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Identification, purification, and expression patterns of chitinase from psychrotolerant Pedobacter sp. PR-M6 and antifungal activity in vitro
Microbial Pathogenesis 107 (2017) 62e68
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Identification, purification, and expression patterns of chitinase from psychrotolerant Pedobacter sp. PR-M6 and antifungal activity in vitro Yong-Su Song, Dong-Jun Seo, Woo-Jin Jung* Department of Agricultural Chemistry, Institute of Environmentally-Friendly Agriculture (IEFA), College of Agricultural and Life Science, Chonnam National University, Gwangju 61186, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 January 2017 Received in revised form 28 February 2017 Accepted 13 March 2017 Available online 19 March 2017
In this study, a novel psychrotolerant chitinolytic bacterium Pedobacter sp. PR-M6 that displayed strong chitinolytic activity on 0.5% colloidal chitin was isolated from the soil of a decayed mushroom. Chitinase activity of PR-M6 at 25 C (C25) after 6 days of incubation with colloidal chitin increased rapidly to a maximum level (31.3 U/mg proteins). Three chitinase isozymes (chiII, chiIII, and chiIV) from the crude enzyme at 25 C (C25) incubation were expressed on SDS-PAGE gels at 25 C. After purification by chitinaffinity chromatography, six chitinase isozymes (chiI, chiII, chiIII, chiIV, chiV, and chiVI) from C25fractions were expressed on SDS-PAGE gels at 25 C. Major bands of chitinase isozymes (chiI, chiII, and chiIII) from C4-fractions were strongly expressed on SDS-PAGE gels at 25 C. Pedobacter sp. PR-M6 showed high inhibition rate of 60.9% and 57.5% against Rhizoctonia solani and Botrytis cinerea, respectively. These results indicated that psychrotolerant Pedobacter sp. PR-M6 could be applied widely as a microorganism agent for the biocontrol of agricultural phytopathogens at low temperatures. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Psychrotolerance Pedobacter sp. Chitinolytic activity Chitinase isozyme
1. Introduction Chitin exists in edible fungi such as Agaricus bisporus, Pleurotus ostreatus, and Lentinula edodes [1], and the covalent link between chitin and b-glucan has been studied in Schizophyllum commune (Basidiomyceta) [2]. Chitin also exists in yeast and filamentous fungi with glucan and chitosan in cell walls [3]. Chitinase obtained from microorganisms is fundamental in the enzymatic production of chitin oligosaccharides for industrial applications. Recently, research of psychrophilic enzymes in biotechnological applications and protein structure-function has increased. The structural properties and function of psychrophilic chitinase have been reported in the yeast strain Glaciozyma antarctica PI12 [4]. The biochemical properties, action mode of oligosaccharide production, and catalytic mechanism of psychrophilic chitinase has been reported in the marine bacteria, Pseudoalteromonas sp. DL-6 [5] and Moritella marina [6]. The purification and optimal production of cold-active endochitinase has been reported in the Antarctic bacterium, Sanguibacter antarcticus KOPRI 21702 [7,8] and this Chi21702 recombinant chitinase was expressed in the
* Corresponding author. E-mail address: [email protected] (W.-J. Jung). http://dx.doi.org/10.1016/j.micpath.2017.03.018 0882-4010/© 2017 Elsevier Ltd. All rights reserved.
methylotrophic yeast, Pichia pastoris, for the mass production of chitinase [9]. Also, a chitinolytic bacterial community has been collected from penguin guano at low temperatures, belonging to Janthinobacterium sp., Stenotrophomonas sp. of g-Proteobacteria, Cytophaga sp. of the Cytophaga-Flexibacter-Bacteroides group, and Streptomyces and Nocardiopsis sp. of Actinobacteria in Antarctica [10]. The antifungal activity of chitinase produced from the Antarctic bacterium, Verticillium lecanii A3, has been reported against some spoiling agents of refrigerated foods, such as Mucor plumbeus, Penicillium verrucosum, Aspergillus versicolor, and Cladosporium cladosporioides [11]. Cold-responsive extracellular chitinase has been characterized in forage bromegrass (Bromus inermis) culture cells to determine its relationship with antifreeze activity [12]. Recently, oligosaccharides produced from chitinase were used in several biotechnological applications, such as the extraction of antioxidants from marine waste using Bacillus sp [13]. and Serratia marcescens [14]. However, limited studies focusing on the characteristics of psychrophilic chitinases produced from bacteria have been conducted. The objectives of this study were to investigate the expression patterns of psychrophilic chitinase on SDS-PAGE gels after chitinase purification, and to estimate the effects of different culture temperatures on the chitinase activity from psychrotolerant Pedobacter sp. PR-M6. To our knowledge, this is the first report to
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Fig. 1. Nine chitinolytic bacteria showed halo formation around their colonies on 0.5% winter mushroom (Flammulina velvtipes) powder agar at 30 C after 3 days of incubation (A). Bacterium No. 6 (circle of red dotted) having the strongest cell growth and mushroomlytic activity incubated on 0.5% colloidal chitin agar medium at 30 C (B) and 25 C (C) for 13 days, and at 4 C for 30 days (D). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
investigate the expression of various extracellular chitinase isozymes in Pedobacter sp. 2. Materials and methods 2.1. Isolation and identification of the chitinolytic bacterium Rot mushroom soils were collected in an arboretum of the Chonnam National University, Gwangju, Korea. The soil samples were grained and inoculated on a 0.5% colloidal chitin (pH 6.0) agar medium at 30 C for 5 days. Nine bacteria among them were inoculated on 0.5% winter mushroom (Flammulina velvtipes) powder agar and then incubated at 30 C for 3 days. One bacterium with the strongest chitinolytic activity was selected and used for further characterization. For identification of the bacterium, polymerase chain reaction (PCR), (GeneAmp 9700, Applied Biosystems, USA) was performed to amplify part of the 16S rRNA gene of the bacterium. The nucleotide sequence of the 16S rRNA gene of PR-M6 was determined using an ABI PRISM Big Dye™ Terminator Cycle Sequencing Kit (Applied Biosystems, USA) and ABI PRISM 3730xl Analyzer (Applied
Biosystems) in Geno Tech Co. (Daejeon, Korea). The nucleotide sequence of the 16S rRNA gene of PR-M6 was compared with published 16S rRNA sequences using a blast search at NCBI [15]. The phylogenetic trees of isolate PR-M6 were constructed using the maximum likelihood method based on the Jukes-Cantor model [16]. Evolutionary analyses were conducted using MEGA7 [17]. 2.2. Determination of chitinase activity The chitinase assay consisted of a mixture of 50 mL sample, 500 mL 0.5% colloidal chitin, and 450 mL 50 mM sodium acetate buffer (pH 5.0) [18]. The mixture including the sample was incubated at 25 C and 37 C for 3 h, and then 200 mL 1 N NaOH was added to stop the enzyme reaction. Next, the sample was briefly centrifuged (10,000g, 5 min), after which 500 mL supernatant was mixed with 1 mL Schales' reagent and then heated in boiling water for 15 min. The absorbance was then immediately measured at 420 nm using a spectrophotometer (Mecasys, Optizen 3220UV, Korea). The amount of reducing sugar was calculated based on a comparison with a standard curve generated from different concentrations of GlcNAc (0e100 mg). One unit of chitinase activity was
Fig. 2. Phylogenetic tree of 16S rRNA from the PR-M6 strain using the maximum likelihood method based on the Jukes-Cantor model. The scale bar indicates 1 base change per 20 nucleotides.
