Research Article A High Diversity in Chitinolytic and Chitosanolytic …downloads.hindawi.com/journals/bmri/2015/857639.pdf · 2019-07-31 · Research Article A High Diversity in

Research ArticleA High Diversity in Chitinolytic and Chitosanolytic Species andEnzymes and Their Oligomeric Products Exist inSoil with a History of Chitin and Chitosan Exposure

Malathi Nampally12 M B Govinda Rajulu13 Dominique Gillet4

T S Suryanarayanan3 and Bruno B Moerschbacher1

1 Institute for Biology and Biotechnology of Plants WWUMunster Schlossplatz 8 48143 Munster Germany2Research and Development Laboratory Sri Biotech Laboratories India Ltd Hyderabad 500 034 India3Vivekananda Institute of Tropical Mycology (VINSTROM) Ramakrishna Mission Vidyapith Chennai 600 004 India4Gillet Chitosan EURL Laurent Bonnevay 17 54100 Nancy France

Correspondence should be addressed to Bruno B Moerschbacher moerschuni-muensterde

Received 11 December 2014 Revised 31 March 2015 Accepted 15 April 2015

Academic Editor Spyridon Ntougias

Copyright copy 2015 Malathi Nampally et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Chitin is one of the most abundant biomolecules on earth and its partially de-N-acetylated counterpart chitosan is one of themost promising biotechnological resources due to its diversity in structure and function Recently chitin and chitosan modifyingenzymes (CCMEs) have gained increasing interest as tools to engineer chitosans with specific functions and reliable performancein biotechnological and biomedical applications In a search for novel CCME we isolated chitinolytic and chitosanolyticmicroorganisms from soils with more than ten-years history of chitin and chitosan exposure and screened them for chitinaseand chitosanase isoenzymes as well as for their patterns of oligomeric products by incubating their secretomes with chitosanpolymers Of the 60 bacterial strains isolated only eight were chitinolytic andor chitosanolytic while 20 out of 25 fungal isolateswere chitinolytic andor chitosanolytic The bacterial isolates produced rather similar patterns of chitinolytic and chitosanolyticenzymes while the fungal isolates produced a much broader range of different isoenzymes Furthermore diverse mixtures ofoligosaccharides were formed when chitosan polymers were incubated with the secretomes of select fungal species Our studyindicates that soils with a history of chitin and chitosan exposure are a good source of novel CCME for chitosan bioengineering

1 Introduction

Shrimp and crab shell wastes are used commercially for theextraction of chitin which can then be converted into itspartially de-N-acetylated forms chitosans Chitosans are afamily of molecules differing with respect to their degreeof polymerisation (DP) degree of acetylation (DA) andpattern of acetylation (PA) Such variations influence thephysicochemical solution properties as well as the biologicalfunctionalities of chitosans [1ndash5] which find use in agricul-tural food and pharmaceutical industries [6] Thereforewell characterized chitosans with broad ranges of specificDPs DAs and PAs are crucial for detailed structure-functionanalyses To this end chitin and chitosanmodifying enzymes

(CCMEs) such as chitinase chitin deacetylase and chi-tosanase could be used to complement the chemical methodscurrently used for this purpose [7ndash9] Furthermore chitosanscould be broken down to soluble derivatives called chi-tooligosaccharides (CHOS) which are vested with desirabletechnological properties [10] Since enzymatic conversion ofchitin to chitosan and CHOS is ecofriendly more specificand a cheaper option compared to the chemical methods [11]and could potentially augment the existing chemicalmethods[12 13] for characterization of chitosans search for novelCCME is a worthwhile exercise With the expectation thatsoils with a long history of exposure to chitin and chitosanwould select organisms elaborating diverse chitinolytic andchitosanolytic enzymes we proceeded with the current work

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 857639 8 pageshttpdxdoiorg1011552015857639

2 BioMed Research International

Although fungi are reported to contribute more thanbacteria to environmental degradation of chitin [14] muchless is known about the fungi involved compared to chiti-nolytic bacteria bacterial CCMEs have been studied inmuch more detail than fungal enzymes In terrestrial soilsthe most prevalent chitin degrading bacteria are speciesof Bacillus Stenotrophomonas Gammaproteobacteria andArthrobacter [15 16] while those inmarine sludges are speciesofActinobacterium Pantoea and Pseudomonas [17 18] Fungisuch as Trichoderma viride [15] and species ofMortierella andFusarium isolated from soil exhibit appreciable chitinolyticactivity in the presence of chitin in the culture medium [19]Here we looked at the diversity of chitinolytic and chi-tosanolytic fungi and bacteria in soils of a chitin and chitosanproducing company in Gujarat India These soils had beenexposed to dry or fresh shrimp shells or to chitin or differenttypes of chitosan for more than ten years In addition tospecies diversity we also analysed the diversity of CCMEpresent in these organisms as well as the diversity of productsproduced by these CCMEs

2 Materials and Methods

21 Soil Samples Seven soil samples were collected fromdifferent sites ofMahtani Chitosan Pvt Ltd Veraval (GujaratIndia) a chitinchitosan producing company from a depth of5 to 10 cm and stored at 4∘C for a maximum of two monthsbefore further processing

22 Preparation of Colloidal Chitin and Chitosans Colloidalchitin was prepared according to the method of Berger andReynolds [20] To 10 g of 120573-chitin 500mL of conc HCl wasadded stirred to get a homogeneous mixture and incubatedat 4∘Covernight Two litres of double-distilledwater was thenadded stirred for 48 hours at 4∘C and then washed withdouble-distilled water until the pH was neutral

Chitosan (average DA 3 average DPn ca 2000) wasdissolved in an aqueous acetic acid (01M) solution andpurified by successive filtration and extensive washing byrepeated precipitation and centrifugation following thischitosans with DA 35 DP 900 and DA 50 DP 820 wereprepared by partial re-N-acetylation using acetic anhydridein 12-propanediol [21]TheDA of the resulting chitosans wasdetermined using 1H NMR spectroscopy [22] and the DPusing HP-SEC coupled to RI and MALLS detectors [3]

