IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VOL.23 (1) 2010 Purification, Characterization and Antifungal Activity of Chitinase from Serratia marcescens Isolated from Fresh Vegetables N. H. Zek i and S. N. Muslim Department of Biology , College of Science , Unive rsity of Al-Mustansiriya, Abstract Seven [35%] and f ive [25%] Serratia marcescens isolates were obtained out of 20 samples of lettuce and 20 samples of sp inach, resp ectively, taken from different locations in a farm in Baghd ad city . The isolate that p roduced chitinase in higher level was chosen to p urify chitinase through several st ages of p urification includin g: ammonium sulfate p recipitation, DEAE- sep hadex ion exchan ge chromatograpgy and sep hadex G-200 gel filtration with 89.5- fold p urification and 30% recovery. The p urified chitinase was characterized and the molecular weight of enzy me was 59000 daltons by using gel filtration chromatography . The optimum p H and temp erature of the p urified chitinase were 6.0 and 50° c, r esp ectively, and the purified enzy me was stable on p H 5-7 up to 50° c. The enzy me was activated by Ca +2 , Cu +2 , M g +2 and inhibited by Hg +2 . In addition , Triton x-100 and n-ethy lmaleimide increased the chitinase activity while EDTA, methanol, ethanol and acetone inhib ited enzy me activity ; and this indicates that chitinase is a metaloenzy me. Chitinase showed stronger inhibitory activity to Fusarium solaini comp ared with Aspergillus flavus with p ercent of inhibition 83 and 69%, resp ectively . Therefore, this research leads to increase interest by using the chitinase as biocontrol agent of p hytop athogenic fungi and insects , p roduction of chito –oligosacch arides,p reparartion of sp haerop last and protop last from y east and fungi and bioconv ersion of chitin wast e to single cell protein for animal feed. Introduction Serratia sp . are gram negative b acteria, classified in the lar ge family of Enterobacteriaceae [1]. The Serratia genus includes different sp ecies, the best characterized sp ecies, and the one most frequently recovered from human , is Serratia marcescens [2]. This common microbe is found as a saprop hy te in soil, water and plants [3]. Serratia is isolated from many p lants such as tomatoes, sp inach, carrots, coconuts, green onions, lettuce and broccoli [4,5]. In addition, Serratia sp ecies frequently have been recovered from diseased or dead insects [5]. Serratia sp . have a good set of exoenzy mes that may be harmful to insects such as p roteases, gelatinase and ch itinase[4]. Chitin a homopolymer of N-acety l-D-glucosamin e (GlcNAc) residues linked by β1-4 bonds is widely distributed in nature as a constituent of insects exo skeleton, shells of crustaceans and fungul and algae cell wa lls [6,7,8]. Chitinase p lays an imp ortant role in the virulence of Serratia for insects and fungi, since this enzy me is active in lysing the cell walls of many insects and fungi that infect the economic p lants [8], in addition inhibit sp ore germination and germ tube elon gation of the phytop athogenic fungi [9,10] . This enzy me has also antimicrob ial and cell lysis activites against many kinds of bacteria [6,11]. Therefore, chitinase has wide range of biotechnolo gical app lications, esp ecially in p roduction of chito-oligosacch arides, p reparation of sp herop last and p rotop last from y east and fungal sp ecies and bioconversion of chitin wast e to single cell p rotein for animals feed [7-12]. IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VOL.23 (1) 2010 For these reasons, the goal of our r esearch was to p urify chitinase, to characterize this enzy me and to study the antifungul activity of chitinase. Materials and Methods A t otal of 40 samples of fresh ve getables, in cluding 20 samples of lett uce (L) and 20 samples of sp inach (S), were collected from d ifferent locations in a farm in Baghd ad city . Each sa mple was analyzed according to the method that described by [13] . Briefly, 25g of each sample was enriched in peptone water for 24h at 35°c. Isolation and identification Serratia m arcescens One loopfull of p lant samples was p lated on blood agar and M acConkeys agar, then inucubated at 30°c for 18-24 h. For isolation of Serratia marcesc ens, several biochemical tests were done and these include