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Fig. 3. Time course of cell growth (A), and protein content (B) of Pedobacter sp. PR-M6 at 0, 1, 2, 3, 4, 5, and 6 days after incubation in 0.5% colloidal chitin medium at 25 C (-B- C25) and 30 C (-C- C30).
Fig. 4. Time course of chitinase activity of Pedobacter sp. PR-M6. The bacterium was grown in 0.5% colloidal chitin medium at 25 C (-B- C25) and 30 C (-C- C30) for 6 days. The chitinase produced from PR-M6 was assayed at 25 C (A) and 37 C (B) as a condition of the enzyme reaction.
2.4. Chitinase purification defined as the amount of enzyme that liberated 1 mmol of GlcNAc per h. The protein concentration was determined using the method described by Bradford [19]. 2.3. Chitinase staining activity on SDS-PAGE gel Pedobacter sp. PR-M6 was incubated on 0.5% colloidal chitin medium at 25 C and 30 C for 6 days. After incubation, the proteins at two culture temperatures were loaded on SDS-PAGE gels to investigate the expression patterns of chitinase isozymes, according to the method described by Laemmli [20]. Electrophoresis was performed using a Bio-Rad Mini-PROTEAN (80 73 1.5 mm), to evaluate the active staining of chitinase, 10% SDS-PAGE containing 0.01% glycol chitin was conducted according to the method described by Trudel and Asselin [21]. The gel was incubated in 50 mM sodium acetate buffer (pH 5.0) containing 1% (v/v) Triton X100 and 1% skim milk at 37 C for 2 h with reciprocal shaking. A subsequent incubation was then conducted overnight under the same conditions, but without skim milk, in buffer solution. The gel was then immersed in 500 mM TriseHCl (pH 8.9) solution containing 0.01% Calcofluor White M2R (Sigma F3397). The lysed zones were visualized and photographed using a UV transilluminator (Daihan Sci. Co., WGD-30, Korea).
For preparation of the chitin affinity column, regenerated chitin from chitosan (deacetylation 92% and viscosity 20 cPs) was obtained using a modified method described by Molano et al. [22]. The regenerated chitin was equilibrated in 20 mM sodium carbonate buffer (pH 8.4), after which the crude enzyme of PR-M6 was loaded onto a regenerated chitin column (4 10 cm) at a flow rate of 0.5 mL/min. The first washing was conducted using 500 mL 20 mM sodium carbonate buffer (pH 8.4) until the eluate was free of proteins based on detection by measuring the absorbance at 280 nm. The second washing was conducted with 100 mL 20 mM sodium acetate buffer (pH 5.3). The proteins from PR-M6 were finally released from the chitin matrix with 20 mM acetic acid (pH 3.2) and then 5 mL was collected in individual tubes to give a total of 100 fractions [23]. 2.5. Mycelial growth inhibition To investigate the inhibition of mycelial growth with strain PRM6, mycelial discs (8 mm in diameter) of test fungi (Rhizoctonia solani KACC40111, Botrytis cinerea KACC40574, Alternaria brassicicola KACC40036, and Fusarium oxysporum KACC47615) grown on PDA plates were cut from the margins of the colony and were
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placed on plates. After incubation at 25 C for 54e160 h, and the distances between the edges of the PRC-5 and fungal mycelium were measured [24]. Control plates consisted of PDA only. The percentage of inhibition was calculated based on the percentage inhibition of radial growth (IRG) as follows: IRG (%) ¼ [(GCeGT)/ GC] 100, where GC and GT represent mycelial growth in the control and treatment, respectively [25]. 3. Results and discussion 3.1. Identification of the chitinolytic bacterium Nine bacteria showing chitinolytic activity were inoculated on 0.5% winter mushroom (F. velvtipes) powder agar and then incubated at 30 C for 3 days. From the incubation of bacteria, one bacterium, No. 6, having the strongest mushroomlytic activity and high cell growth was finally selected (Fig. 1A). Chitinolytic activity of the PR-M6 strain showed a stronger clear zone at 25 C than at 30 C incubation after 13 days on 0.5% colloidal chitin plates (Fig. 1B and C). In addition, cell growth and chitinolytic activity was clearly shown at 4 C after incubation for 30 days (Fig. 1D). Recombinant chitinase ChiA from the psychrophilic bacterium, Pseudoalteromonas sp. DL-6, showed optimal chitinolytic activity at 20 C incubation for 24 h on 4MU-(GlcNAc)2 as a substrate [5]. The recombinant ChiA exhibited 75% at 4 C and 79% at 10 C of its maximal activity, respectively. On the basis of the nucleotide sequence of a conserved segment of the 16S rRNA gene, the bacterium was identified as Pedobacter sp. and named Pedobacter sp. PR-M6. The PR-M6 strain showed 99% homology with Pedobacter sp. Mal11-5, which showed 99% homology with P. steynii WB2.3-45 (Accession No. NR042605) [26]. Based on its 16S rRNA sequence and its position in the phylogenetic tree (Fig. 2), this bacterium was designated as Pedobacter sp. PR-M6, which is taxonomically close to Pedobacter sp. Mal11-5. Pedobacter sp. PR-M6 was taxonomically closer to P. caeni than to P. steynii. The Pedobacter genus in the Sphingobacteriaceae family is characterized by Gram-negative, aerobic, and rod-shaped bacteria [27]. P. caeni sp. nov. was isolated from a nitrifying inoculum [28], and
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P. arcticus sp. nov. A12T was originally isolated from tundra soil [29]. Psychrotolerant Pedobacter sp. KFC76 was isolated from soil from the Kafni Glacier, Himalaya [30].