23 Preparation of Chitin and Chitosan Agar Plates Forvisualizing chitinolytic activity Petri dishes withM9minimalmedium amendedwith 05 of colloidal chitin as sole carbonsource were used Petri dishes with Luria-Bertani (LB) agarmedium with 09 chitosan (DA 3) as the sole carbonsource were used to identify chitosanolytic activity Theappearance of a clear zone around the colony of a bacteriumor fungus growing on M9 and LB medium indicated chiti-nolytic and chitosanolytic activity respectively

24 Isolation of Chitinolytic and Chitosanolytic Fungi andBacteria from Soil Samples Fungi were isolated from the soilsamples by dilution plating and Warcup soil-plate methods

[23] For dilution plate 2 g of soil was suspended in 1mL ofsterile distilledwater and tenfold dilutions of this were spreadon PDA (Difco Potato Dextrose Agar medium Becton andDickinson Sparks USA) plates containing chloramphenicol(150mgL) to obtain individual fungal colonies For soilplates 2 g of soil was placed in a sterile Petri dish cooled PDAmedium (15mL) was added and soil particles were spread inthe medium All plates had replicates and were incubated at28∘C for 20 days to obtain fungal colonies The fungi wereisolated and subcultured in PDA slants Slide cultures of theseisolated fungi were then prepared stained and observedunder microscope to identify them based on standard keys[24]

Bacteria were isolated using a modified serial dilutionmethod of Maltseva and Oriel [25] Initially 10 g of soil wasinoculated in M9 minimal medium amended with colloidalchitin (05) to enrich chitinolytic bacteria a few mL of theenriched cultures was spread on LB agar plates and incubatedat 37∘C for isolating bacteria Pure cultures were obtained byrestreaking the colonies several times until single colonieswere obtained

25 Preparation of Samples for Zymography and Thin LayerChromatography Each fungal isolate was grown in potatodextrose broth for 5 days as static culture at 28∘C and 100mLof this culture filtrate (secretome) was dialyzed (MWCO1000 kDa) for 24 h against distilled water at 4∘C 10mg of thelyophilized secretome was mixed in 1mL of 50mM sodiumacetate buffer (pH 52) and centrifuged at 16000 g for 5min(20∘C) An aliquot (5ndash10120583L) of sample was used for dotactivity assay or zymography Bacterial isolates were grownin 10mL of LB medium for 48 h at 37∘C centrifuged andthe secretomes were lyophilised Lyophilised samples weredissolved in 1mL of 5mM sodium acetate buffer (pH 50) andused for assessing enzyme activities

26 Detection of Chitinase and Chitosanase Activity byDot Assay and Zymography Chitinolytic and chitosanolyticenzyme activities were detected using a dot activity assayas described previously [26] Briefly 5 120583L from a fungalsecretome preparation was applied on the gels prepared withglycol-chitin (03mgmL) or chitosan DA 35 (01mgmL)Gels were incubated at 37∘C overnight and then stained l withCalcofluor White A dark spot under UV transilluminationon the gel indicated enzyme activity

For detecting isoenzymes seminative SDS-PAGE (12)was run in gels containing 03mgmL of glycol-chitin forchitinase or 01mgmL of either of two chitosans (DA 50 orDA 35) [27] After electrophoresis (50mA for 4 h) the gelwas washed twice for 20min each in 50mM sodium acetatebuffer (pH 52 with 1TritonX-100) followed by twowashesin buffer without Triton X-100 The gel was incubated at37∘C for 12 h under shaking in 50mM sodium acetate buffer(pH 52) and stained with 001 Calcofluor White (SigmaSteinheim Germany) in 05M TrisHCl-buffer (pH 89) for5min and finally washed in deionized water for 1 h Theisozymes were visualized on a UV transilluminator A crudeextract of a known chitinolytic strain of Bacillus licheniformis[28] was run as a positive control

BioMed Research International 3

Alternatively zymography was done using isoelectricfocusing (IEF) over the pH range 3ndash10 in a polyacrylamidegel containing Ampholine (Amersham Bioscience UppsalaSweden) followed by activity staining using overlay gelscontaining 01mgmL of either of the chitosans mentionedabove After incubation in 50mM ammonium acetate buffer(pH 52) overnight at 37∘C overlay gels were stained withCalcofluor White as described above

27 Detection of Chitosan Oligomers by Thin LayerChromatography A sample (20 120583L) of secretome of selectedfungal isolates was mixed with 20120583L of chitosan DA 35solution (1mgmL) and incubated overnight at 37∘C in50mM sodium acetate buffer (pH 55) Samples wereconcentrated under reduced pressure to scale downthe volume and aliquots of 10 120583L were applied onTLC plates (Merck Berlin Germany) run in butanol methanol ammonia water (5 4 2 1 vvvv) and stainedusing aniline-diphenylamine reagent (4mL of aniline 4 gof diphenylamine 200mL of acetone and 30mL of 85phosphoric acid) Oligomers were visualised by heating theplate at 180∘C for 3ndash5min Oligomers were compared withauthentic N-acetyl-D-glucosamine (DP 1 2 3 5 6) andD-glucosamine (DP 1 3 4) standards (Seikagaku TokyoJapan)

28 16S-rDNA Analysis of Bacterial Isolates PCR was per-formed on the bacterial soil isolates using bacterial uni-versal primers forward primer (51015840AGAGTTTGATC(AC)-TGGCTCAG31015840) reverse primer (51015840AAGGAGGTGATC-CA(AGCT)CC(AG)CA31015840) [29] Amplicons were cloned intoPCRII-TOPO vector and sequenced at MWG EbersbergGermany Blast analyses were performed with the sequencesin the NCBI database Sequences obtained were depositedin NCBI under Gene Bank with sequence idrsquos JN593073ndashJN593080