the following tests: inability to ferment lactose, a negative o xidase, p ositive results to catalase, DNAase,lysine decarboxy lase, orn ithine decarbo xy lase, growt h at 40°c and motility tests [5,14]. Besides to API 20E sy st em to differentiate Serratia marcesc ens from the other typ es. Isolation of fungi Aspergillus Flavus and Fusarium solani wer e obtained from the labor atory of Agricu lture College in Baghdad University and they were identified according to [15]. Detection of chitinolytic activity on plates Ten µl of Serratia marcescens culture was p laced into wells (5 mm in diameter) p repared in sy nthetic medium agar (SM ) that contained 2.0g colloidal ch itin, 0.5 g p eptone 0.5g y east extract, 0.1g KH2PO4, 0.01g M gSO4.7H2O (p H=6.5) and 1.7%w\w agar in 100ml of water and incub ated at 30 0 c. Aft er 18-24 h, the chitinoly tic activity was indicated by the formation of clear halos around the wells [12,16]. Chitinase assay Chitinase activity was conducted by a modified method described by [7,10,12]. 0.5 ml of enzy me solution was added to 1 ml of 0.1 M p hosp hate buffer (p H=6.0) as blank. The reaction initiated by the addition of 0.5ml of enzy me solution to 1ml of 0.2%(wt /vol) colloidal chitin in the same buffer and incubated at 50 0 c for 15 min, the reaction was st opp ed by centrifugation (5000xg) for 10min and the addition of 1ml of dinitrosalicylate (DNS) reagent. The reducing sugars released (GLcNAc) were measured by observing the absorbancy at 410nm and returning to st andared curve for Gl cNAc (N-acety l-D-glucosa mine). One unit of chitinase activity was defined as t he amount of enzy me of that released 1µmol of reducing sugars (GLcNAc) in 1 min. Protein assay The protein concentration was measured by usin g the method of [17] by sp ectrop hotometeric assay at 600nm in each st age of chitinase p urification and usin g bovine serum albumin as st andard. Purification of chitinase Serratia marcescens chitinase was p urified by a modification of the method [12]. Cells were grown in M 9 medium containin g the followin g: 0.7% Na2HPO4, 0.3%KH2PO4, 0.1% NH4Cl, 0.05% NaCl, 0.5% soluble st arch and 0.5% colloidal chitin (p H=7.0) in a 500ml flask and incubated at 30°c for 24h [7]. Sup ernatant was carefully removed after centrifugation at 10000 xg for 30min at 4°C and filtered through 0.22µm millip ore filters. Chitinase activity in sup ernatant was assay ed. The p urification of chitinase was carried out in three step s. The sup ernatant was p recipitated with ammonium sulfate 40-75% saturation for 1h with gentle stirrin g. The precipitate was collected by centrifugation at 10000xg for 30min.It was dissolved in 25 mM t ris-HCl buffer (p H=7.5) and dialysed against the same buffer. IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VOL.23 (1) 2010 The dialysed p rotein was subjected to ion exchan ger, DEAE- Sephadex column (2.5 by 25cm). The adsorbed chitinase was eluted with gr adient of NaCl fro m 0.2 to 0.5M in the same buffer. Fractions (5ml)were collected and assay ed for chitinase activity . In the final st ep, the active fractions were p ooled, dialyzed against 25mM tris-HCl buffer (p H=7.5) and loaded on sep hadex G-200 column (2.0 by 80cm) containing 100ml of sep hadex G-200 which had been equilibrated and washed with 10mM tris-HCl buffer (pH=7.5), then the elution was done by the same buffer. The fractions (5ml) were collected and assay ed for chitinase activity . Characterization of purified chitinase -Evaluation of the molecular weight : Accordin g to the p rinciples that were described by [18] the molecular weight of chitinase was evaluated by gel filtration chromatography by using sep hadex G-200 column. This column was equilibr ated in 25 mM tris-HCl buffer. The void volume ( Vo) was d etermined by using blue dextran. Elution volumes (Ve) of p roteins of known molecular mass (Bovine serum albu mine [66kDa], ovalbumin [45kDa], tryp sin [23kDa] and lysozyme [14kDa] dissolved in 25mM tris- HCl buffer) were measured and used as refer ence st andards in chitinase n ative molecular mass determination. The relationship between (Ve/Vo) and log molecular weight for standard p roteins was p lotted to obtain the standard curve.The molecu lar weight for chitinase was evaluated from inciden ce ( Ve/Vo) value for ch itinase on the st