3.2. Cell growth and chitinase activity Pedobacter sp. PR-M6 was incubated in a 0.5% colloidal chitin medium at 25 C and 30 C for 6 days; cell growth of Pedobacter sp. PR-M6 was higher at 25 C than 30 C (Fig. 3A). Cell growth of PRM6 gradually increased for 5 days and decreased slightly on day 6 when cultured at 25 C on 0.5% colloidal chitin liquid medium. The protein content was measured after six days of incubation. The protein content of the Pedobacter sp. PR-M6 medium was higher at 30 C than 25 C after three days of incubation (Fig. 3B), and was maintained at a higher amount at 25 C than that at 30 C. The protein content of PR-M6 showed a maximum amount (133.5 mg/ mL) at 25 C after 5 days of incubation. For the time course of chitinase activity of Pedobacter sp. PR-M6, the bacterium was grown in 0.5% colloidal chitin medium at 25 C (-B- C25) and 30 C (-C- C30) for 6 days (Fig. 4). Chitinase activity from PR-M6 measured at 25 and 37 C as a condition of the enzyme reaction. Chitinase activity of PR-M6 after 6 days of incubation at 25 C (C25) with colloidal chitin increased rapidly to a maximum level (31.3 U/mg proteins) (Fig. 4A). Chitinase activity of PR-M6 at 30 C incubation (C30) was < 10 U/mg proteins after 6 days. Chitinase activity of PR-M6 after 6 days of incubation at 25 C (C25) with colloidal chitin increased rapidly to a maximum level (21.0 U/ mg proteins) by 37 C treatment as a condition of the enzyme reaction (Fig. 4B). Chitinase activity of PR-M6 after 5 days of incubation at 30 C (C30) with colloidal chitin increased slightly with 17 U/mg proteins by 37 C treatment as a condition of the enzyme reaction. The optimum temperature of chitinase activity was 15 C in cold-adapted chitinase (chiII) from Glaciozyma antarctica PI12 [31], and 28 C in the marine psychrophilic bacterium, Moritella marina [6]. Especially, the optimum temperature of bacterial culture was shown at 30 C for the cold-adapted chitinase B of Alteromonas sp. O-7 [32], and chitinase A of Vibrio sp. Fi:7 [33].
Fig. 5. Expression patterns of chitinase activity on SDS-PAGE gels after M2R staining with different temperatures. Pedobacter sp. PR-M6 strain was grown in 0.5% colloidal chitin medium at 25 C (C25) and 30 C (C30) for 6 days. The chitinase (25 mL of culture broth) produced from PR-M6 expressed at 25 C (A and C) and 37 C (B and D) as a condition of the enzyme reaction on SDS-PAGE gels. Lanes D0, D1, D2, D3, D4, D5, and D6 are 0e6 days after incubation respectively. M: Protein marks.
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Fig. 6. Expression patterns of chitinase from PR-M6 grown in 0.5% colloidal chitin medium at 25 C for 5 days. Chitinase activity of the fraction solutions (C25-fractions) collected from the chitin-affinity column expressed on SDS-PAGE gels at different temperatures of 25 C (A) and 37 C (B) as a condition of the enzyme reaction. The crude enzyme of the PRM6 strain after 5 days of incubation with LB medium at 25 C (LB), and colloidal chitin medium at 30 C (C30) and 25 C (C25). Protein marks (M).
3.3. Properties of chitinase isozymes Pedobacter sp. PR-M6 was grown in 0.5% colloidal chitin medium at 25 C (C25) and 30 C (C30) for 6 days. The expression pattern of chitinase from PR-M6 was examined on SDS-PAGE gels (Fig. 5). Chitinase isozyme bands were shown as chiII, chiIII, and chiIV on
SDS-PAGE gels at 25 C as a condition of the enzyme reaction (Fig. 5A and C). Chitinase isozyme bands were not detected on the SDS-PAGE gels at 37 C as a condition of the enzyme reaction (Fig. 5B and D). From these results, chitinase activity was strongly inhibited at 37 C. Chitinase isozymes were slightly expressed on SDS-PAGE gels at day 1 (chiIII, 40 kDa) and day 2 (chiII, 47 kDa)
Fig. 7. Expression patterns of chitinase from PR-M6 grown in 0.5% colloidal chitin medium at 4 C for 14 days. Chitinase activity of the fraction solutions (C4-fractions) collected from the chitin-affinity column expressed on SDS-PAGE gels at different temperatures of 4 C (A), 25 C (B) and 37 C (C) as a condition of the enzyme reaction. The crude enzyme of PR-M6 incubated with 0.5% colloidal chitin medium at 25 C (C25) and 30 C (C30) for 5 days and 4 C (C4) for 14 days. Protein marks (M).
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isozyme (chiII) activity in the C4 crude enzyme was clearly inhibited at 37 C, and chitinase isozymes (chiIII) activity was weak (Fig. 7C). Purified chitinase isozymes (chiIII) from C4-fractions were strongly expressed 37 C (Fig. 7C). 3.4. Antifungal activity of PR-M6 against phytophathogens
Fig. 8. Inhibition on mycelial radial growth of four phytopathogens [R. solani (A0 and A1) at 72 h, B. cinerea (B0 and B1) at 144 h, A. brassicicola (C0 and C1) at 144 h, and F. oxysporum (D0 and D1) at 144 h] by Pedobacter sp. PR-M6 (A). Inhibition rate for fungal growth of phytopathogens (B). Values are means ± SE of three replicates. Bars carrying different letters are significantly different at p < 0.05 [Tukey's Studentized Range (HSD) test].
