isolationandcharacterizationof chitinase-producingbacillusandpaenibacillus...

7/23/2019 IsolationandCharacterizationof Chitinase-ProducingBacillusandPaenibacillus StrainsfromSaltedandFermented Shrimp

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Isolation and Characterization ofChitinase-ProducingBacillusandPaenibacillusStrains from Salted and FermentedShrimp,Acetes japonicusKook-Il Han, Bharat BhusanPatnaik, YongHyun Kim,Hyun-Jung Kwon, Yeon Soo Han, and Man-DeukHan

Abstract: Chitinases catalyze the conversion of chitin and are produced by a wide range of bacteria. The biological

applications of these enzymes have been exploited in food and pharmaceutical industries. We isolated 2 halophilic

chitinase-producing novel strains of bacteriaSCH-1 and SCH-2 from Saeu-jeot, a traditional Korean salted and fermented

food made with shrimp (Acetes japonicus). The isolated strains- SCH-1 and SCH-2 were Gram-positive, rod-shaped,

endospore-forming facultative anaerobes, with strain SCH-2 showing peritrichous flagella. Molecular characterization of

the 16S rRNA gene identified the strains SCH-1 and SCH-2 as Bacillussp. and Paenibacillussp. respectively. Basic Local

Alignment Search Tool and subsequent phylogenetic analysis of strain SCH-1 showed an identity of 97.83% with Bacillus

cereus ATCC 14579 (NR_074540), whereas strain SCH-2 showed an identity of 99.16% with Paenibacillus lautus JCM

9073 (NR_040882). Furthermore, the SCH-1 strain could use glucose, N-acetyl glucosamine, esculin, and maltose as

carbon source substrates. Cellular fatty acid analysis showed that iso-C15:0 and anteiso-C15:0 are the major acids in strain

SCH-1 and SCH-2, respectively. The SCH-1 strain showed a higher chitinase activity at 15.71 unit/mg protein comparedwith SCH-2 strain. Chitinase isozymes ofBacillussp. SCH-1was expressed as 2 bands having sizes of 41 and 50 kDa, and

as 4 bands with sizes of 30, 37, 45.7, and 50 kDa in Paenibacillussp. SCH-2. The rich chitinase activity with the isozyme

profiles of the isolated Bacillus and Paenibacillus strains provide advancement in the study of fermentation and may play

putative functions in the chitin bioconversion of sea crustacean foods.

Keywords: Bacillus sp., cellular fatty acids, chitinase, isozyme profile, Paenibacillus sp., salted and fermented shrimp,

16S rRNA gene

Practical Application: This is the 1st report for the isolation of chitinolyticBacillusand Paenibacillussp. from the Korean

traditional food, thejeotgal, made of salted and fermented shrimp (SFS), Acetes japonicus. The novel isolates available now

under Korean Collection for Type Cultures (KCTC) strains 33049 and 33051 could be beneficial for starter culture design

and preservation of SFS.

IntroductionChitinase, the chitin degrading enzyme have been found dis-

tributed in organisms as diverse as fungi, plants, insects, crus-

taceans, and bacteria and is involved in the process of producing

mono- and oligosaccharides from chitin (Ajit and others 2006;

Song and others 2012). Chitinase-producing marine bacteria play

an important role in the degradation of chitin in the oceans

(Orikoshi and others 2005). Fungi and bacteria are thought to be

important degraders of chitin in soil and thereby contribute towardthe recycling of carbon and nitrogen resources in soil ecosystems.

In bacteria, the primary role of the chitinase is thought to be the

digestion and utilization of chitin as a carbon and energy source

(Cohen-Kupiec and Chet 1998).

MS 20131559 Submitted 10/28/2013, Accepted 1/7/2014. Authors K.-I. Han,Kim, Kwon, and M.-D. Han are with Dept. of Biology, Soonchunhyang Univ., Asan,Chungnam, 336-745, Republic of Korea. Authors Patnaik and Y.S. Han are withDiv. of Plant Biotechnology, College of Agriculture and Life Science, Chonnam Natl.Univ., Gwangju, 500-757, Republic of Korea. Direct inquiries to author M.-D. Han(E-mail: [email protected]).

Chitinase genes have been cloned from diverse bacterial groups

(Shekhar and others 2006; Song and others 2012). Bacterial chiti-

nase members have been subdivided into 3 groups (group A, B,

and C), based on the amino acid sequence similarity in the C-

terminal catalytic domain (Suzuki and others 1999). They have a

size range of approximately 20 to 60 kDa and are typically smaller

than the plant (approximately 25 to 40 kDa) and insect chitinases

(approximately 40 to 85 kDa). Their stability over a wide range

of temperature (approximately 28 to 80

C) and pH (4.5 to 10),make them excellent candidates for applications under different

conditions. The foremost application have been in inhibiting the

phytopathogenic fungal growth by disorganization of their cell

walls, serving as biocontrol agents in agriculture (Jung and oth-

ers 2003). Transgenic technologies that include the expression of

bacterial chitinase genes into cereal crops have provided success in

resistance to common phytopathogenic fungal species (Barboza-

Corona and others 2003). The biotechnological applications of

Bacillus thuringiensis for the control of pests and fungi have beenrichly explored with the engineering of heterologous chitinase

genes from wide bacterial resources (Ramirez-Reyes and others

2004). Other major applications of bacterial and viral chitinases

C 2014Instituteof FoodTechnologists R

doi: 10.1111/1750-3841.12387 Vol. 79, Nr. 4, 2014 Journal of Food Science M665Further reproductionwithout permissionis prohibited


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Chitinase-rich isolates from shrimp food . . .

have been their inherent property to dissolve the chitin-containing

cell wall (Barboza-Corona and others 2003; Oh and others 2013),

and the acceleration of protoplast generation leading to the devel-

opment of economically viable strains for industrial use (Shimosaka

and others 2001). Furthermore, generation of bio-pharmaceutical

products as chitobiose and N-acetyl d-glucosamine by bacterial

chitinases has been seriously explored (Felse and Panda 2000).