andard curve. -Effect of temperature on the activity and stability of chitinase Chitinase activity was assay ed at different temp eratures ranging from 10-70°c at p H 6.0 in sodium p hosp hate buffer (50mM ). To determine thermost ability , chitinase p rep aration was incubated at t he same ran ge of temperatures for 2h then chitinase activity was assay ed. -Effect of pH on the activity and stability of chitinase Chitinase activity was assay ed at different p H values (p H 3 to 10) by using different buffers 50mM such as cirate p hosp hate buffer (p H 3-6), sodium p hosp hate buffer (p H 7-8) and glycine of buffer (p H 9-10). To determine pH stability , chitinase p rep arations in buffer at the same range of pHs were incubater at 50°c for 2h then chitinase activity was assay ed. -Effect of various chemicals on chitinase activity The effect of various chemicals (CuSO4, CaSO4, CoCl2, NiSO4, Triton x-100, M gSO4, HgCl2, EDTA, n-ethy lmaleimide, methanol, ethanol and acetone) on the enzy me ativity were invest igated by p reincubating the enzy me with chemicals in 50mM sodium phosp hate buffer (p H 6.0) for 1 h at 50°c and then measurin g the residual activity of chitinase. -Antifungal activity of chitinase Agar p lates containin g p otato dextrose agar (PDA) and 10% p urified enzy me were p repared for findin g the antifungal activity of chitinase , and the same agar p late (without enzy me)was used as a control. Six (6)mm diameter mycelial discs of Aspergillus flavus and Fusarium solani grown on PDA p lates at 28°c for 3 days were p laced at the center of the p lates. The radial diameter of the colonies was measured after 5 days at 28°c then the p ercent of inhibition was calcu lated[10,19]. Results and Discussion Serratia marcescens was isolated from 35% [7] of lettuce samples (L1-L7) and 25% [5] of sp inach samples (S1-S5). Giri et.al.,2004 [1] found that cap ry llate thallous agar was the best for selectin g Serratia. DT C (deoxy ribonuclease – toludine blue- cep halothin)agar was used for isolating Serratia, while DNase medium for isolating Serratia marcescens. Vegetables used in salads might bring Serratia st rains to hosp itals and contamin ate the p atients digestive tract. Serratia marcescens , Serratia liquefaciens and Serratia marinorubra were found in 29%, 28% IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VOL.23 (1) 2010 and 11% (r esp ectively) in vegetable salad sample[5]. Gri mont&Grimont, 1998[4] reported that Serratia was isolated fro m mushrooms, t omatoes, leeks, l ett uce, gr een on ions, grass, and sp inach and the origin of Serratia found on p lants is p robably the soil. Chitinolytic activity on plates Twelve (12) Serratia marcesc ens were tested for chitinase p roduction by measuring the diameter of clear zone of lysis in sy nthetic medium agar (SM ) Figur e(1). In this fi gur e, only nine (9) isolates p roduced chitinase enzy me and Serratia marcescens L5 produced chitinase in higher level amon g the producer isolates, hence this isolate was selected for further study . Green et.al.,2004[20] reported that the optimum conditions (p H 7.0, 32.5°c and 1.0%(w/v) substrate) induced a high er level of enzy me activity . Also [21] showed that the maximum p roduction of Serratia marcescens chitinase was between pH 6 and 7 at 30 0 c and the optimal shaking sp eed at 150 rp m. Chitinase p roduction was reduced by 50% at p H 8.5 of p roduction medium [7]. In a st udy done by [22] found that among 102 Serratia marcescens strains screened, 57st rains showed chitinase activity . Purification of chitinase The chitinase was p urified by using st andard techniques such as ammonium sulf ate p recipitation (40-75%), DEAE-sephadex ion exchange chro matogr aphy and sep hadex G-200 gel filrtation chromatogr aphy . When cell free sup ernatant was subjected to fractional ammoniu m sulfate precipitation, Chitinase activity was p recipitated in 40-75% salt saturation The y ield of chitinase was 71% with a p urification fold of 3.1 and sp ecific activity of 6.2 U mg -1 p rotein. The dialy zed protein was loaded on DEAE ion exchanger. After elution with 0.2 to 0.5M NaCl gradient three major p eaks of p roteins were observed but chitinase activity was observed only in the first p eak figure (2). Here the y ield of chitinase was 42% with a p urification