(Fig. 5A and C). Especially, chitinase isozyme bands in C25 incubation were expressed strongly at days 5 and 6 of incubation on SDS-PAGE gels at 25 C (Fig. 5A). This result coincided with the protein content at days 5 and 6 of incubation (Fig. 4A). The molecular weight of psychrophilic chitinase from G. antarctica PI12 was about 39 and 50 kDa, respectively [30]. Pedobacter sp. PR-M6 was incubated in 0.5% colloidal chitin medium at 25 C for 5 days. To investigate the PR-M6 chitinase activity, LB, C30, and C25 crude enzymes were loaded on SDS-PAGE gels. After partial purification of the C25 crude enzyme by a chitinaffinity column, the C25-fraction solutions were loaded on SDSPAGE gels. The chitinase isozymes were expressed on SDS-PAGE gels at different temperatures of 25 C (A) and 37 C (B) as a condition of the enzyme reaction (Fig. 6). Chitinase isozymes (chiI, chiV, and chiVI) from C25-fractions were clearly expressed on SDSPAGE gels at 25 C (Fig. 6A). In Fig. 6A, chitinase isozymes from C25fractions were expressed as major bands (chiI, chiII, chiIII, and chiIV) and minor bands (chiV and chiVI). Chitinase isozymes (chiIII, chiV, and chiVI) from C25-fractions were expressed on SDS-PAGE gels at 37 C (Fig. 6B). Chitinase isozymes (chiI and chiII) from C4-fractions were expressed more clearly than chiIII on SDS-PAGE gels at 4 C (Fig. 7A). In Fig. 7A, chitinase isozymes chiIII were expressed in C25 and C30 crude enzymes on SDS-PAGE gels and but were not expressed in the C4 crude enzyme. Chitinase isozyme (chiI and chiII) activity of C4-fractions was strongly inhibited at 4 C. However, the chitinase isozymes (chiI and chiII) were slightly expressed from fraction No. 55 (F55). Chitinase isozyme (chiII and chiIII) activity in the C4 crude enzyme was expressed clearly at 25 C (Fig. 7B). In Fig. 7B, the chitinase isozyme (chiIII) in C25 and C30 crude enzymes was expressed as a major band. Three chitinase isozymes (chiI, chiII, and chiIII) from the C4-fractions (F45-F80) were expressed strongly on SDS-PAGE gels at 25 C (Fig. 7B). Chitinase
From antifungal activity against phytopathogens, inhibition rate was measured at mycelial radical growth of phytopathogens on PDA plates. Rhizoctonia solani was measured at 72 h after treatment of Pedobacter sp. PR-M6. Botrytis cinerea, Alternaria brassicicola, and Fusarium oxysporum were measured at 144 h after treatment. Pedobacter sp. PR-M6 showed high inhibition rate of 60.9% and 57.5% against R. solani and B. cinerea, respectively (Fig. 8A1 and 8B1). Pedobacter sp. PR-M6 showed low inhibition rate of 31.4% and 39.1% against A. brassicicola and F. oxysporum, respectively (Fig. 8C1 and 8D1). Yin et al. [34] reported that Pedobacter sp. showed low inhibition rate of 34.4% against R. solani. De Boer et al. [35] reported that treatment of combination of Pedobacter V48 and Pseudomonas AD21 showed high antifungal activity against R. solani. Also Pedobacter sp. plays an important role for defense mechanism of amphibian against fungi [36]. From this result, the chitinase produced from Pedobacter sp. PR-M6 could be used as a strong biocontrol agent for phytophathogens. In future the chitinaseproducing bacterium, psychrotolerant Pedobacter sp.PR-M6 could be used in the development of commercial products. 4. Conclusion In this work, the psychrotolerant Pedobacter sp. PR-M6 was isolated, partially purified, and characterized. Chitinase produced from Pedobacter sp. PR-M6 is possibly a useful enzyme, with strong chitinase activity and cell growth at 25 C. Therefore, Pedobacter sp. PR-M6 could be used to improve the efficiency of industrial processes for chito-oligomer products, and develop antimicrobial agents for the biocontrol of phytopathogens using bacterial culture systems in cold environments. Acknowledgement This work was supported by a grant from the Korea Institute of Planning & Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (iPET) through the Agri-Bioindustry Technology Development Program, funded by the Ministry of Agriculture, Food and Rural Affairs (no. 316032-5). References [1] R.A. Muzzarelli, J. Boudrant, D. Meyer, N. Manno, M. DeMarchis, M.G. Paoletti, Current views on fungal chitin/chitosan, human chitinases, food preservation, glucans, pectins and inulin: a tribute to Henri Braconnot, precursor of the carbohydrate polymers science, on the chitin bicentennial, Carbohydr. Polym. 87 (2) (2012) 995e1012. [2] J. Sietsma, J. Wessels, Evidence for covalent linkages between chitin and bglucan in a fungal wall, Microbiology 114 (1) (1979) 99e108. [3] R. Muzzarelli, F. Tanfani, M. Emanuelli, Chelating ability of chitinous materials from Streptomyces, Mucor rouxii, Phycomyces blakesleeanus, and Choanephora cucurbitarum, J. Appl. Biochem. 3 (4) (1981) 322e327. [4] A.N.M. Ramli, N.M. Mahadi, M.S. Shamsir, A. Rabu, K.H. Joyce-Tan, A.M.A. Murad, R.M. Illias, Structural prediction of a novel chitinase from the psychrophilic Glaciozyma Antarctica PI12 and an analysis of its structural properties and function, J. Comput. Aided Mol. Des. 26 (8) (2012) 947e961. [5] X. Wang, Y. Zhao, H. Tan, N. Chi, Q. Zhang, Y. Du, H. Yin, Characterisation of a chitinase from Pseudoalteromonas sp. DL-6, a marine psychrophilic bacterium, Int. J. Biol. Macromol. 70 (2014) 455e462. [6] E. Stefanidi, C.E. Vorgias, Molecular analysis of the gene encoding a new chitinase from the marine psychrophilic bacterium Moritella marina and biochemical characterization of the recombinant enzyme, Extremophiles 12 (4) (2008) 541e552.