Chitinase also play an important role in the bioconversion of shell-

fish waste to obtain value-added products (Wang and others 2006).

For the conversion of shrimp shell into commercially valued foodand chitosan products, it is imperative to isolate study chitinases

from high-salt inhabiting bacterial populations and their activity

in the degradation of marine crustaceans.

Salted and fermented food, either as an additive or as food in

itself, forms a delight in Korean cuisine. It is made by adding 20%

to 30% (w/w) salt to various types of seafood such as shrimp,

oyster, shellfish, fish, fish eggs, and fish intestines and becomes

palatable through subsequent preservation and fermentation. The

jeotgal is a traditional recipe from Korea that is made from tiny

shrimps (Acetes japonicus) and rock salt (Guan and others 2011).Salted and fermented shrimp (SFS) constitutes chitin-rich food

that serves good protein and chitosan sources, due to the decom-

position of shrimp shells and its body by chitinase-producing bac-teria from oceans (Ghorbel-Bellaaj and others 2012; Halder and

others 2012). To gain rich insights into commercially valuable food

preservation industry, isolation of chitinase and efficient bacteria

from SFS is necessary. To our knowledge, there are no available

reports on identification of such bacterial isolates toward elucidat-

ing the microbial community dynamics and chitinase-producing

bacteria from SFS. In addition, such microbial chitinases could

provide for broad-spectrum applications in industrial and scientific

environments. An earlier report has isolatedPaenibacillus tyramini-

gens sp. from Myeolchi-jeotgal, a traditional salted and fermented

anchovy (Engraulis japonicus), having high tyramine activity (Mahand others 2008). In this study, phenotypic, molecular, and bio-

chemical characterization of novel chitinase-rich bacterial strains

have been reported from salted and fermented food made with

small prawns (A. japonicus). These chitinases would be able to effi-

ciently degrade shrimp shell to obtain useful chitosan for humans.

This is the 1st description for chitinase-producing Bacillus andPaenibacillus sp. from Korean traditional food, jeotgal. The strains

SCH-1 and SCH-2 have been deposited to Korean Collection for

Type Cultures (KCTC) with No. 33049 and 33051, respectively.

Materials and Methods

Samples, culture, and isolation of chitinase-producingbacterial strains

Fresh shrimp (A. japonicus), fermented with 20% to 25% salt for

12 wk at 15 C were collected, serially diluted, and spread on 0.5%colloidal chitin marine agar (CCMA) (Difco, Mich., U.S.A.). The

composition of modified CCMA agar (per liter) was as follows:

peptone 5.0 g, yeast extract 1.0 g, ferric citrate 0.1 g, NaCl

19.45 g, MgCl2 8.8 g, Na2SO4 3.24 g, CaCl2 1.8 g, KCl

0.55 g, NaHCO3 0.16 g, KBr 80 mg, SrCl2 34 mg, H3BO3 22 mg, Na2SiO3 4 mg, NaF 2.4 mg, NH4NO3 1.6 mg,

Na2HPO4 8 mg, 0.5% (w/v) colloidal chitin, 20 g agar at pH 7.0

(Roberts and Selitrennikoff 1988). After 5 d of incubation at 37 C,

the isolates capable of degrading chitin with distinct zone of clear-

ance on CCMA were selected. All experiments for the enzymatic

tests were replicated 3 times. Typically, 5 different types of colonies

were collected from each plate based on differences in their mor-

phological, biochemical, and genetic characteristics. The collected

colonies were selected by successive transfer on CCMA medium.

Morphological and phenotypic characterizationsThe chitinase-rich bacterial isolates were treated according to

the procedure described by Weise and Rheinheimer (1978) and

Novitsky and MacSween (1989). Bacterial motility tests were

observed using a phase-contrast microscope. For morphological

characterization, the strains were cultivated for 24 h at 37 C

on marine agar medium and were subsequently prefixed in 2.5%glutaraldehyde for 1 h. The prefixed samples were washed and

postfixed in 1% osmium tetroxide (pH 7.2, 0.1 M phosphate

buffer) solution for 90 min. Following postfixation, the samples

were dehydrated in a graded ethanol series (60%, 70%, 80%,

90%, and 100% with each change for 10 min), and subsequently

in hydroxymexamethyldisilazane. After drying, the g rains were

attached to Scanning Electron Microscope stubs using double-

sided conductive tape and sputter coated with gold. The samples

were examined using Hitachi S-4700 Field Emission Scanning

Electron Microscope (Hitachi High-Technologies Corp., Japan)

with an acceleration tension of 40 kV.