3 Results

31 Isolation and Screening for Chitinolytic or ChitosanolyticBacteria and Fungi A total of sixty bacterial strains wereisolated from seven soil samples collected from different sitesof the chitinchitosan producing company On a minimalmedium with 05 colloidal chitin as the sole carbon sourceeight strains consistently produced clear zones aroundtheir colonies indicating a chitinolytic activity (Figure 1(a))Microscopic observations indicated that all of them wereBacillus species differing in their motility sporulation andarrangement of sporesThe overall 16S-rDNA sequence iden-tity between these Bacillus strains ranged from 995 to 10016S-rDNA sequence identities of 99 and 997 corroboratedthis to the cereusanthracisthuringiensis group of Bacillus

A total of 25 fungal strains were isolated from twodifferent soil samples Many of themwereAspergillus speciesother genera included Acremonium Aureobasidium Cla-dosporium Curvularia Drechslera Fusarium Penicilliumand Sporormiella (Table 1) Of the seven randomly chosenisolates from these two produced clearing zones on chitinmedium (Figure 1(b)) and two others on chitosan medium

Table 1 Chitinase and chitosanase activity of the fungal isolatesscreened from soil samples

Isolatenumber Name of the fungus Activity in dot assays

Chitinaselowast Chitosanaselowast

1 Fusarium sp + minus

2 Unidentified minus +3 Penicillium sp + +4 Aspergillus sp + minus

5 Acremonium sp minus minus

6 Cladosporium sp + +7 Cladosporium sp minus +8 Aureobasidium pullulans + minus

9 Aureobasidium pullulans + minus

10 Unidentified + +11 Unidentified minus +12 Unidentified minus +13 Unidentified + minus

14 Aspergillus sp minus minus

15 Curvularia sp minus minus

16 Unidentified minus minus

17 Aspergillus sp + +18 Aspergillus niger + +19 Sporormiella intermedia minus minus

20 Cladosporiumcladosporioides minus +

21 Drechslera sp + +22 Drechslera sp + minus

23 Aspergillus sp minus +24 Acremonium sp + +25 Unidentified minus +lowastGlycol-chitin and chitosan DA 36 were used as substrates to detectchitinase and chitosanase activity respectively + = positive minus = negative

(Figure 1(c)) Of the 25 isolates 13 were positive for chiti-nase 14 were positive for chitosanase and 7 produced boththe enzymes as visualized by dot assay (Table 1) Amongthe 20 fungal isolates which were chitinolytic andor chi-tosanolytic 14 identified fungi belong to seven differentgenera namely Acremonium Aspergillus AureobasidiumCladosporium Drechslera Fusarium and Penicillium whichare common saprotrophs found in soils [30]

32 Chitinolytic and Chitosanolytic Enzymes of Bacterial andFungal Isolates Thecrude extract ofB licheniformis (control)showed activity on all three substrateswhereas the secretomesfrom the different soil bacterial strains showed differencesin their activities Isolates 2 3 5 and 7 showed the sametwo high-molecular weight chitinases as B licheniformiswhile the extracts from isolates 1 4 6 and 8 were notactive on glycol-chitin (Figure 2(a)) All isolates including Blicheniformis produced one high MW isoenzyme degradingchitosan (DA 56) and isolates 2 4 and possibly 7 possessedan additional isoenzyme with a MW between 50 and 75 kDa


(a) (b) (c)

Figure 1 Chitinolytic and chitosanolytic activities in bacterial and fungal isolates from soil samples (a) Bacterial strains showing clearingzones on minimal medium agar plates containing colloidal chitin one strain showing weak chitinolytic activity (top marked with circle) wasexcluded from further studies as it did not show the activity consistently (b) and (c) examples of fungal strains showing clearing zones onagar plates containing colloidal chitin in minimal medium (b) or chitosan DA 3 in LB medium (c)

250150100

75

50

37

1 M 2 3 4 5 6 7 8 BL

(a)

250150100

75

50

37

1 M2 3 4 5 6 7 8 BL

(b)

250150100

75

50

37

1 M 2 3 4 5 6 7 8 BL

(c)

Figure 2 Seminative SDS-PAGE of crude extracts of the bacterialsoil isolates (1ndash8) followed by zymography using glycol-chitin (a)chitosan DA 56 (b) or chitosan DA35 (c) as a substrate Aknown chitinolytic strain of Bacillus licheniformis (BL) was used asa positive controlThe positions of marker proteins (M) are given onthe sides of the gels

capable of degrading this chitosan (Figure 2(b)) All of thestrains including B licheniformis had isoenzymes degradingchitosan DA 35 (Figure 2(c)) Isolates 1 and 5 producedfewer and weakly active chitosanase isoforms and isolates 4and 7 produced chitosanase of highest MW Considering theactivities on all three substrates it was clear that all eightisolates differ from each other and from B licheniformis intheir chitinolytic and chitosanolytic isoenzymes but theirdiversity was limited

PCR was performed on genomic DNA of the eightbacterial soil isolates using primers designed from conservedregions of known Bacillus chitosanases Amplicons wereobserved at 13 Kb only in strains 1 3 6 and 7 (data notshown) the other strains did not show any amplificationBlast results showed that the sequences were identical tothe known chitosanase sequence of Bacillus sp strain KCTC0377BP [31]

To analyse the chitosanolytic isoenzymes of fungi crudeextracts of fungal isolates which were positive in the dotassay with chitosan DA 35 as a substrate were subjected toseminative SDS-PAGE in a gel containing chitosan DA 35(Figure 3) Isoenzyme activity was observed in all isolatesthe isolates differed in the number of isoforms and in theiroverall activity The number of isoenzymes ranged fromone to three and their MW ranged from very low to veryhigh Isolates 3 10 17 18 and 23 (Penicillium and all threechitosanolytic Aspergillus isolates) had a strong activity atMW of ca 250 kDa two of the three Cladosporium isolates(7 and 20) showed one sharp band around 50 kDa isolates 1112 and 18 had one or two low MW isoforms between 10 and20 kDa