fold 13.2 and sp ecific activity 26.6 Umg -1 . Gel f iltration chromatography with sep hadex G-200 was the last st ep of chitinase p urification. The eluted fractions of this step contained the chitinase two p rotein p eaks,only the second p eak contained the chitinase activity figure (3). The y ield of chitinase was 30% with a p urification fold 89.5 and sp ecific activity 179.1 Umg -1 . The results of chitinase p urification were summarized in table(1). The increase in the sp ecific activity for chitinase may refer to the efficiency of the purification process and the decrease of the contamin ating materials. Chitinase was p urified from the culture filtrate of Enterobacter , Fusarium and Sreptomyces and p urified by usin g ion- exchan ge chromatography and gel filtration as mentioned at [9, 12, 23]. On the other hand, S erratia sp . chitinase was p urified by usin g DEAE Bio-Gel, chromatofocusin g with PBE 96 and gel filtration with sep hacry l S-200 [24]. In addition, Serratia marcescens chitinase was p urified by affinity adsorp tion, hy droxy lap atite and sep hadex G-200 [25]. Serratia chitinase showed h igh sequence ho molo gy with chitinase fro m Bacillus and Streptomyces [26]. Characterization of purified chitinase -Evaluation of the molecular weight of chitinase By using gel filtration with sep hadex G-200, the molecular weight of p urified chitinase was evaluated. The r esult revealed that p urified chitinase had app roximately 59000 daltons fi gure (4). The molecu lar weight of Enterobacter sp . chitinase was estimated to be 60kDa by SDS-PAGE and sephadex G-200 gel filtration, su ggesting that the purified chitinase is a monomer ty p e (12). Wan g& Chan ge, 1997. Frankowski et.al., 2001[6,27] found the mo lecular mass for Pseudomonas IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VOL.23 (1) 2010 aeruginosa and Serratia plymuthica were 60 and 60.5kDa, resp ectively. In a study done by [28] reported that Serratia marcescens strains grown in the p resence of M itomy cin C revealed the p resence of four extracellular p roteins with chitinase activity and mo lecular masses of 21, 38, 52 and 58kDa. -Effect temperature on activity and s tability of chitinase The effect of temp erature on the activity of chitinase with colloid chitin as subst rate of various temp eratures ranging from 10 to 70 0 c figur e (5) . The enzy me showed a good activity between 40-60 0 c with maximu m activity at 50 0 c. Considerable decrease in the chitinoly tic activity was observed for lowest and highest temp eratures, reaching 25, 38 and 20% activity at 10, 20 and 70 0 c. Thermostability of the enzyme was observed in the temp eratures up to 50 0 c after its t hermal exp osure for 2h figure(5). Incubations above 50 0 c p romoted inactivation of this enzy me. The chitinase p roduced by Enterobacter sp ., Bacillus sp ., Serratia marcescens showed op timum activity at 45, 45-55 and 40 0 c, resp ectively[12,29,30] .M oreover, the chitinase p roduced by Serratia sp . and Bacillus sp . were more thermostable than Fusarium sp . chitinase[9, 25, 29]. -Effect of pH on activity and s tability of chitinase The chitinase activity was evaluated at different p H values at 500c by using colloid chitin as substrate figure (6). The op timum activity for colloid chitin hydrolyisis reached at p H 6.0. Chitinase mantained 88 and 80% of its initial activity when incubated for 2h at p H 5 and 7, with a decrease in chitinase activity at p H lower and high er than the above p H values. The enzy me was st able at PH 5 to 7 f igure(6). The chitinase p roduced by Serratia marcescens and S tretomyces exh ibited maximum activity at pH 6.0 and 5.0, resp ectively [30]. In other st udy done by [31] reported that the chitinase produced by Serratia sp . was an exochitinase and exhibited a greater p H range [5.0-10.0]. -Effect of various chemicals on chitinase activity Chitinase of Serratia marcescens was treated with many chemicals some of these chemicals showed activation effect, others had inhibition effect. Co +2 , Ni +2 , Hg +2 inhibited chitinase activity to 82, 70 and 23% resp ectively, also EDT A, methanol,ethanol and acetone were inhibited the activity to 30, 45, 68 and 44%, resp ectively table(2). On t he other hand, the other metal ions Ca +2 , Cu +2 , and M g +2 increased chitinase activitor