The bacterial isolates were phenotypically characterized by us-

ing the API 50CH system tests (bioMerieux, Inc, Hazelwood,Mo., U.S.A.) as described by Logan and Berkeley (1984). The

API 50CH strips were inoculated with 2 McFarland standard sus-

pensions of bacterial cells in CHB/E medium (bioMerieux, Inc,

Durham, N.C., U.S.A.) as recommended by the manufacturer

and incubated at 37 C for 2 d. Carbohydrate fermentation test

was performed as described previously (Wauters and others 1998).

Other phenotypic tests included catalase activity and the effect of

salinity on growth (Snibert and Krieg 1994).

Phylogenetic analysis of 16S rRNA sequencesMolecular procedures were carried out as described by

Sambrook and Rusell (2001). Genomic DNA of the isolates was

extracted using genomic DNA preparation Kit (SolGent, Daejeon,

Korea), according to manufacturers instructions. The 16S rRNA

gene was polymerase chain reaction (PCR)-amplified using the

universal primers, 27f (5-AGAGTTTGATCCTGGCTCAG-3)

and 1492r (5-GGTTACCTTGTTACGACTT-3) in an UNO

II Thermo cycler (Biometra, Gottingen, Germany). The PCR

reaction mixture consisted of the template DNA, 0.5 mM of

each primer, 1 U of Taq polymerase (SolGent, Daejeon, Korea),

100 mM dNTPs, and 2.5 mM MgCl2. Samples were preheated

for 15 min at 95 C and then amplified for 30 cycles at 95 C

for 20 s, 50 C for 40 s, and 72 C for 90 s. Subsequent to PCR

amplification, 5 mL of each reaction was run on a 1% agarose

gel, and the DNA was visualized by UV illumination followed

with ethidium bromide staining. The amplified PCR products

were purified using the DNA clean up system (SolGent, Daejeon,Korea) according to manufacturers instructions. DNA sequences

were determined directly from the purified PCR products with

automated fluorescentTaqcycle sequencing using an ABI 3730XL

DNA Analyzer (Applied Biosystems, Foster City, Calif., U.S.A.).

The primers for sequencing used in this study were 27f and 1492r

(Johnson 1994).

Small-subunit rRNA sequences of Bacillus and Paenibacillus

reference strains were obtained from GenBank and the Ribo-

somal Database Project (RDP; Maidak and others 1999). The

16S rRNA sequence similarities of the chitinase-rich new isolates

were inferred by comparison with other gene sequences ofBacillus

and Paenibacillus sp., using the Basic Local Alignment Search

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Tool program at Natl. Center for Biotechnology Information.

Multiple-sequence alignment of 16S rRNA was conducted using

the Clustal W2 editor at www.ebi.ac.uk/tools/msa/clustalw2/.

The 5- and 3-gaps were edited using the BioEdit program

at (http://www.mbio.ncsu.edu/BioEdit/bioedit.html/). On the

basis of the molecular identification of the isolated strains SCH-1

and SCH-2, the nucleotide sequence information was submitted

to GenBank with accession nr. KC878876 and KC878877,

respectively.

Neighbor-joining (NJ) analysis (Saitou and Nei 1987) was per-formed using PHYLIP suite to find the phylogenetic positions

of the isolates. Evolutionary distances were calculated by using

the MEGA 5.0 Software (Tamura and others 2011) and bootstrap

analysis was used to evaluate the NJ tree topology, performing

1000 replicates and marked into branching points. The evolution-

ary distance matrix was estimated using the Kimuras 2-parameter

method (Kimura 1980).

Whole-cell fatty acid (FAME) analysesFor fatty acid compositional analysis (Van der Velde and oth-

ers 2006), the chitinase-rich isolates were grown on marine agar

(Difco, Becton, Dickinson Co., N.J., U.S.A.) and incubated for

24 h in an oxygen incubator. Approximately, 50 mg of the cellmass were harvested and transferred to a Teflon-lined screw cap

tube (13 100 mm, Coning, Inc, Corning, N.Y., U.S.A.) using

inoculation loops. To release the fatty acids, 1 mL of saponification

reagent (sodium hydroxide 45 g, methanol 150 mL, distilled

water 150 mL) was added, and the tubes were heated at 100 C

for 5 min. The methyl ester formation was cooled and 2 mL of

methylation reagent (6N hydrochloric acid 325 mL, methanol

27 mL) was added. The mixture was then heated for 10 min

at 80 C and after cooling, the fatty acid methyl esters (FAMEs)

were extracted by adding 1.25 mL of extraction solvent (hexane

20 mL, methyl-tert butyl ether 200 mL). After mixing for

10 min, the aqueous layer was removed and the organic layer was

washed with 3 mL of base (sodium hydroxide 10.8 g, distilled wa-

ter 900 mL). After mixing the samples for 5 min, the upper layer

was transferred to a gas chromatograph vial. A total of 2 L of the

FAMEs were then analyzed using a HP6890 gas chromatograph

(Agilent Technologies, Inc. Wilmington, Del., U.S.A.), with 5%

phenyl methyl silicone capillary column, 25 m 0.2 mm (Ag-

ilent Technologies, Diegem, Belgium) and Sherlock Microbial

Identification System version 6.1 (MIS, Microbial ID, Newark,

Del., U.S.A.). Sherlock MIS uses fatty acids of 9 to 20 carbons

in length. The peaks are automatically named and quantitated by

the system. The parameter settings of the gas chromatograph were

as follows: injection volume of 2 L, column split ratio 1:100,

injection port temperature 250 C, detector temperature 300 C,

column temperature 170 to 270 C at 5 C/min, and run time

of 22 min. Identification and comparison were made using theAerobe (TSBA version 3.9) database of the Sherlock MIS.