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biochem. J. 24 (1991) 121e126. [24] W.J. Jung, J.H. Kuk, K.Y. Kim, T.H. Kim, R.D. Park, Purification and characterization of chitinase from Paenibacillus illinoisensis KJA-424, J. Microbiol. Biotechnol. 15 (2005) 274e280. [25] E.M. Soylu, S. Soylu, S. Kurt, Antimicrobial activities of the essential oils of various plants against tomato late blight disease agent Phytophthora infestans, Mycopathologia 161 (2006) 119e128. [26] L. Yang, H. Maeda, T. Yoshikawa, G.-q. Zhou, Algicidal effect of bacterial isolates of Pedobacter sp. against cyanobacterium Microcystis aeruginosa, Water Sci. Eng. 5 (4) (2012) 375e382. [27] P. Steyn, P. Segers, M. Vancanneyt, P. Sandra, K. Kersters, J. Joubert, Classification of heparinolytic bacteria into a new genus, Pedobacter, comprising four species: Pedobacter heparinus comb. nov., Pedobacter piscium comb. nov., Pedobacter africanus sp. nov. and Pedobacter saltans sp. nov. proposal of the family Sphingobacteriaceae fam. nov, Int. J. Syst. Evol. Microbiol. 48 (1) (1998) 165e177. [28] B. Vanparys, K. Heylen, L. Lebbe, P. De Vos, Pedobacter caeni sp. nov., a novel species isolated from a nitrifying inoculum, Int. J. Syst. Evol. Microbiol. 55 (3) (2005) 1315e1318. [29] Y. Yin, G. Yue, Q. Gao, Z. Wang, F. Peng, C. Fang, X. Yang, L. Pan, Genome sequence of Pedobacter arcticus sp. nov., a sea ice bacterium isolated from tundra soil, J. Bacteriol. 194 (23) (2012) 6688. ˛ tecki, D. Go _ rniak, [30] M.K. Zdanowski, M.J. Zmuda-Baranowska, P. Borsuk, A. Swia D. Wolicka, K.M. Jankowska, J. Grzesiak, Culturable bacteria community development in postglacial soils of ecology glacier, king george island, Antarctica, Polar Biol. 36 (4) (2013) 511e527. [31] A.N. Ramli, N.M. Mahadi, A. Rabu, A.M. Murad, F.D. Bakar, R.M. Illias, Molecular cloning, expression and biochemical characterisation of a cold-adapted novel recombinant chitinase from Glaciozyma Antarctica PI12, Microb. Cell Fact. 10 (1) (2011) 1e13. [32] H. Orikoshi, N. Baba, S. Nakayama, H. Kashu, K. Miyamoto, M. Yasuda, Y. Inamori, H. Tsujibo, Molecular analysis of the gene encoding a novel coldadapted chitinase (ChiB) from a marine bacterium, Alteromonas sp. strain O-7, J. Bacteriol. 185 (4) (2003) 1153e1160. [33] A. Bendt, H. Hüller, U. Kammel, E. Helmke, T. Schweder, Cloning, expression, and characterization of a chitinase gene from the Antarctic psychrotolerant bacterium Vibrio sp. strain Fi: 7, Extremophiles 5 (2) (2001) 119e126. [34] C. Yin, S.H. Hulbert, K.L. Schroeder, O. Mavrodi, D. Mavrodi, A. Dhingra, Role of bacterial communities in the natural suppression of Rhizoctonia solani bare patch disease of wheat (Triticum aestivum L.), Appl. Environ. Microbiol. 79 (2013) 7428e7438. [35] W. De Boer, A.-M. Wagenaar, P.J.K. Gunnewiek, J.A. Van Veen, In vitro suppression of fungi caused by combinations of apparently non-antagonistic soil bacteria, FEMS Microbiol. Ecol. 59 (2007) 177e185. [36] A. Lauer, M.A. Simon, J.L. Banning, B.A. Lam, R.N. Harris, Diversity of cutaneous bacteria with antifungal activity isolated from female four-toed salamanders, ISME J 2 (2008) 145e157.
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Short communication
Identification, purification, and expression patterns of chitinase from psychrotolerant Pedobacter sp. PR-M6 and antifungal activity in vitro Yong-Su Song, Dong-Jun Seo, Woo-Jin Jung* Department of Agricultural Chemistry, Institute of Environmentally-Friendly Agriculture (IEFA), College of Agricultural and Life Science, Chonnam National University, Gwangju 61186, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 January 2017 Received in revised form 28 February 2017 Accepted 13 March 2017 Available online 19 March 2017
In this study, a novel psychrotolerant chitinolytic bacterium Pedobacter sp. PR-M6 that displayed strong chitinolytic activity on 0.5% colloidal chitin was isolated from the soil of a decayed mushroom. Chitinase activity of PR-M6 at 25 C (C25) after 6 days of incubation with colloidal chitin increased rapidly to a maximum level (31.3 U/mg proteins). Three chitinase isozymes (chiII, chiIII, and chiIV) from the crude enzyme at 25 C (C25) incubation were expressed on SDS-PAGE gels at 25 C. After purification by chitinaffinity chromatography, six chitinase isozymes (chiI, chiII, chiIII, chiIV, chiV, and chiVI) from C25fractions were expressed on SDS-PAGE gels at 25 C. Major bands of chitinase isozymes (chiI, chiII, and chiIII) from C4-fractions were strongly expressed on SDS-PAGE gels at 25 C. Pedobacter sp. PR-M6 showed high inhibition rate of 60.9% and 57.5% against Rhizoctonia solani and Botrytis cinerea, respectively. These results indicated that psychrotolerant Pedobacter sp. PR-M6 could be applied widely as a microorganism agent for the biocontrol of agricultural phytopathogens at low temperatures. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Psychrotolerance Pedobacter sp. Chitinolytic activity Chitinase isozyme
1. Introduction Chitin exists in edible fungi such as Agaricus bisporus, Pleurotus ostreatus, and Lentinula edodes [1], and the covalent link between chitin and b-glucan has been studied in Schizophyllum commune (Basidiomyceta) [2]. Chitin also exists in yeast and filamentous fungi with glucan and chitosan in cell walls [3]. Chitinase obtained from microorganisms is fundamental in the enzymatic production of chitin oligosaccharides for industrial applications. Recently, research of psychrophilic enzymes in biotechnological applications and protein structure-function has increased. The structural properties and function of psychrophilic chitinase have been reported in the yeast strain Glaciozyma antarctica PI12 [4]. The biochemical properties, action mode of oligosaccharide production, and catalytic mechanism of psychrophilic chitinase has been reported in the marine bacteria, Pseudoalteromonas sp. DL-6 [5] and Moritella marina [6]. The purification and optimal production of cold-active endochitinase has been reported in the Antarctic bacterium, Sanguibacter antarcticus KOPRI 21702 [7,8] and this Chi21702 recombinant chitinase was expressed in the
* Corresponding author. E-mail address: [email protected] (W.-J. Jung). http://dx.doi.org/10.1016/j.micpath.2017.03.018 0882-4010/© 2017 Elsevier Ltd. All rights reserved.