to 120,150 and 170% resp ectively, hence these metals can be evaluated as activator comp ounds for chitinase activity . In addition, n- ethy lmaleimid e and Triton x-100 increased the activity to 110 and 140%, resp ectively. The results may p rove that chitinase was metaloenzy me. Serratia plymuthica chitinase activity was stimulated by 120, 150 and 240% in presence of mM Ca +2 , Co +2 or M n +2 and inhibited by 80% in presence of 10 mM Cu +2 [27]. Chitinase from Enterobacter sp . was stimulated by Ca +2 , K +2 and M g +2 strongly inhibited by Hg +2 , Ag +1 , Cu +2 and Co +2 [12]. In a st udy done by [29] found t hat Ca+2 , Ni+2 an d T rit on x-100 stimulat ed t he activit y up t o 20% whereas Ag +1 and Hg +2 inhibit ed the act ivit y up to 50 %. - Antifungal activity of chitinase The p urified chitinase showed inhib itory activity against Aspergillus flavus and Fusarium solani. Chitinase showed much st ronger inhibitory activity to Fusarium solani comp ared with Aspergillus flavus figur e (7 and 8). The p ercent of inhibition for Fusarium solani was 83% while 64% for Aspergillus flavus table (3). This exp eriment incr eased the benefit by using the chitinase as biocontrol agent against p hy top athogenic fun gi. El-Katatny , et.al.,2005 [10] found that the p urified endochitinase of Trichoderma has shown antifungal activity against Sclerotium rolfsii and the inhibition of Sclerotium rolfsii depended on the carbon sourc e used and correlated with the level of chitinase activity . In addition, the chitinase was able to induce morphological altration in both Aspergillus flavus and Fusarium moniliforme. In a study done by [32] reported that chitinase of Serratia marcescens showed antifungal activity against Sclerotium rolfsii. IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VOL.23 (1) 2010 Re ferences 1- Giri, A.V.; Anandlkumar,N.;M uthukumaran,G. and p ennathur, G. (2004). A novel medium for the enhan ced cell growt h and p roduction of p rodigiosin from S errratia marcescens isolated from soil. BM C M icrobiol.4:11-21. 2- Collee,J.G.; Fraser,A.G; M armion,B.P. and Simmons,A. (1996). M acke & M cCartney p ractical medical microbiolo gy . 14 th ed.p:371-373. Churchill Livingst one Singapore. 3- Anone. 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Phy top athol. 78:84-88. 2010) 1( 23مجلة ابن الھیثم للعلوم الصرفة والتطبیقیة المجلد نتج من مال لمضادة للفطریات النزیم الكایتینیزعزل وتوصیف ودراسة الفعالیة ا المعزولة من الخضراوات الطازجة Serratia marcescensبكتریا نهایة حكمت زكي و ساهرة نصیف مسلم الجامعة المستنصریة ، كلیة العلوم، سم علوم الحیاةق الخالصة من مجمـوع Serratia marcescensتعود لبكتریا %)35بنسبة (عزالت 5 و ،)%35بنسبة (عزالت 7تم الحصول على .ة بغدادنمأخوذة من مواقع مختلفة في مزرعة في مدی، على التوالي، عینة لنبات السبانج 20و ، عینة لنبات الخس 20 نــزیم الكــایتینیز بعــدة مراحــل تتضــمن الترســیب اقیــة نأختیــرت العزلــة التــي انتجــت الكــایتینیز بــأعلى مســتوى الســتخالص وت G-200 Sephadexوالترشـــیح الهالمـــي باســــتخدام ، DEAE-Sephadexوالتبـــادل االیـــوني بأســـتخدام ، بكبریتـــات االمونیـــوم . %30وبحصیلة نهائیة 89.5بعدد مرت تنقیة . دالتـون باسـتخدام كروماتوغرافیـا الترشـیح الهالمـي 59000تم توصیف انزیم الكایتینیز المنقى ووجد أن وزنـه الجزیئـي بحـدود 50و 6.0ة الحــرارة المثلـى لفعالیــة االنــزیم المنقـى هــي جـوقـد وجــد أن الـرقم الهیــدروجیني االمثــل ودر 0 ووجــد أن ،علـى التــوالي، م 50ودرجـة الحـرارة تصـاعدیا الـى 5-7النـزیم المنقـى مسـتقر فـي الـرقم الهیـدروجیني المتـراوح بــین ا 0 تـم تنشـیط أنـزیم الكــایتینیز . م Mبوجــــود g +2 Cu +2 , Ca +2 ــــت فعالیتــــه بوجـــــود ، Hgوثبطـ +2 ـــد أن " فضـــــال. -nو x-100 Trironعــــن ذلـــــك وجــ ethy lmaleimide أمــا ، نیز ادت الـى زیــادة فعالیــة انـزیم الكــایتیEDTA ،االیثــانول واالســیتون فـأدت الــى تثبــیط ، المیثـانول كــذلك اظهـــر انـــزیم الكــایتینیز اعلـــى فعالیــة تثبیطـــة لفطـــر . الفعالیــة وهـــذه النتیجــة تؤكـــد أن انــزیم الكـــایتینیز هـــو انــزیم معـــدني Fusarium solani مقارنة مع فطر لباAspergillus Flavus لذا أدى هـذا البحـث . التواليعلى % 69و 83وبنسبة تثبیط انتــاج ســكریات بســیطة ، الــى زیــادة الفائــدة باســتخدام الكــایتینیز عامــل ســیطرة بایولوجیــة للفطریــات والحشــرات الممرضــة للنیــات وتحویــل المخلفــات الكایتینیــة الــى بــروتین احــادي الخلیــة تحضــیر البروتوبالســت والسفیروبالســت مــن الخمــائر واعفــان، كایتینیــة .ة الحیواناتلتغذی