Growth kineticsThe cell growth was monitored at 620 nm using an UV-

spectrophotometer (Libra S12 UV spectrophotometer, Biochrom

Ltd, Cambridge, UK) to determine the temperature and pH range

of growth. Test strains were routinely grown using marine agar at

37 C, and maintained as a glycerol suspension (10%, w/v) at

80 C. The isolated strains were cultured at 37 C for 6 d in

colloidal chitin (CMB) medium (having 0.5% colloidal chitin).

Samples were collected daily for a period of 7 d for studying the

cell growth, pH, total protein, and chitinase enzyme activity.

Preparation of colloidal chitinColloidal chitin was prepared by partial hydrolysis of chitin

(Sigma-Aldrich, St. Louis, Mo., U.S.A.) using 10 N HCl and

left at 4 C overnight (Roberts and Selitrennikoff 1988). Sub-

sequently, the mixture was added to 95% ethanol and kept at

20 C overnight. The precipitate was collected by centrifugation

at 4000 g for 20 min at 4 C. The colloidal chitin was washedseveral times with sterile distilled water till pH 7.0. It was freeze-

dried to powder and stored at 4 C. A total of 2% colloidal chitin

was used for the chitinase activity experiments.

Determination of chitinase activityChitinase assay mixture consisted of 0.6 mL of sample and

0.4 mL of 2% colloidal chitin in 50 mM sodium acetate buffer

(pH 5.0) (Monreal and Reese 1969). The reaction was maintained

at 37 C for 30 min. Subsequently, the mixture was heated in boil-

ing water bath for 10 min and centrifuged at 10000 gfor 1 min,to remove the insoluble chitin. The resultant adduct of reducing

sugar was calculated using the dinitrosalicylic acid method (Miller

1959). The standard curve was generated from known concentra-

tions of GlcNAc (0 to 100 g). One unit of chitinase activity was

defined as the amount of enzyme that released 1 mol of GlcNAc

per hour. Protein concentrations were measured using the PierceBCA assay kit (Pierce Biotechnology, Rockford, Ill., U.S.A.).

Activity staining of chitinase using gel electrophoresisBacillussp. strain SCH-1 andPaenibacillussp. strain SCH-2 were

incubated in marine agar medium containing 0.5% colloidal chitin

at 37 C for 4 d. To investigate the expression patterns of chitinase

isozymes, the culture media were loaded on 12% sodium dodecyl

sulfatepolyacrylamide gel electrophoresis (SDSPAGE) gels and

electrophoresis was conducted using a Bio-Rad Mini-PROTEAN

gel system (Bio-Rad Laboratories, Hercules, Calif., U.S.A.), ac-

cording to the method described by Laemmli (1970). For assessing

the active staining of chitinase, a 12% SDSPAGE gel containing

0.01% glycol chitin was used (Trudel and Asselin 1989). After theelectrophoresis step, the gel was incubated in 100 mM sodium ac-

etate buffer (pH 5.0), containing 1% (v/v) Triton X-100 and 1%

skim milk at 37 C for 2 h. This was followed with incubation un-

der the same conditions, but without skim milk. Subsequently, the

gel was stained in a solution of 500 mM Tris-HCl (pH 8.9) con-

taining 0.01% Calcofluor white M2R (Daihan Sci. Co., WGD-30,

Korea).

Results

Isolation of chitinase-producing bacterial strainsA total of 63 morphologically different chitinolytic bacterial

colonies were isolated from 10 samples of SFS. On the basis of

colloidal chitin degradation and zone of clearance (>0.2 cm) onColloidal Chitin Agar plates, 2 colonies were selected for sec-

ondary screening in broth media. A parallel assessment was con-

ducted for the testing of enzyme activity. These potential isolates,

named as SCH-1 and SCH-2 had the maximum chitinolytic activ-

ity on agar medium containing 0.5% (w/v) colloidal chitin, show-

ing clear zones around colonies. The morphological, biochemical,

genetic, and fatty acid analysis of the isolates were investigated.

Initial observations suggested a mixed culture; thus, cells were

separated by streaking them into marine agar plates and finally bac-

teria displaying 2 different colony morphologies were obtained.

Scanning electron microscopic investigations revealed the mor-

phology of the isolated strains (Figure 1). No major differences

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Table 1Taxonomic characteristics of the isolated chitinolytic

strains from salted and fermented shrimp.

Bacillussp. SCH-1Paenibacillus sp.

SCH-2

Colony shape Round Round

Colony color Cream CreamShape of cell Rod RodCell size (m) (2.77 to 3.69) (1.23

to 1.36)(3.38 to 3.76) (0.63

to 0.66)

Motility +

Gram stain + +

were observed between SCH-1 and SCH-2 isolates, with respect

to colony shape and color, although the rod-shaped cells of SCH-

2 isolate were longer in size as compared with SCH-1 isolate

(Table 1; Figure 1A and 1B). SCH-2 isolate showed peritrichous

flagellation and was motile (Figure 1C and 1D). CCMA agar plates

were the sole carbon source and after incubation for 7 d at 37 C

on the medium, the strain colonies turned into circular form.