methylotrophic yeast, Pichia pastoris, for the mass production of chitinase [9]. Also, a chitinolytic bacterial community has been collected from penguin guano at low temperatures, belonging to Janthinobacterium sp., Stenotrophomonas sp. of g-Proteobacteria, Cytophaga sp. of the Cytophaga-Flexibacter-Bacteroides group, and Streptomyces and Nocardiopsis sp. of Actinobacteria in Antarctica [10]. The antifungal activity of chitinase produced from the Antarctic bacterium, Verticillium lecanii A3, has been reported against some spoiling agents of refrigerated foods, such as Mucor plumbeus, Penicillium verrucosum, Aspergillus versicolor, and Cladosporium cladosporioides [11]. Cold-responsive extracellular chitinase has been characterized in forage bromegrass (Bromus inermis) culture cells to determine its relationship with antifreeze activity [12]. Recently, oligosaccharides produced from chitinase were used in several biotechnological applications, such as the extraction of antioxidants from marine waste using Bacillus sp [13]. and Serratia marcescens [14]. However, limited studies focusing on the characteristics of psychrophilic chitinases produced from bacteria have been conducted. The objectives of this study were to investigate the expression patterns of psychrophilic chitinase on SDS-PAGE gels after chitinase purification, and to estimate the effects of different culture temperatures on the chitinase activity from psychrotolerant Pedobacter sp. PR-M6. To our knowledge, this is the first report to
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Fig. 1. Nine chitinolytic bacteria showed halo formation around their colonies on 0.5% winter mushroom (Flammulina velvtipes) powder agar at 30 C after 3 days of incubation (A). Bacterium No. 6 (circle of red dotted) having the strongest cell growth and mushroomlytic activity incubated on 0.5% colloidal chitin agar medium at 30 C (B) and 25 C (C) for 13 days, and at 4 C for 30 days (D). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
investigate the expression of various extracellular chitinase isozymes in Pedobacter sp. 2. Materials and methods 2.1. Isolation and identification of the chitinolytic bacterium Rot mushroom soils were collected in an arboretum of the Chonnam National University, Gwangju, Korea. The soil samples were grained and inoculated on a 0.5% colloidal chitin (pH 6.0) agar medium at 30 C for 5 days. Nine bacteria among them were inoculated on 0.5% winter mushroom (Flammulina velvtipes) powder agar and then incubated at 30 C for 3 days. One bacterium with the strongest chitinolytic activity was selected and used for further characterization. For identification of the bacterium, polymerase chain reaction (PCR), (GeneAmp 9700, Applied Biosystems, USA) was performed to amplify part of the 16S rRNA gene of the bacterium. The nucleotide sequence of the 16S rRNA gene of PR-M6 was determined using an ABI PRISM Big Dye™ Terminator Cycle Sequencing Kit (Applied Biosystems, USA) and ABI PRISM 3730xl Analyzer (Applied
Biosystems) in Geno Tech Co. (Daejeon, Korea). The nucleotide sequence of the 16S rRNA gene of PR-M6 was compared with published 16S rRNA sequences using a blast search at NCBI [15]. The phylogenetic trees of isolate PR-M6 were constructed using the maximum likelihood method based on the Jukes-Cantor model [16]. Evolutionary analyses were conducted using MEGA7 [17]. 2.2. Determination of chitinase activity The chitinase assay consisted of a mixture of 50 mL sample, 500 mL 0.5% colloidal chitin, and 450 mL 50 mM sodium acetate buffer (pH 5.0) [18]. The mixture including the sample was incubated at 25 C and 37 C for 3 h, and then 200 mL 1 N NaOH was added to stop the enzyme reaction. Next, the sample was briefly centrifuged (10,000g, 5 min), after which 500 mL supernatant was mixed with 1 mL Schales' reagent and then heated in boiling water for 15 min. The absorbance was then immediately measured at 420 nm using a spectrophotometer (Mecasys, Optizen 3220UV, Korea). The amount of reducing sugar was calculated based on a comparison with a standard curve generated from different concentrations of GlcNAc (0e100 mg). One unit of chitinase activity was
Fig. 2. Phylogenetic tree of 16S rRNA from the PR-M6 strain using the maximum likelihood method based on the Jukes-Cantor model. The scale bar indicates 1 base change per 20 nucleotides.
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Fig. 3. Time course of cell growth (A), and protein content (B) of Pedobacter sp. PR-M6 at 0, 1, 2, 3, 4, 5, and 6 days after incubation in 0.5% colloidal chitin medium at 25 C (-B- C25) and 30 C (-C- C30).
Fig. 4. Time course of chitinase activity of Pedobacter sp. PR-M6. The bacterium was grown in 0.5% colloidal chitin medium at 25 C (-B- C25) and 30 C (-C- C30) for 6 days. The chitinase produced from PR-M6 was assayed at 25 C (A) and 37 C (B) as a condition of the enzyme reaction.
2.4. Chitinase purification defined as the amount of enzyme that liberated 1 mmol of GlcNAc per h. The protein concentration was determined using the method described by Bradford [19]. 2.3. Chitinase staining activity on SDS-PAGE gel Pedobacter sp. PR-M6 was incubated on 0.5% colloidal chitin medium at 25 C and 30 C for 6 days. After incubation, the proteins at two culture temperatures were loaded on SDS-PAGE gels to investigate the expression patterns of chitinase isozymes, according to the method described by Laemmli [20]. Electrophoresis was performed using a Bio-Rad Mini-PROTEAN (80 73 1.5 mm), to evaluate the active staining of chitinase, 10% SDS-PAGE containing 0.01% glycol chitin was conducted according to the method described by Trudel and Asselin [21]. The gel was incubated in 50 mM sodium acetate buffer (pH 5.0) containing 1% (v/v) Triton X100 and 1% skim milk at 37 C for 2 h with reciprocal shaking. A subsequent incubation was then conducted overnight under the same conditions, but without skim milk, in buffer solution. The gel was then immersed in 500 mM TriseHCl (pH 8.9) solution containing 0.01% Calcofluor White M2R (Sigma F3397). The lysed zones were visualized and photographed using a UV transilluminator (Daihan Sci. Co., WGD-30, Korea).
For preparation of the chitin affinity column, regenerated chitin from chitosan (deacetylation 92% and viscosity 20 cPs) was obtained using a modified method described by Molano et al. [22]. The regenerated chitin was equilibrated in 20 mM sodium carbonate buffer (pH 8.4), after which the crude enzyme of PR-M6 was loaded onto a regenerated chitin column (4 10 cm) at a flow rate of 0.5 mL/min. The first washing was conducted using 500 mL 20 mM sodium carbonate buffer (pH 8.4) until the eluate was free of proteins based on detection by measuring the absorbance at 280 nm. The second washing was conducted with 100 mL 20 mM sodium acetate buffer (pH 5.3). The proteins from PR-M6 were finally released from the chitin matrix with 20 mM acetic acid (pH 3.2) and then 5 mL was collected in individual tubes to give a total of 100 fractions [23]. 2.5. Mycelial growth inhibition To investigate the inhibition of mycelial growth with strain PRM6, mycelial discs (8 mm in diameter) of test fungi (Rhizoctonia solani KACC40111, Botrytis cinerea KACC40574, Alternaria brassicicola KACC40036, and Fusarium oxysporum KACC47615) grown on PDA plates were cut from the margins of the colony and were
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placed on plates. After incubation at 25 C for 54e160 h, and the distances between the edges of the PRC-5 and fungal mycelium were measured [24]. Control plates consisted of PDA only. The percentage of inhibition was calculated based on the percentage inhibition of radial growth (IRG) as follows: IRG (%) ¼ [(GCeGT)/ GC] 100, where GC and GT represent mycelial growth in the control and treatment, respectively [25]. 3. Results and discussion 3.1. Identification of the chitinolytic bacterium Nine bacteria showing chitinolytic activity were inoculated on 0.5% winter mushroom (F. velvtipes) powder agar and then incubated at 30 C for 3 days. From the incubation of bacteria, one bacterium, No. 6, having the strongest mushroomlytic activity and high cell growth was finally selected (Fig. 1A). Chitinolytic activity of the PR-M6 strain showed a stronger clear zone at 25 C than at 30 C incubation after 13 days on 0.5% colloidal chitin plates (Fig. 1B and C). In addition, cell growth and chitinolytic activity was clearly shown at 4 C after incubation for 30 days (Fig. 1D). Recombinant chitinase ChiA from the psychrophilic bacterium, Pseudoalteromonas sp. DL-6, showed optimal chitinolytic activity at 20 C incubation for 24 h on 4MU-(GlcNAc)2 as a substrate [5]. The recombinant ChiA exhibited 75% at 4 C and 79% at 10 C of its maximal activity, respectively. On the basis of the nucleotide sequence of a conserved segment of the 16S rRNA gene, the bacterium was identified as Pedobacter sp. and named Pedobacter sp. PR-M6. The PR-M6 strain showed 99% homology with Pedobacter sp. Mal11-5, which showed 99% homology with P. steynii WB2.3-45 (Accession No. NR042605) [26]. Based on its 16S rRNA sequence and its position in the phylogenetic tree (Fig. 2), this bacterium was designated as Pedobacter sp. PR-M6, which is taxonomically close to Pedobacter sp. Mal11-5. Pedobacter sp. PR-M6 was taxonomically closer to P. caeni than to P. steynii. The Pedobacter genus in the Sphingobacteriaceae family is characterized by Gram-negative, aerobic, and rod-shaped bacteria [27]. P. caeni sp. nov. was isolated from a nitrifying inoculum [28], and
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P. arcticus sp. nov. A12T was originally isolated from tundra soil [29]. Psychrotolerant Pedobacter sp. KFC76 was isolated from soil from the Kafni Glacier, Himalaya [30].