Samples which differed clearly in their isoenzyme spec-trum were selected and their proteins were separated byisoelectric focusing (IEF) For zymography polyacrylamidegels containing different chitosans with DA 35 and DA 56were overlaid on the IEF gel after the run (Figure 4) Gels


250150100755037

(kDa)

201510

2 3 6 7 10 M 11 12 13 14 16 17 18 20 21 22 23 24 25 M

Figure 3 Seminative SDS-PAGE of crude extracts of selected fungal soil isolates (numbers correspond to Table 1) followed by zymographyusing chitosan DA 35 as a substrate The positions of marker proteins (M) are given on the right side

Chitosan DA 56Chitosan DA 35(a) (b)

PH 3

PH 1

0

3 7 10 11 12 17 18 20 21 23 3 7 10 12 17 18 20 23

Figure 4 Isoelectric focusing of crude extracts of selected fungal soil isolates (numbers correspond to Table 1) followed by zymographyusing overlay gels containing chitosan DA 35 (a) or chitosan DA 56 (b) as a substrate The pH range of the gels is indicated at the rightside

were incubated at 37∘Covernight and stainedwith CalcofluorWhite to detect chitosanolytic activity All fungal isolates hadone to four chitosanolytic isoenzymes with isoelectric pointsranging from pH 4 to pH 8 While few differences wereseen between the two substrates tried clear differences wereobvious between the different isolates

33 Chitosan Oligomers Produced by Chitosanolytic Enzymesof Select Fungal Isolates Secretomes from fungal isolateswhich showed a single dominant isoenzyme in zymography(isolates 3 7 10 12 17 20 and 23) were incubated with chi-tosan DA 35 overnight at 37∘C and the chitosan oligomersproduced were analysed using TLC (Figures 5(a) and 5(b))This preliminary analysis showed that different oligomermixtures were produced by each fungal isolate ranging fromthe monomers GlcN and GlcNAc (isolates 3 23) to a mixtureof small oligomers ranging in degree of polymerization from2 to 6 (isolates 7 10 12 and 20) Isolate 17 produced only largeroligomers

4 Discussion

We argued that soils with a long history of exposure tochitin and chitosan would select microbes with an ability

to degrade chitin andor chitosan Hence we studied thesoils of a chitin and chitosan producing company which hasbeen processing ca 5000 tons of fresh and dried shrimpannually since the year 1995 Earlier our collaborators fromIndia reported that Gammaproteobacteria were dominantin these soils [16] We found eight different Bacillus speciesbelonging to the cereusanthracisthuringiensis group whichis well known for their potential to degrade chitin andchitosan [32ndash35] With regard to the CCME chitosanaseswere more diverse than chitinases in these species Usingdegenerate primers of known Bacillus chitosanases [36] wecould amplify a chitosanase gene from four of the eight strainswhich was identical to a chitosanase gene from Bacillus spstrain KCTC 0377BP [31] We have now set up a pooledgenomic DNA library of these strains and are screening it forchitinase and chitosanase genes

Although fungi with CCME activities have been reportedfrom soils [37ndash39] to our knowledge this is the first reporton fungal diversity in soils with a history of chitinchitosanexposure We identified the fungi based on spore morphol-ogy but amolecular approachwill be essential to authenticatetheir identity at the species level Though fungi producechitinolytic enzymes per se for cell wall remodelling duringtheir developmental processes [40] the diversity of these


A1

A2

A3

A5

A6

D1

D3D4

An Dn 3c 3s 7c 7s 10c 10s 12c 12s co

(a)

A1

A2

A3

A5A6

D1

D3D4

An Dn 17c 17s 20c 20s 23c 23s co

(b)

Figure 5 (a) and (b) TLC analysis of the products of chitosan (DA 35) incubation with crude extracts of selected fungal soil isolates(numbers correspond to Table 1) Crude extracts were incubated with (samples labeled 3s 7s 10s 12s 17s 20s and 23s) or without chitosan(samples labeled 3c 7c 10c 12c 17c 20c and 23c) as a substrate Chitosan incubated without any crude extract (co) was used as a controland oligomers of GlcNAc (An) and GlcN (Dn) were used as standards The DP of the standards is given on the left sides of the plates

enzymes could be higher in fungi present in soils withspent chitin material since the fungi here could possiblybe utilizing chitin and chitosan polysaccharides as carbonand nitrogen source [41] This could be the reason whyalmost all of the fungi screened here were positive for chitinandor chitosan degrading enzymes 13 were chitinolytic 14were chitosanolytic and 7 isolates were both chitinolyticand chitosanolytic Furthermore isolates belonging to thesame genus differed significantly in their chitinolytic andchitosanolytic potential A typical case is the genusAspergilluswhich dominated the fungal isolates Of the five Aspergillusisolates one was chitinolytic but not chitosanolytic one waschitosanolytic but not chitinolytic two were both chitinolyticand chitosanolytic and one was neither chitinolytic norchitosanolytic Similarly of the twoAcremonium isolates onewas negative for both activities and one was positive for bothactivities all three Cladosporium isolates were chitosanolyticbut only onewas also chitinolytic both theDrechslera isolateswere chitinolytic and one was also chitosanolytic Thus soilswith a history of chitin and chitin exposure appear to be apromising source of fungi with high CCME diversity usefulfor technological exploitation This assumption was furthersubstantiated by the isoenzyme patterns of chitosanasesdiscerned based on size and isoelectric point Most fungalisolates produced more than one chitosanolytic isoenzymeIEF was superior to native PAGE for visualizing isozymes as

more isoforms were visible for isolates 3 10 12 17 and 20 inthe latter method of detection A few fungal isolates whichtested positive for chitosanase in the dot activity assay (withchitosan DA 35 as substrate) showed weak activities in thezymograms with this substrate possibly owing to improperrenaturation of the enzymes when the SDS was washed outIn spite of the multiplicity of enzymes chitosan DA 35 wasnot fully degraded to monomers or very small oligomers byany of the crude enzyme preparations pentamers and largeroligomers were produced by isolates 7 10 12 17 and 20