Phenotypic characterization of the isolates

In further investigating the identity of the isolates, we con-ducted a complete series of biochemical tests that includes catalase

test, oxidase test, methyl red test, VogesProskauer test, gelatin

liquefaction test, nitrate reduction test, and litmus milk reaction

(Table 2). The isolate Bacillus sp. SCH-1 showed a positive re-sponse with catalase, oxidase, Vogesproskauer, and gelatin hy-

drolysis test with stormy fermentation in the litmus milk reaction

and showed negative response to methyl red and nitrate reduction

tests. The isolatePaenibacillussp. SCH-2 was positive in the cata-

Table 2Phenotypic characteristics of the isolated chitinolytic

strains from salted and fermented shrimp.

Bacillussp. SCH-1 Paenibacillus sp. SCH-2

Catalase + +Oxidase +

Methyl red +

Voges proskauer + +Gelatin hydrolysis + Litmus Stormy fermentation Alkaline reaction

Nitrate reduction

Salt tolerance (%) 7 6.5

lase, methyl red, Vogesproskauer tests with alkaline reaction in the

litmus milk reaction test, although it was negative with the oxidase,

gelatin hydrolysis, and nitrate reduction tests. Furthermore, bio-

chemical characteristics of the isolated strains were complemented

with studies on the utilization of a variety of substrates as carbon

source. The strain SCH-1 (Bacillus sp.) was able to utilize glu-

cose, N-acetylglucosamine, esculin, and maltose as a substrate for

their growth. The SCH-2 strain (Paenibacillussp.) showed propen-sity to utilize a variety of carbon sources for its growth including

arabinose, glucose, N-acetylglucosamine, starch, and other sugars

(Table 3).

Molecular identity and phylogenetics of the isolatesMolecular identification of the isolated strains showing chiti-

nolytic activity was carried out based on 16S rDNA sequence

analysis. The nearly complete 16S rDNA sequence for SCH-1

(1450 bases; GenBank accession nr. KC878876) and SCH-2 strains

(1453 bases; GenBank accession nr. KC878877) were determined.

Figure 1Scanning electron micrographs of the isolated chitinase-producing strains SCH-1 (A and B) and SCH-2 (C and D).



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Table 3Physiological and biochemical characteristics of the isolates Bacillus sp. SCH-1 and Paenibacillus sp. SCH-2 based on theAPI 50 CH system and additional tests described in Materials and Methods section.

Carbohydrates Bacillus sp. SCH-1 Paenibacillus sp. SCH-2 Carbohydrates Bacillus sp. SCH-1 Paenibacillus sp. SCH-2

Erythritol Salicin +

d-Arabinose + Cellobiose +

l-Arabinose + Maltose + +

Ribose + Lactose +

d-Xylose + Melibiose +

l-Xylose Sucrose +

Adonitol Trehalose +Methyl--d-xylopyranoside + Inulin Galactose + Melezitose +

Glucose + + Raffinose +

Fructose + Starch +Mannose + Glycogen +Sorbose Xylitol

Rhamnose Gentiobiose +

Dulcitol d-Turanose +Inositol d-Lyxose Mannitol + d-Tagatose

Sorbitol d-Fucose

Methyl-, d-mannopyranoside l-Fucose +Methyl-, d-glucoside + d-Arabitol N-Acetyl-glucosamine + + l-Arabitol

Amygdalin + Gluconate

Arbutin + 2-keto-Gluconate

Esculin + + 5-keto-Gluconate

+, positive; , negative.

The sequence match option from the RDP and Basic Local Align-

ment Search Tool analysis of the GenBank was used to identify

the most similar sequences available in the database. Phylogenetic

trees based on the 16S rDNA sequence data of SCH-1 and SCH-2

strains were constructed (Figure 2). The tree topology inferred

that the SCH-1 strain belonged to the genus Bacillus, whereas

the SCH-2 strain was classified within the Paenibacillus genus.Figure 2A shows the relationship between strain SCH-1 and rep-

resentatives of the genusBacillus. Strain SCH-1 closely resembled

its nearest neighbors, Bacillus cereus ATCC 14579 (NR_074540),Bacillus thuringiensis IAM 12077 (NR_043403), and BacillusweihenstephanensisDSM 11821 (NR_024697) with sequence sim-

ilarities of 97.83%, 97.69%, and 97.48% respectively. Strain SCH-2

formed a highly significant clade with the members of the genus

Paenibacillus, and closely resembled Paenibacillus lautus JCM 9073

(NR_040882) with a sequence similarity of 99.16% (Figure 2B).

The SCH-2 strain also has about 97.57% and 96.81% similarity

with Paenibacillus glucanolyticus DSM 5162 and Paenibacillus lactis

MB1871, respectively. The Paenibacillusclade is also supported bya high bootstrap value of 95%. On the basis of pairwise 16S rDNA

gene similarities, it was evident that chitinolytic strains SCH-1 and

SCH-2 represent novel genomic species in the genus BacillusandPaenibacillus, respectively. This is the 1st report of identification of

novel Bacillus and Paenibacillus strains showing chitinase activity;isolated from the Korean traditional foodthejeotgal.

Hence, the morphological, physio-biochemical and molecular

evidence presented in the study, classifies the strains SCH-1 and

SCH-2 to be belonging to the genus Bacillus and Paenibacillus,

respectively.