3.2. Cell growth and chitinase activity Pedobacter sp. PR-M6 was incubated in a 0.5% colloidal chitin medium at 25 C and 30 C for 6 days; cell growth of Pedobacter sp. PR-M6 was higher at 25 C than 30 C (Fig. 3A). Cell growth of PRM6 gradually increased for 5 days and decreased slightly on day 6 when cultured at 25 C on 0.5% colloidal chitin liquid medium. The protein content was measured after six days of incubation. The protein content of the Pedobacter sp. PR-M6 medium was higher at 30 C than 25 C after three days of incubation (Fig. 3B), and was maintained at a higher amount at 25 C than that at 30 C. The protein content of PR-M6 showed a maximum amount (133.5 mg/ mL) at 25 C after 5 days of incubation. For the time course of chitinase activity of Pedobacter sp. PR-M6, the bacterium was grown in 0.5% colloidal chitin medium at 25 C (-B- C25) and 30 C (-C- C30) for 6 days (Fig. 4). Chitinase activity from PR-M6 measured at 25 and 37 C as a condition of the enzyme reaction. Chitinase activity of PR-M6 after 6 days of incubation at 25 C (C25) with colloidal chitin increased rapidly to a maximum level (31.3 U/mg proteins) (Fig. 4A). Chitinase activity of PR-M6 at 30 C incubation (C30) was < 10 U/mg proteins after 6 days. Chitinase activity of PR-M6 after 6 days of incubation at 25 C (C25) with colloidal chitin increased rapidly to a maximum level (21.0 U/ mg proteins) by 37 C treatment as a condition of the enzyme reaction (Fig. 4B). Chitinase activity of PR-M6 after 5 days of incubation at 30 C (C30) with colloidal chitin increased slightly with 17 U/mg proteins by 37 C treatment as a condition of the enzyme reaction. The optimum temperature of chitinase activity was 15 C in cold-adapted chitinase (chiII) from Glaciozyma antarctica PI12 [31], and 28 C in the marine psychrophilic bacterium, Moritella marina [6]. Especially, the optimum temperature of bacterial culture was shown at 30 C for the cold-adapted chitinase B of Alteromonas sp. O-7 [32], and chitinase A of Vibrio sp. Fi:7 [33].
Fig. 5. Expression patterns of chitinase activity on SDS-PAGE gels after M2R staining with different temperatures. Pedobacter sp. PR-M6 strain was grown in 0.5% colloidal chitin medium at 25 C (C25) and 30 C (C30) for 6 days. The chitinase (25 mL of culture broth) produced from PR-M6 expressed at 25 C (A and C) and 37 C (B and D) as a condition of the enzyme reaction on SDS-PAGE gels. Lanes D0, D1, D2, D3, D4, D5, and D6 are 0e6 days after incubation respectively. M: Protein marks.
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Fig. 6. Expression patterns of chitinase from PR-M6 grown in 0.5% colloidal chitin medium at 25 C for 5 days. Chitinase activity of the fraction solutions (C25-fractions) collected from the chitin-affinity column expressed on SDS-PAGE gels at different temperatures of 25 C (A) and 37 C (B) as a condition of the enzyme reaction. The crude enzyme of the PRM6 strain after 5 days of incubation with LB medium at 25 C (LB), and colloidal chitin medium at 30 C (C30) and 25 C (C25). Protein marks (M).
3.3. Properties of chitinase isozymes Pedobacter sp. PR-M6 was grown in 0.5% colloidal chitin medium at 25 C (C25) and 30 C (C30) for 6 days. The expression pattern of chitinase from PR-M6 was examined on SDS-PAGE gels (Fig. 5). Chitinase isozyme bands were shown as chiII, chiIII, and chiIV on
SDS-PAGE gels at 25 C as a condition of the enzyme reaction (Fig. 5A and C). Chitinase isozyme bands were not detected on the SDS-PAGE gels at 37 C as a condition of the enzyme reaction (Fig. 5B and D). From these results, chitinase activity was strongly inhibited at 37 C. Chitinase isozymes were slightly expressed on SDS-PAGE gels at day 1 (chiIII, 40 kDa) and day 2 (chiII, 47 kDa)
Fig. 7. Expression patterns of chitinase from PR-M6 grown in 0.5% colloidal chitin medium at 4 C for 14 days. Chitinase activity of the fraction solutions (C4-fractions) collected from the chitin-affinity column expressed on SDS-PAGE gels at different temperatures of 4 C (A), 25 C (B) and 37 C (C) as a condition of the enzyme reaction. The crude enzyme of PR-M6 incubated with 0.5% colloidal chitin medium at 25 C (C25) and 30 C (C30) for 5 days and 4 C (C4) for 14 days. Protein marks (M).