5 Conclusions

In conclusion we observed strong and diverse chitinolyticand chitosanolytic activities among the microorganismspresent in soil samples with a history of chitinchitosanexposure and the diversity in fungal species and their CCMEhere was higher than the bacterial diversity Analysing thebiodiversity of microorganisms in an environmental sampleby screening for the diversity of isoenzymes and for theoligomers produced by these enzymes is a novel but promis-ing approach The high diversity found is of biotechnologicalrelevance as isolated bacterial and fungal chitinases andchitosanases [42 43] as well as the oligomers produced bypurified or crude chitinolytic and chitosanolytic enzymes


[31 44 45] have interesting and diverse biological activitiesand may thus be useful in a wide range of applications

Conflict of Interests

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

Acknowledgments

The research leading to these results has received fundingfrom the European Union Sixth Framework Programme([FP62002ndash2006]) in the framework of the PolyModEproject under Grant Agreement KBBE-2007-3-3-07 coordi-nated by BMM Further financial support came from travelgrants to M B Govinda Rajulu and T S Suryanarayanan inthe framework of a joint project of the German Federal Min-istry of Education and Research BMBF and the Departmentof Biotechnology Govt of India (BTINFRG09TSS2007)and a PhD fellowship to Malathi Nampally by the Ger-man Academic Exchange Service DAAD The authors thankMr Kamlesh Fofandi Mahtani Chitosan Pvt Ltd Veraval362266 India for support Dr Nour Eddine El Gueddari forhelp with the generation and characterisation of chitosansDr Ratna Singh for help with 16S-rDNA phylogenetic treesand Ursula Fassin for technical assistance

References

[1] N E El Gueddari U Rauchhaus B M Moerschbacher and HB Deising ldquoDevelopmentally regulated conversion of surface-exposed chitin to chitosan in cell walls of plant pathogenicfungirdquo New Phytologist vol 156 no 1 pp 103ndash112 2002

[2] N E El Gueddari and B M Moerschbacher ldquoA bioactivitymatrix for chitosans as elicitors of disease resistance reactionsin wheatrdquo Advances in Chitin Science vol 7 pp 56ndash59 2004

[3] G Lamarque J M Lucas C Viton and A Domard ldquoPhysic-ochemical behavior of homogeneous series of acetylated chi-tosans in aqueous solution role of various structural parame-tersrdquo Biomacromolecules vol 6 no 1 pp 131ndash142 2005

[4] B M Moerschbacher ldquoBio-activity matrices of chitosans inplant protectionrdquo in Emerging Trends in Plant-Microbe Inter-actions S S Gnanamanickam R Balasubramanian and NAnand Eds pp 186ndash190 University ofMadras Chennai India2005

[5] A Domard ldquoA perspective on 30 years research on chitin andchitosanrdquo Carbohydrate Polymers vol 84 no 2 pp 696ndash7032011

[6] W Arbia L Arbia L Adour andA Amrane ldquoChitin extractionfrom crustacean shells using biological methodsmdasha reviewrdquoFoodTechnology andBiotechnology vol 51 no 1 pp 12ndash25 2013

[7] S-K Kim and N Rajapakse ldquoEnzymatic production and bio-logical activities of chitosan oligosaccharides (COS) a reviewrdquoCarbohydrate Polymers vol 62 no 4 pp 357ndash368 2005

[8] N E El Gueddari A Schaaf M Kohlhoff C Gorzelanny andB M Moerschbacher ldquoSubstrates and products of chitin andchitosan modifying enzymesrdquo Advances in Chitin Science vol10 pp 119ndash126 2007

[9] M Kohlhoff N E El Gueddari C Gorzelanny et alldquoBio-engineering of chitosans with non-random patterns of

acetylationmdasha novel sequence-specific chitosan hydrolase gen-erating oligomers with block-PArdquo Advances in Chitin Sciencevol 11 pp 463ndash468 2009

[10] W Xia P Liu J Zhang and J Chen ldquoBiological activities ofchitosan and chitooligosaccharidesrdquoFoodHydrocolloids vol 25no 2 pp 170ndash179 2011

[11] I Tsigos A Martinou D Kafetzopoulos and V BouriotisldquoChitin deacetylases new versatile tools in biotechnologyrdquoTrends in Biotechnology vol 18 no 7 pp 305ndash312 2000

[12] M Chen X Zhu Z Li X Guo and P Ling ldquoApplication ofmatrix-assisted laser desorptionionization time-of-flight massspectrometry (MALDI-TOF-MS) in preparation of chitosanoligosaccharides (COS) with degree of polymerization (DP)5-12 containing well-distributed acetyl groupsrdquo InternationalJournal of Mass Spectrometry vol 290 no 2-3 pp 94ndash99 2010

[13] BMMoerschbacher F Bernard andN E El Gueddari ldquoEnzy-maticmass spectrometric fingerprinting of partially acetylatedchitosansrdquo Advances in Chitin Science vol 11 pp 185ndash191 2011

[14] M Swiontek-Brzezinska E Lalke-Porczyk and W DonderskildquoChitinolytic activity of bacteria and fungi isolated from shrimpexoskeletonsrdquo Oceanological and Hydrobiological Studies vol36 no 3 pp 101ndash111 2007