Cellular fatty acid analysis of the strainsThe cellular fatty acids composition in both the aerobic,

endospore-forming, rod-shaped strains isolated from SFS were

determined using the gas chromatograph. Table 4 lists the fatty

acid compositions of the isolates, and the reference type strains ( B.

cereus JCM 2152 and P. lautus NRRL NRS-666). Most impor-

tantly, the fatty acids profile for both SCH-1 and SCH-2 strain

shows saturated iso- and anteiso-methyl-branched fatty acids. The

most often encountered fatty acids and with structures of this type

have 14 to 18 carbons in the chain are common constituents in

bacteria. Four fatty acids were present in abundant amounts in the

gas chromatograms profile of SCH-1 strain: iso-C15:0, iso-C16:0,

C16:0, and iso-C17:0fatty acid. The predominant cellular fatty acid

of theBacillusstrain SCH-1 was the 15:0 iso fatty acid (31.92%),

followed with the 16:0 iso fatty acid (16.31%). The most abundantfatty acid in the Paenibacillus strain SCH-2 was the C15:0 anteiso

fatty acid (39.39%), followed by C16:0iso fatty acid (16.79%). This

is indicative of the genusPaenibacillus(Shida and others 1997). Thefatty acids iso-C12:0, iso-C13:0, anteiso-C13:0, anteiso A-C17:0, and

iso-C17:1 w5c were not found in the gas chromatograph profile

of thePaenibacillus SCH-2 strain. These were detected in a lowersignificant percentage in the Bacillus SCH-1 strain. The unsatu-

rated fatty acids were found in trace amounts (>0.2%) in both

the strains. The differences in the levels of the C 14:0, iso-C15:0,

anteiso-C15:0, iso-C16:0, and C16:0 were sufficient to differentiate

the 2 novel strains.

Growth kinetics and chitinase activityThe isolated strainsBacillus sp. SCH-1 and Paenibacillus sp.

SCH-2- were grown aerobically in CMB medium (0.5% colloidal

chitin) at 37 C for 6 d to assess the cell growth, optimum pH

conditions, total protein content, and the chitinase activity. The

time course profile for the cell growth, pH, and total protein

content for Bacillus sp. SCH-1 and Paenibacillus sp. SCH-2 has

been depicted in Figure 3A and 3B, respectively. The Bacillus

strain SCH-1 grew rapidly for 2 d and subsequently showed a

marginal decline toward the end of the cultivation time. The pH

of the medium for the optimal growth of the strain was about 6.0

to 7.0. In the same medium, the protein content was found to

get declined as the growth rates of the strain increased. At 2 d of



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cultivation time, the total protein declined appreciably from the

start of the culture. In case of the Paenibacillus strain SCH-2, the

cell growth was high till 1 d of cultivation but thereafter showed a

steady decline till 2 d of cultivation, followed by a marked decline

at the later stages of cultivation. The optimal pH for growth of

the strain was 6.0 to 7.0. The total protein content of the medium

decreased with the cultivation time ofPaenibacillussp.

The chitinase activity of the Bacillussp. strain SCH-1 increased

along with the cell growth and reached maximum (12.52 unit/mg

protein) when the cell growth reached stationary phase at about 4 dof incubation. The chitinase activity for the Paenibacillussp. SCH-

2 strain also increased with the cell growth and reached maximum

(5.12 unit/mg protein), when the cell growth reached death phase

at 4 d of incubation (Figure 4). The chitinase isozymes in chitin

medium showed 2 bands at 41 and 50 kDa for theBacillussp. strainSCH-1. In contrast we observed 4 chitinase isozymic bands with

sizes of 30, 37, 45.7, and 50 kDa with the Paenibacillussp. SCH-2(Figure 5).

DiscussionSixty-three chitinase-producing bacterial colonies were isolated

from traditional Korean SFS on the selective medium contain-

ing colloidal chitin. Strains KC878876 (SCH-1) and KC878877(SCH-2) were selected for further study of chitinase activity as

they formed the largest clear zone on the chitin agar plate. We

confirmed the strains by studying their morphological, biochemi-

cal, and 16S rRNA-based molecular characteristics. Strain SCH-1

was identified as Bacillus sp. and strain SCH-2 was identified asPaenibacillussp. It is known that the Gram-positive bacterial genusBacillussecretes a number of degradative enzymes (Schallmey andothers 2004). Therefore, it was not surprising to isolate chitinase-

rich proteolytic strains ofBacillus in our study. Also in agreement

to our study, a Gram-positive, rod-shaped, endospore-forming

strain ofP. tyraminigens sp. has been characterized from traditional

Korean salted and fermented anchovy (E. japonicus) (Mah and

others 2008). It is expected that SFS includes other halotolerant

and/or halophilic bacteria along with the spore-forming bacte-ria. But the chitin degrading activity of other bacterial isolates in

this study was not considered to be significant. Some earlier re-

ports have isolatedSalimicrobiums andLactobacillus from the salted

and fermented anchovy (Lee and others 2012; Belfiore and others

2013). Paenibacillus chitinolyticus strain MP-306 has been isolatedfrom the cast-off shells of cicads with strong chitinase activity

(Song and others 2012). A proteolytic but chitinase-deficient mi-

crobial culture has been isolated from shrimp shell waste that was

characterized as Bacillus licheniformis (Waldeck and others 2006).

Apart from the above, chitinase-producing marine bacteria have

been isolated that play important roles in degradation of chitin in

oceans (Annamalai and others 2010, 2011).

