1. Urea has also beenreported as an important constituent for chitinaseproduction by Pantoea dispersa30. Several studies haveidentified yeast extract, peptone, ammonium sulfate etc.to have significantly increased chitinase and/or cellulaseproduction by Paenibacillus sp.D1, Paenibacillussabina strain JD2, Geobacillus sp.31-33. In this study,both organic and inorganic nitrogen sources enhancedmycolytic enzyme production by Bacillus subtilis.

Effect of surfactants and metal ions on mycolytic enzymeproduction

Effect of various surfactants and metal ions testedis summarized in Fig. 3 and 4. The enzyme production

Fig.1Effect of different carbon sources on mycolytic enzymeproduction of B.subtilis

Fig. 2Effect of different nitrogen sources (organic and inorganic)on mycolytic enzyme production of B.subtilis

Fig.3Effect of metal ions on mycolytic enzyme production ofB.subtilis

175SHIVAKUMAR et al: OPTIMIZATION OF MYCOLYTIC ENZYMES BY BACILLUS SUBTILIS

showed varied response to the supplementation ofdifferent metal ions. Addition of CaCl2 and CoCl2enhanced chitinase production by 3.74 and 3.47 foldsrespectively and was repressed by MgCl2, CuSO4, HgCl2and FeSO 4; glucanase production was positivelyinfluenced by CoCl2, FeSO4 and CaCl2 with 6.06, 8.19and 2.13 fold increase respectively and repressed byMgCl2, CuSO 4 , ZnSO 4 and FeSO 4; and cellulaseproduction was increased by addition of FeSO4 (1.66),CuSO4 (1.53) and MgSO4 (1.25) folds respectively andrepressed by ZnSO4. Overall, addition of CoCl2 andCaCl2 enhanced the production of all the three mycolyticenzymes by B.subtilis.Effect of metal ions on chitinaseproduction by bacteria has not been studied in detail.Chitinase production by Paenibacillus sp. D1 wasenhanced by FeCl3 addition34. Addition of FeCl3.6H2Ohighly induced chitinase production by Bacillusamyloliquefaciens22. CaCl2 was an important mediaconstituent for chitinase production by Pantoeadispersa30, while it affected chitinase production inPaenibacillus sabina31.

Detergents Triton X100, CTAB, Tween 20 andTween 80 had positive effect on chitinase production,while SDS had an inhibitory effect, TritonX100,Tween 20 and Tween 80 positively influenced glucanaseproduction while CTAB and SDS had slight inhibitoryeffect. Cellulase production was positively influencedonly by Triton X100 addition while all other detergentsnegatively affected the production. Triton X100 wasidentified as best surfactant supplement with an increasein chitinase production (5.49 folds); glucanase (7.39 folds)and cellulase (1.91 folds) respectively. Surfactants areknown to alter the porosity of cell membrane resulting inleakage of enzyme into the external milieu. Addition of

surfactant in the medium can, therefore, improve theenzyme production. Our observations are in accordancewith other similar studies20,35.

Effect of pH and temperature on mycolytic enzyme productionMaximum production of all three enzymes by

B.subtilis was observed when initial pH of the mediumwas set at 7.0 at 30°C (Fig. 5 & 6). Chitinase productionwas seen in the range from pH 4-7 and considerablydropped at alkaline range. Glucanase production wasobserved in the range pH 4-9 with increase in productionat the alkaline range. Cellulase production was alsoobserved in the pH range of 4-7 with optimum productionat pH 7. The pH of the culture medium plays an importantrole in chitinase production. Majority of the bacteria arereported to produce maximum level of chitinase at neutralor slightly acidic pH and whereas fungi mostly secret itin acidic conditions36-39. In contrast, B. laterosporusMML2270 produced highest chitinase at pH 8.0 andinterestingly it failed to produce chitinase at pH 4.0. Similar

Fig. 4Effect of surfactants on mycolytic enzyme production ofB.subtilis

Fig. 5Effect of medium pH on mycolytic enzyme production ofB.subtilis

Fig. 6Effect of incubation temperature on mycolytic enzymeproduction of B.subtilis

176 J SCI IND RES VOL 72 MARCH 2013

optimum pH of 8.0 for chitinase production was reportedin B. pabuli K140. B. laterosporus produced highchitinase activity at 35oC. Paenibacillus sp. D1 isolateexhibited chitinase production over a wide temperature(25 - 45°C) and pH (6 - 9) range34. In contrast to ourobservation, Clostridium acetobutylicum showedoptimal cellulase production at pH 9 41 . Cellulaseproduction has also been reported at acidic 42 and neutralpH43,44. Comparison of process parameters showed thatusing one factor approach all three enzyme activitiesincreased as follows- chitinase (5.49 folds); β-1,3-glucanase (7.39 folds) and cellulase (1.91 folds)respectively (Fig. 6a).

Statistical analysisANOVA (One-way) was performed for each of the

media components affecting the production of the threemycolytic enzymes by B.subtilis using the one-factorapproach. P- value was found to be significant for carbonsources, nitrogen sources, metal ions, surfactants, pHand temperature (P≤ 0.05 level of significance) indicatingsignificant influence of these varying factors on enzymeproduction.

Assay optimizationEffect of assay pH, temperature and metal ions on mycolyticenzyme activity of B.subtilis

Chitinase of B.subtilis showed two pH optima atpH 6.0 and pH 11 indicating the presence of twoisoforms. Glucanase was active from pH 4-6 and showeda pH optima at pH 6. Cellulase was active from pH 4-6and showed optima at pH 5. Several workers havereported broad range of pH and temperature optima forchitinase, from 4.5 to 7.5 for Bacillus cereus chitinase45,

pH 5.0 to 8.0 for Aeromonas hydrophila H-233046, pH7.5 to 9.0 for Bacillus sp. BG-118. Bacillus chitinasesalso work better at an acidic or near neutral pH47. Allthree enzymes showed temperature optima of 55°C.According to some previous studies, the optimal pH was5.0-6.5 for those from Bacillus strains48. The optimumtemperature of purified cellulase was lower than someof other Bacillus strains [65°C (CH43) and 70°C(RH68)]49, but similar to Mucor circinelloides (55°C)50.None of the enzymes showed requirement of any specificmetal ions for their activity though FeSO4 showedstimulatory effect on chitinase activity by increasing theactivity 1.44 folds.

ConclusionMicrobial production of mycolytic enzymes has

captured the worldwide attention of both industrial,agricultural and scientific environments, not only becauseof its wide spectrum of applications but also for the lacunaof an effective production method47. The present studyidentified the optimum cultivation conditions using onefactor approach for increased production of the threemycolytic enzymes- chitinase, β-1,3 and β-1,4 glucanaseby a single strain of Bacillus subtilis, a potential biocontrolagent. This strain with increased mycolytic enzymes canbe developed into a potential biocontrol agent which canmediate antagonism of phytopathogens through its lyticenzymes. Moreover, low cost cellulosic materials can beused as carbon source for these enzymes production bythe isolate for industrial applications as well.

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