[15] N A Manucharova A N Vlasenko G M Zenova T GDobrovolrsquoskaya and A L Stepanov ldquoMethodological aspectsof assessing chitin utilization by soil microorganismsrdquo BiologyBulletin vol 35 no 5 pp 549ndash553 2008

[16] S N Das P V S R N Sarma C Neeraja N Malati andA R Podile ldquoMembers of Gammaproteobacteria and Bacillirepresent the culturable diversity of chitinolytic bacteria inchitin-enriched soilsrdquo World Journal of Microbiology and Bio-technology vol 26 no 10 pp 1875ndash1881 2010

[17] A C Metcalfe M Krsek G W Gooday J I Prosser and EM HWellington ldquoMolecular analysis of a bacterial chitinolyticcommunity in an upland pasturerdquo Applied and EnvironmentalMicrobiology vol 68 no 10 pp 5042ndash5050 2002

[18] V Gohel T Chaudhary P Vyas and H S Chhatpar ldquoIsolationand identification of marine chitinolytic bacteria and theirpotential in antifungal biocontrolrdquo Indian Journal of Experimen-tal Biology vol 42 no 7 pp 715ndash720 2004

[19] W de Boer S Gerards P J A Klein Gunnewiek and R Mod-derman ldquoResponse of the chitinolytic microbial community tochitin amendments of dune soilsrdquo Biology and Fertility of Soilsvol 29 no 2 pp 170ndash177 1999

[20] L R Berger and DM Reynolds ldquoColloidal chitin preparationrdquoMethods in Enzymology vol 161 pp 140ndash142 1988

[21] L Vachoud N Zydowicz and A Domard ldquoFormation andcharacterisation of a physical chitin gelrdquoCarbohydrate Researchvol 302 no 3-4 pp 169ndash177 1997

[22] A Hirai H Odani and A Nakajima ldquoDetermination of degreeof deacetylation of chitosan by 1HNMR spectroscopyrdquo PolymerBulletin vol 26 no 1 pp 87ndash94 1991

[23] J HWarcup ldquoThe soil-plate method for isolation of fungi fromsoilrdquo Nature vol 166 no 4211 pp 117ndash118 1950

[24] R W Riddell ldquoPermanent stained mycological preparationsobtained by slide culturerdquoMycologia vol 42 no 2 pp 265ndash2701950

[25] O Maltseva and P Oriel ldquoMonitoring of an alkaline 246-trichlorophenol-degrading enrichment culture by DNA finger-printing methods and isolation of the responsible organismhaloalkaliphilic Nocardioides sp Strain M6rdquo Applied and Envi-ronmental Microbiology vol 63 no 11 pp 4145ndash4149 1997


[26] M B G Rajulu N Thirunavukkarasu T S Suryanarayanan JP Ravishankar N E El Gueddari and B M MoerschbacherldquoChitinolytic enzymes from endophytic fungirdquo Fungal Diver-sity vol 47 no 1 pp 43ndash53 2011

[27] J Trudel and A Asselin ldquoDetection of chitinase activity afterpolyacrylamide gel electrophoresisrdquo Analytical Biochemistryvol 178 no 2 pp 362ndash366 1989

[28] C Songsiriritthigul S Lapboonrueng P Pechsrichuang PPesatcha and M Yamabhai ldquoExpression and characterizationof Bacillus licheniformis chitinase (ChiA) suitable for biocon-version of chitin wasterdquo Bioresource Technology vol 101 no 11pp 4096ndash4103 2010

[29] J C Hogg and M J Lehane ldquoIdentification of bacterial speciesassociated with the sheep scab mite (Psoroptes ovis) by usingamplified genes coding for 16S rRNArdquo Applied and Enviro-nmental Microbiology vol 65 no 9 pp 4227ndash4229 1999

[30] N Satish S Sultana and V Nanjundiah ldquoDiversity of soil fungiin a tropical deciduous forest in Mudumalai southern IndiardquoCurrent Science vol 93 no 5 pp 669ndash677 2007

[31] Y J Choi E J Kim Z Piao Y C Yun and Y C Shin ldquoPurifica-tion and characterization of chitosanase from Bacillus sp strainKCTC0377BP and its application for the production of chitosanoligosaccharidesrdquoApplied and EnvironmentalMicrobiology vol70 no 8 pp 4522ndash4531 2004

[32] R M Cody ldquoDistribution of chitinase and chitobiase in Bacil-lusrdquo Current Microbiology vol 19 no 4 pp 201ndash205 1989

[33] T Nishijima K Toyota and M Mochizuki ldquoPredominant cul-turable Bacillus species in Japanese arable soils and their poten-tial as biocontrol agentsrdquo Microbes and Environments vol 20no 1 pp 61ndash68 2005

[34] L D Alcaraz G Moreno-Hagelsieb L E Eguiarte V SouzaL Herrera-Estrella and G Olmedo ldquoUnderstanding the evo-lutionary relationships and major traits of Bacillus throughcomparative genomicsrdquoBMCGenomics vol 11 no 1 article 3322010

[35] N Ivanova A Sorokin I Anderson et al ldquoGenome sequenceof Bacillus cereus and comparative analysis with Bacillusanthracisrdquo Nature vol 423 no 6935 pp 87ndash91 2003

[36] C X Su D M Wang L M Yao and Z L Yu ldquoPurifica-tion characterization and gene cloning of a chitosanase fromBacillus species strain S65rdquo Journal of Agricultural and FoodChemistry vol 54 no 12 pp 4208ndash4214 2006

[37] A A Sherief M M A Elsawah and M A A Elnaby ldquoSomeproperties of chitinase produced a potent Aspergillus carneusstrainrdquo Applied Microbiology and Biotechnology vol 35 no 2pp 228ndash230 1991

[38] E F Sharaf ldquoApotent chitinolytic activity ofAlternaria alternataisolated from Egyptian black sandrdquo Polish Journal of Microbiol-ogy vol 54 no 2 pp 145ndash151 2005