Physiological tests (API) (bioMerieux, Inc., Hazelwood, Mo.,U.S.A.), 16S rRNA gene sequence comparison (Goto and oth-

ers 2000), as well as microscopic and macroscopic investigations,

revealed differences between the bacterial isolates SCH-1 and

SCH-2. Our observations can be considered reliable, as we have

Figure 2Phylogenetic tree based on 16S rRNA sequence showing the position of strains SCH-1 (A) and SCH-2 (B) and their relationships within theBacillusandPaenibacillusgroup. The tree was constructed using the neighbor-joining method with Kimura 2-parameter distance matrix and pairwisedeletion. Numbers at nodes representthe bootstrap percentage (based on 1000 replicates).The scale bar indicates 0.005 substitutions per nucleotide

position.



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Table 4Fatty acid composition of the isolates Bacillus sp. SCH-1 and Paenibacillussp. SCH-2 and the reference test species (BacilluscereusJCM 2152T and Paenibacillus lautus NRRL NRS-666T).

Fatty acid composition (%) Bacillus sp. SCH-1 Bacillus cereusJCM 2152T Paenibacillus sp. SCH-2 Paenibacillus lautus NRRL NRS-666T

Saturated acids

C13:0 Tr 0.8 Tr Tr C14:0 3.62 3.1 1.08 1.1

C15:0 ND 4.9 ND 0.3C16:0 11.22 2.4 15.67 15.6C17:0 0.46 Tr 0.27 Tr

C18:0 Tr Tr 0.37 Tr Unsaturated acids

C16:1w7c alcohol Tr Tr Tr Tr C16:1w9c Tr 1.1 Tr Tr C16:1w11c Tr 4.4 Tr 2.0

Branched acids

iso-C12:0 0.75 Tr Tr Tr iso-C13:0 2.25 7.8 Tr Tr anteiso-C13:0 0.67 0.6 Tr Tr

iso-C14:0 3.90 2.4 2.03 0.8iso-C15:0 31.92 48.7 7.20 1.5anteiso-C15:0 4.06 3.8 39.39 57.3iso-C16:0 16.31 2.7 16.79 7.4

anteiso A-C17:1 0.85 Tr Tr Tr iso-C17:0 10.03 6.2 6.03 1.2

anteiso-C17:0 3.33 0.7 10.88 9.7iso-C17:1 w5c 1.04 Tr Tr Tr

iso-C17:1 w10c Tr 2.8 Tr 0.2iso-C18:0 Tr ND 0.29 ND

Values are percentages of total fatty acids.Tr, trace (


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conducted 16S rRNA gene sequence comparisons in conjunc-

tion with a number of phenotypic and phylogenetic properties.

It is important to note that 2 distinct species may have identical

16S rRNA gene sequences, and therefore are unreliable without

other evidences (Stackerbrandt and others 2002).

The SCH-1 strain has shown highest homology with B.cereus ATCC 14579 (NR_074540) and also substantially high

relatedness with other Bacillus sp. B. cereus is a Gram-positive,

facultative anaerobic rod-shaped bacterium seen ubiquitously in

soil and also found in many raw and processed foods such as

rice, milk, dairy products, spices, and vegetables (de Vries and

others 2004). The mixed culture consisting of 2 B. licheniformis

strains, isolated from shrimp shell waste were found to encode a

frameshift mutated chitinase, thus showing the absence of chiti-

nolytic activities (Waldeck and others 2006). Phylogenetic analysis

for SCH-2 strain revealed its existence within the Paenibacillus

Figure 4Determination of chitinase activity byBacillussp.SCH-1 andPaenibacillussp. SCH-2 cultivated in CMB mediumat 37 C for 6 d. ()Bacillussp. SCH-1; ()Paenibacillussp.SCH-2.

Figure 5Chitinase activity staining ofBacillussp. strain SCH-1 andPaenibacillusstrainSCH-2 on SDSPAGE after M2R staining. Thebacterial isolates were grown in marine agarmedium containing 0.5% colloidal chitin.



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clade and showed highest 16S rRNA gene similarities with P.lautus strain JCM 9073 (NR_040882). P. lautus JCM 9073 have

been isolated from sources that include soil, water, plant rhizo-

sphere, plant materials, food, and fodder (Daane and others 2002;

Lee and others 2013). The P. tyraminigens strain earlier reported

from E. japonicus also formed a highly significant monophyleticclade with the members of the genus Paenibacillus(Mah and oth-

ers 2008). The bootstrap value (95%) shown was in agreement

with our study. The genus Paenibacillus is phenotypically related

to the Bacillus, and is generally characterized as rod-shaped andendospore-forming bacteria with DNA G+C content of 39 to 54

mol% (Saha and others 2005).

Cellular fatty acid analysis falls under the category chemotaxon-

omy and has been applied for the bacterial identification process.

We utilize the gas chromatograph to understand fatty acid profile

of both the chitinolytic strains. The major cellular fatty acid was

iso-C15:0in Bacillussp. and the anteiso-C15:0in Paenibacillussp. Wealso found other iso- and anteiso- forms of branched cellular fatty

acids. As per the literature, the composition ofBacillus sp. seems

to be complex with typically 7 or more iso- or anteiso- forms

(Ehrhardt and others 2010). In case of B. cereus, large amounts

of C16:0, iso-C15:0, iso-C15:0, iso-C17:0, iso-C14:0, C15:0, anteiso-

C15:0, and anteiso-C13:0 fatty acids are found (Lawrence and oth-ers 1991). Bacillus strain SCH-1 characterized in our study also

showed a higher proportion of C16:0 iso and iso-C17:0 that agrees

to the available report. Chromatographic analysis of FAMEs has

also been developed that gives an added advantage, as over 1500

species of bacteria, includingB. anthrax, used as potential agents in

biological terrorism have been identified (Teska and others 2001).