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isozyme (chiII) activity in the C4 crude enzyme was clearly inhibited at 37 C, and chitinase isozymes (chiIII) activity was weak (Fig. 7C). Purified chitinase isozymes (chiIII) from C4-fractions were strongly expressed 37 C (Fig. 7C). 3.4. Antifungal activity of PR-M6 against phytophathogens
Fig. 8. Inhibition on mycelial radial growth of four phytopathogens [R. solani (A0 and A1) at 72 h, B. cinerea (B0 and B1) at 144 h, A. brassicicola (C0 and C1) at 144 h, and F. oxysporum (D0 and D1) at 144 h] by Pedobacter sp. PR-M6 (A). Inhibition rate for fungal growth of phytopathogens (B). Values are means ± SE of three replicates. Bars carrying different letters are significantly different at p < 0.05 [Tukey's Studentized Range (HSD) test].
(Fig. 5A and C). Especially, chitinase isozyme bands in C25 incubation were expressed strongly at days 5 and 6 of incubation on SDS-PAGE gels at 25 C (Fig. 5A). This result coincided with the protein content at days 5 and 6 of incubation (Fig. 4A). The molecular weight of psychrophilic chitinase from G. antarctica PI12 was about 39 and 50 kDa, respectively [30]. Pedobacter sp. PR-M6 was incubated in 0.5% colloidal chitin medium at 25 C for 5 days. To investigate the PR-M6 chitinase activity, LB, C30, and C25 crude enzymes were loaded on SDS-PAGE gels. After partial purification of the C25 crude enzyme by a chitinaffinity column, the C25-fraction solutions were loaded on SDSPAGE gels. The chitinase isozymes were expressed on SDS-PAGE gels at different temperatures of 25 C (A) and 37 C (B) as a condition of the enzyme reaction (Fig. 6). Chitinase isozymes (chiI, chiV, and chiVI) from C25-fractions were clearly expressed on SDSPAGE gels at 25 C (Fig. 6A). In Fig. 6A, chitinase isozymes from C25fractions were expressed as major bands (chiI, chiII, chiIII, and chiIV) and minor bands (chiV and chiVI). Chitinase isozymes (chiIII, chiV, and chiVI) from C25-fractions were expressed on SDS-PAGE gels at 37 C (Fig. 6B). Chitinase isozymes (chiI and chiII) from C4-fractions were expressed more clearly than chiIII on SDS-PAGE gels at 4 C (Fig. 7A). In Fig. 7A, chitinase isozymes chiIII were expressed in C25 and C30 crude enzymes on SDS-PAGE gels and but were not expressed in the C4 crude enzyme. Chitinase isozyme (chiI and chiII) activity of C4-fractions was strongly inhibited at 4 C. However, the chitinase isozymes (chiI and chiII) were slightly expressed from fraction No. 55 (F55). Chitinase isozyme (chiII and chiIII) activity in the C4 crude enzyme was expressed clearly at 25 C (Fig. 7B). In Fig. 7B, the chitinase isozyme (chiIII) in C25 and C30 crude enzymes was expressed as a major band. Three chitinase isozymes (chiI, chiII, and chiIII) from the C4-fractions (F45-F80) were expressed strongly on SDS-PAGE gels at 25 C (Fig. 7B). Chitinase
From antifungal activity against phytopathogens, inhibition rate was measured at mycelial radical growth of phytopathogens on PDA plates. Rhizoctonia solani was measured at 72 h after treatment of Pedobacter sp. PR-M6. Botrytis cinerea, Alternaria brassicicola, and Fusarium oxysporum were measured at 144 h after treatment. Pedobacter sp. PR-M6 showed high inhibition rate of 60.9% and 57.5% against R. solani and B. cinerea, respectively (Fig. 8A1 and 8B1). Pedobacter sp. PR-M6 showed low inhibition rate of 31.4% and 39.1% against A. brassicicola and F. oxysporum, respectively (Fig. 8C1 and 8D1). Yin et al. [34] reported that Pedobacter sp. showed low inhibition rate of 34.4% against R. solani. De Boer et al. [35] reported that treatment of combination of Pedobacter V48 and Pseudomonas AD21 showed high antifungal activity against R. solani. Also Pedobacter sp. plays an important role for defense mechanism of amphibian against fungi [36]. From this result, the chitinase produced from Pedobacter sp. PR-M6 could be used as a strong biocontrol agent for phytophathogens. In future the chitinaseproducing bacterium, psychrotolerant Pedobacter sp.PR-M6 could be used in the development of commercial products. 4. Conclusion In this work, the psychrotolerant Pedobacter sp. PR-M6 was isolated, partially purified, and characterized. Chitinase produced from Pedobacter sp. PR-M6 is possibly a useful enzyme, with strong chitinase activity and cell growth at 25 C. Therefore, Pedobacter sp. PR-M6 could be used to improve the efficiency of industrial processes for chito-oligomer products, and develop antimicrobial agents for the biocontrol of phytopathogens using bacterial culture systems in cold environments. Acknowledgement This work was supported by a grant from the Korea Institute of Planning & Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (iPET) through the Agri-Bioindustry Technology Development Program, funded by the Ministry of Agriculture, Food and Rural Affairs (no. 316032-5). References [1] R.A. Muzzarelli, J. Boudrant, D. Meyer, N. Manno, M. DeMarchis, M.G. Paoletti, Current views on fungal chitin/chitosan, human chitinases, food preservation, glucans, pectins and inulin: a tribute to Henri Braconnot, precursor of the carbohydrate polymers science, on the chitin bicentennial, Carbohydr. Polym. 87 (2) (2012) 995e1012. [2] J. Sietsma, J. Wessels, Evidence for covalent linkages between chitin and bglucan in a fungal wall, Microbiology 114 (1) (1979) 99e108. [3] R. Muzzarelli, F. Tanfani, M. Emanuelli, Chelating ability of chitinous materials from Streptomyces, Mucor rouxii, Phycomyces blakesleeanus, and Choanephora cucurbitarum, J. Appl. Biochem. 3 (4) (1981) 322e327. [4] A.N.M. Ramli, N.M. Mahadi, M.S. Shamsir, A. Rabu, K.H. Joyce-Tan, A.M.A. Murad, R.M. Illias, Structural prediction of a novel chitinase from the psychrophilic Glaciozyma Antarctica PI12 and an analysis of its structural properties and function, J. Comput. Aided Mol. Des. 26 (8) (2012) 947e961. [5] X. Wang, Y. Zhao, H. Tan, N. Chi, Q. Zhang, Y. Du, H. Yin, Characterisation of a chitinase from Pseudoalteromonas sp. DL-6, a marine psychrophilic bacterium, Int. J. Biol. Macromol. 70 (2014) 455e462. [6] E. Stefanidi, C.E. Vorgias, Molecular analysis of the gene encoding a new chitinase from the marine psychrophilic bacterium Moritella marina and biochemical characterization of the recombinant enzyme, Extremophiles 12 (4) (2008) 541e552.
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