[39] A A Shindia and K A El-Sherbiny ldquoChitosanase productionusing some fungi optimization of fermentation conditions ofchitosanase produced by Aspergillus ornatusrdquo in Proceeding ofthe 2nd Scientific Environmental Conference pp 97ndash113 ZagazigUniversity 2007

[40] D J Adams ldquoFungal cell wall chitinases and glucanasesrdquoMicrobiology vol 150 no 7 pp 2029ndash2035 2004

[41] E Battaglia I Benoit J van den Brink et al ldquoCarbohydrate-active enzymes from the zygomycete fungus Rhizopus oryzaea highly specialized approach to carbohydrate degradationdepicted at genome levelrdquo BMC Genomics vol 12 article 382011

[42] N Dahiya R Tewari and G S Hoondal ldquoBiotechnologicalaspects of chitinolytic enzymes a reviewrdquo Applied Microbiologyand Biotechnology vol 71 no 6 pp 773ndash782 2006

[43] C Neeraja K Anil P Purushotham et al ldquoBiotechnologicalapproaches to develop bacterial chitinases as a bioshield againstfungal diseases of plantsrdquo Critical Reviews in Biotechnology vol30 no 3 pp 231ndash241 2010

[44] C Y Cheng and Y-K Li ldquoAn Aspergillus chitosanase withpotential for large-scale preparation of chitosan oligosaccha-ridesrdquo Biotechnology and Applied Biochemistry vol 32 no 3 pp197ndash203 2000

[45] T-W Liang Y-J Chen Y-H Yen and S-L Wang ldquoTheantitumor activity of the hydrolysates of chitinous materialshydrolyzed by crude enzyme from Bacillus amyloliquefaciensV656rdquo Process Biochemistry vol 42 no 4 pp 527ndash534 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

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Zoology


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GenomicsInternational Journal of


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Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Although fungi are reported to contribute more thanbacteria to environmental degradation of chitin [14] muchless is known about the fungi involved compared to chiti-nolytic bacteria bacterial CCMEs have been studied inmuch more detail than fungal enzymes In terrestrial soilsthe most prevalent chitin degrading bacteria are speciesof Bacillus Stenotrophomonas Gammaproteobacteria andArthrobacter [15 16] while those inmarine sludges are speciesofActinobacterium Pantoea and Pseudomonas [17 18] Fungisuch as Trichoderma viride [15] and species ofMortierella andFusarium isolated from soil exhibit appreciable chitinolyticactivity in the presence of chitin in the culture medium [19]Here we looked at the diversity of chitinolytic and chi-tosanolytic fungi and bacteria in soils of a chitin and chitosanproducing company in Gujarat India These soils had beenexposed to dry or fresh shrimp shells or to chitin or differenttypes of chitosan for more than ten years In addition tospecies diversity we also analysed the diversity of CCMEpresent in these organisms as well as the diversity of productsproduced by these CCMEs

2 Materials and Methods

21 Soil Samples Seven soil samples were collected fromdifferent sites ofMahtani Chitosan Pvt Ltd Veraval (GujaratIndia) a chitinchitosan producing company from a depth of5 to 10 cm and stored at 4∘C for a maximum of two monthsbefore further processing

22 Preparation of Colloidal Chitin and Chitosans Colloidalchitin was prepared according to the method of Berger andReynolds [20] To 10 g of 120573-chitin 500mL of conc HCl wasadded stirred to get a homogeneous mixture and incubatedat 4∘Covernight Two litres of double-distilledwater was thenadded stirred for 48 hours at 4∘C and then washed withdouble-distilled water until the pH was neutral

Chitosan (average DA 3 average DPn ca 2000) wasdissolved in an aqueous acetic acid (01M) solution andpurified by successive filtration and extensive washing byrepeated precipitation and centrifugation following thischitosans with DA 35 DP 900 and DA 50 DP 820 wereprepared by partial re-N-acetylation using acetic anhydridein 12-propanediol [21]TheDA of the resulting chitosans wasdetermined using 1H NMR spectroscopy [22] and the DPusing HP-SEC coupled to RI and MALLS detectors [3]

23 Preparation of Chitin and Chitosan Agar Plates Forvisualizing chitinolytic activity Petri dishes withM9minimalmedium amendedwith 05 of colloidal chitin as sole carbonsource were used Petri dishes with Luria-Bertani (LB) agarmedium with 09 chitosan (DA 3) as the sole carbonsource were used to identify chitosanolytic activity Theappearance of a clear zone around the colony of a bacteriumor fungus growing on M9 and LB medium indicated chiti-nolytic and chitosanolytic activity respectively

24 Isolation of Chitinolytic and Chitosanolytic Fungi andBacteria from Soil Samples Fungi were isolated from the soilsamples by dilution plating and Warcup soil-plate methods

[23] For dilution plate 2 g of soil was suspended in 1mL ofsterile distilledwater and tenfold dilutions of this were spreadon PDA (Difco Potato Dextrose Agar medium Becton andDickinson Sparks USA) plates containing chloramphenicol(150mgL) to obtain individual fungal colonies For soilplates 2 g of soil was placed in a sterile Petri dish cooled PDAmedium (15mL) was added and soil particles were spread inthe medium All plates had replicates and were incubated at28∘C for 20 days to obtain fungal colonies The fungi wereisolated and subcultured in PDA slants Slide cultures of theseisolated fungi were then prepared stained and observedunder microscope to identify them based on standard keys[24]

Bacteria were isolated using a modified serial dilutionmethod of Maltseva and Oriel [25] Initially 10 g of soil wasinoculated in M9 minimal medium amended with colloidalchitin (05) to enrich chitinolytic bacteria a few mL of theenriched cultures was spread on LB agar plates and incubatedat 37∘C for isolating bacteria Pure cultures were obtained byrestreaking the colonies several times until single colonieswere obtained