This was attributed to the unique cellular fatty acid profile ofB.anthracisthat distinguished it from other related Bacillus sp. The

anteiso-C15:0 fatty acid was also the predominant fatty acid in the

Paenibacillussp. isolated from salted and fermented anchovy that is

very relevant to our findings (Mah and others 2008). The anteiso-

C15:0 value was lower at 39.39% in our study, although it can be

useful to differentiate the bacterial strains.

We also made a study on the cell growth characteristics and pH

development of the 2 chitinolytic strainsSCH-1 and SCH-2-

isolated from our SFS samples. The total protein in the media

at the start of the cultivation period and its utilization for cell

growth has also been observed. Both the Bacillus and Paenibacillus

sp. cell growth increased rapidly for 2 d and then maintained

in the medium although at a lower level. Similar observations

have been recorded forP. chitinolyticus grown in Luria Bertani

(LB) medium with 0.5% colloidal chitin (Song and others 2012).

The pH of the culture dropped during the exponential phase

of growth, but subsequently increased, although the cell growth

declined over the cultivation period. This decrease in pH can be

attributed to the fermentation of glucose as subsequently when the

glucose level depleted, the pH of the culture started rising again.Upon the substrate and protein exhaustion from the medium, the

cells entered the stationary phase and may form aggregates. Cell

aggregation is a special event within the B. cereus group and may

play a role in the transfer of genetic material (Helgason and others

2000). The growth characteristics and the sporulation process in

B. cereusATCC 14579 have been extensively studied (de Vries and

others 2004).

Chitinase activity ofBacillusstrain SCH-1 andPaenibacillusstrain

SCH-2 increased rapidly and was maximum at about 4 d of cultiva-

tion in colloidal chitin medium. The trend of chitinase activity for

both the isolated strains were found similar, althoughBacillusstrain

SCH-1 would have a stronger chitinolytic activity than thePaeni-

bacillusstrain SCH-2. It is known that under lower environmen-

tal pH and depleted protein, the cell growth stopped increasing.

With a subsequent upsurge in pH, a sharp increment of chiti-

nase secretion is observed, indicating an alteration to secondary

metabolism (Yan and others 2011). The reasons for decreased pro-

duction may be the lack of nutrients in the medium resulting in

the inactivation of the enzyme synthesis machinery (Nochur and

others 1993). Furthermore, according to the adaptations (alkaline

and acidic conditions) of the isolated strains, the difference in the

chitinase enzyme activity is noticed. Bacillus sp. K2914 showedoptimal chitinase production in 0.5% concentration of colloidal

chitin and crustacean waste powder in combination after 8 d of

cultivation (Uria and others 2005). Paenibacillussp. D1 showed the

highest chitinase activity in pH 5.0 at 50 C (Singh and Chhat-

par 2011). Previous reports have suggested that B. laterosporous

(Shanmugaiah and others 2008) and Aeromonas punctata (Kuddus

and Ahmed 2013) are capable for producing highest chitinase at

alkaline conditions. It is known that chitinases from B. cereuspro-

vide the best activity at an acidic or near neutral pH (Wang and

others, 2001; Chang and others 2003). Chitinase activity in B.

cereus SV1 was found maximum at 55 C and pH 7.0 in me-

dia containing shrimp shell powder (Ghorbel-Bellaaj and others

2012). In an earlier report, chitinases produced from B. amyloliq-uefaciens have been reported to show a pH optimum close to 7

(Wang and others 2002). Other bacterial chitinases isolated from

marine sources, show broader pH optima, with maximal activity

in neutral or slightly alkaline conditions (Wang and Chang 1997;

Park and others 2000). In our study on the chitinase isozymes, we

observed 4 bands of chitinase from Paenibacillus strain, in contrastto 2 bands from Bacillus strain isolated in this study. Recent re-

port has suggested differential expression patterns ofP. chitinolyticus

MP-306 chitinase isozymes in both colloidal chitin and LB media.

Three chitinase isozyme bands were observed irrespective of the

medium in Native-gel (Song and others 2012). Three chitinase

isozymes (63, 54, and 38 kDa) were observed in a SDSPAGE

gel forP. illinoisensis KJA-424 having antifungal activity (Jung and

others 2005).

ConclusionsWe have noticed that in recent years scientific thrust on chitinase

purification and production has led to exploring the Korean tradi-

tional fermented food, including the vegetable kimchi, soybean

paste and most recently the salted and beneficial microorganisms.

The results of this study, advances our knowledge on the iso-

lated bacterial strains having high chitinase activity from SFS. This

would be extremely useful in the development of starter culture

design and preservation of SFS. These novel identified bacterial

isolates would be useful in improving the traditional fermented

food.

AcknowledgmentsThis work was supported in part by the Soonchunhyang Univ.

Research Fund.

Author ContributionsKook-Il Han performed the experiments, analyzed the data, and

wrote the paper. Bharat Bhusan Patnaik wrote the paper and ana-

lyzed the data. Yong Hyun Kim and Hyun-Jung Kwon performed

the experiments. Yeon Soo Han provided technical details. Man-

Deuk Han designed the experiments, analyzed the data, provided

technical details, and wrote the paper.



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