25 Preparation of Samples for Zymography and Thin LayerChromatography Each fungal isolate was grown in potatodextrose broth for 5 days as static culture at 28∘C and 100mLof this culture filtrate (secretome) was dialyzed (MWCO1000 kDa) for 24 h against distilled water at 4∘C 10mg of thelyophilized secretome was mixed in 1mL of 50mM sodiumacetate buffer (pH 52) and centrifuged at 16000 g for 5min(20∘C) An aliquot (5ndash10120583L) of sample was used for dotactivity assay or zymography Bacterial isolates were grownin 10mL of LB medium for 48 h at 37∘C centrifuged andthe secretomes were lyophilised Lyophilised samples weredissolved in 1mL of 5mM sodium acetate buffer (pH 50) andused for assessing enzyme activities

26 Detection of Chitinase and Chitosanase Activity byDot Assay and Zymography Chitinolytic and chitosanolyticenzyme activities were detected using a dot activity assayas described previously [26] Briefly 5 120583L from a fungalsecretome preparation was applied on the gels prepared withglycol-chitin (03mgmL) or chitosan DA 35 (01mgmL)Gels were incubated at 37∘C overnight and then stained l withCalcofluor White A dark spot under UV transilluminationon the gel indicated enzyme activity

For detecting isoenzymes seminative SDS-PAGE (12)was run in gels containing 03mgmL of glycol-chitin forchitinase or 01mgmL of either of two chitosans (DA 50 orDA 35) [27] After electrophoresis (50mA for 4 h) the gelwas washed twice for 20min each in 50mM sodium acetatebuffer (pH 52 with 1TritonX-100) followed by twowashesin buffer without Triton X-100 The gel was incubated at37∘C for 12 h under shaking in 50mM sodium acetate buffer(pH 52) and stained with 001 Calcofluor White (SigmaSteinheim Germany) in 05M TrisHCl-buffer (pH 89) for5min and finally washed in deionized water for 1 h Theisozymes were visualized on a UV transilluminator A crudeextract of a known chitinolytic strain of Bacillus licheniformis[28] was run as a positive control





3 Results






















(a) (b) (c)


250150100

75

50

37

1 M 2 3 4 5 6 7 8 BL

(a)

250150100

75

50

37

1 M2 3 4 5 6 7 8 BL

(b)

250150100

75

50

37

1 M 2 3 4 5 6 7 8 BL

(c)







250150100755037

(kDa)

201510

2 3 6 7 10 M 11 12 13 14 16 17 18 20 21 22 23 24 25 M



PH 3

PH 1

0

3 7 10 11 12 17 18 20 21 23 3 7 10 12 17 18 20 23




4 Discussion





A1

A2

A3

A5

A6

D1

D3D4

An Dn 3c 3s 7c 7s 10c 10s 12c 12s co

(a)

A1

A2

A3

A5A6

D1

D3D4

An Dn 17c 17s 20c 20s 23c 23s co

(b)




5 Conclusions






Acknowledgments


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





3 Results






















(a) (b) (c)


250150100

75

50

37

1 M 2 3 4 5 6 7 8 BL

(a)

250150100

75

50

37

1 M2 3 4 5 6 7 8 BL

(b)

250150100

75

50

37

1 M 2 3 4 5 6 7 8 BL

(c)







250150100755037

(kDa)

201510

2 3 6 7 10 M 11 12 13 14 16 17 18 20 21 22 23 24 25 M



PH 3

PH 1

0

3 7 10 11 12 17 18 20 21 23 3 7 10 12 17 18 20 23




4 Discussion





A1

A2

A3

A5

A6

D1

D3D4

An Dn 3c 3s 7c 7s 10c 10s 12c 12s co

(a)

A1

A2

A3

A5A6

D1

D3D4

An Dn 17c 17s 20c 20s 23c 23s co

(b)




5 Conclusions






Acknowledgments


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b) (c)


250150100

75

50

37

1 M 2 3 4 5 6 7 8 BL

(a)

250150100

75

50

37

1 M2 3 4 5 6 7 8 BL

(b)

250150100

75

50

37

1 M 2 3 4 5 6 7 8 BL

(c)







250150100755037

(kDa)

201510

2 3 6 7 10 M 11 12 13 14 16 17 18 20 21 22 23 24 25 M



PH 3

PH 1

0

3 7 10 11 12 17 18 20 21 23 3 7 10 12 17 18 20 23




4 Discussion





A1

A2

A3

A5

A6

D1

D3D4

An Dn 3c 3s 7c 7s 10c 10s 12c 12s co

(a)

A1

A2

A3

A5A6

D1

D3D4

An Dn 17c 17s 20c 20s 23c 23s co

(b)




5 Conclusions






Acknowledgments


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


250150100755037

(kDa)

201510

2 3 6 7 10 M 11 12 13 14 16 17 18 20 21 22 23 24 25 M



PH 3

PH 1

0

3 7 10 11 12 17 18 20 21 23 3 7 10 12 17 18 20 23




4 Discussion





A1

A2

A3

A5

A6

D1

D3D4

An Dn 3c 3s 7c 7s 10c 10s 12c 12s co

(a)

A1

A2

A3

A5A6

D1

D3D4

An Dn 17c 17s 20c 20s 23c 23s co

(b)




5 Conclusions






Acknowledgments


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


A1

A2

A3

A5

A6

D1

D3D4

An Dn 3c 3s 7c 7s 10c 10s 12c 12s co

(a)

A1

A2

A3

A5A6

D1

D3D4

An Dn 17c 17s 20c 20s 23c 23s co

(b)




5 Conclusions






Acknowledgments


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





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

Research Article A High Diversity in Chitinolytic and Chitosanolytic …downloads.hindawi.com/journals/bmri/2015/857639.pdf · 2019-07-31 · Research Article A High Diversity in