And Xylanse-Producing Catenovulum Jejuensis A28-5 from Coastal Seawater of Jeju Island, Korea

Microbiol. Biotechnol. Lett. (2017), 45(2), 168–177 http://dx.doi.org/10.4014/mbl.1703.03003 pISSN 1598-642X eISSN 2234-7305 Microbiology and Biotechnology Letters

Identification and Characterization of an Agarase- and Xylanse-producing Catenovulum jejuensis A28-5 from Coastal Seawater of Jeju Island, Korea

Da Som Kim, Ga Ram Jeong, Chang Hwan Bae, Joo-Hong Yeo, and Won-Jae Chi* Biological and Genetic Resources Assessment Division, National Institute of Biological Resources, Incheon 22689, Republic of Korea

Received: March 31, 2017 / Revised: June 8, 2017 / Accepted: June 8, 2017

Strain A28-5, which can degrade xylan and agar in solid medium, was isolated from a coastal seawater sample collected from Jeju Island, South Korea. This strain was found to be a gram-negative, Na+-requiring bacterial strain with a polar flagellum for motility. Additionally, the strain was tolerant to antibiotics such as ampicillin and thiostrepton. The G+C content of the genome was 43.96% and menaquinone-7 was found to be the predominant quinone. Major fatty acids constituting the cell wall of the strain were C16:1 ω7c/iso-C15:0 2-OH (23.32%), C16:0 (21.83%), and C18:1 ω7c (17.98%). The 16S rRNA gene sequence of the strain showed the highest similarity (98.94%) to that of Catenovulum agarivorans YM01, which was demonstrated by construct- ing a neighbor-joining phylogenetic tree. A28-5 was identified as a novel species of the genus Catenovulum via DNA-DNA hybridization with Catenovulum agarivorans YM01, and thus was named as Catenovulum jejuensis A28-5. The formation of tetramers and hexamers of xylooligosaccharides and (neo)agarooligosaccharides, respectively, were confirmed by thin-layer chromatography analysis using an enzyme reaction solution containing xylan or agarose with two crude enzymes prepared from the liquid culture of the strain.

Keywords: Agarase, xylanase, Catenovulum, characterization, xylooligosaccharide, agarooligosaccharide

Introduction Agarose is a polymer that is linked by repeating units of 3,6-anhydro-α-L-galactose and β-D-galactose. Depend- Plant biomass is the most abundant and widely ing on the method of degradation, agarooligosaccharide spread source of bioproducts and biofuels. Hydrolysis of (produced by α-agarase or acid hydrolysis) and neoaga- the plant biomass is the most important step for extract- rooligosaccharide (produced by β-agarase) are produced. ing marine plant-derived polysaccharides (e.g., agar, These oligosaccharides can be used in various indus- carrageenan, and alginate) and terrestrial plant-derived tries, including the food industry, for properties such as polysaccharides (e.g., cellulose, and hemicelluloses), temperature-stability, intestinal regulation, and low cal- which exist in enormous amounts in nature. During ories; the pharmaceutical industry, for their therapeutic hydrolysis, distinctive oligosaccharides with various and preventive effects against obesity and diabetes; and physiological activities are produced [1]. the cosmetic industry, for their whitening, moisturizing, Agar is composed of agaropectin and agarose, which and antioxidative effects [2]. are the main constituents of the cell wall of macroalgae. Xylan is a hemicellulose, the main constituent that accounts for 20−40% of the cell wall constituents of ter- *Corresponding author restrial plants, and has a complex structure composed of Tel: +82-32-590-7113, Fax: +82-32-590-7472 β-1,4-linked xylose monomers substituted with arabino- E-mail: [email protected] © 2017, The Korean Society for Microbiology and Biotechnology syl, acetyl, and glucuronosyl groups and can be hydro-

http://dx.doi.org/10.4014/mbl.1703.03003 Catenovulum Jejuensis A28-5 Isolated from Coastal Seawater of Jeju Island 169

lyzed by various xylanases. Xylanases are required in Jeju Island and diluted to a series of concentrations various processes such as in pulp-prebleaching to (10-1−10-5). It was smeared on marine agar (MA) 2216 remove the hemicelluloses in pulp, in stimulation of (MA, Difco, USA) and incubated at 37℃ for 2 d. Colonies digestion of animal feed, as food and baking additives, in formed on the this plate were inoculated on a new culture processing of fruits and vegetables, in ethanol and xylitol plate with media containing azurine-crosslinked(AZCL)- production, and in paper manufacturing. Research on xylan (Megazyme, Ireland), and were incubated at 37℃ the properties of xylanases that are suitable for use in for 2 d. The colonies exhibiting activation of xylanase or each industry is essential. Furthermore, xylooligosac- agarase were selected for further studies. Xylanase acti- charides (XOSs), the xylan hydrolysates produced by vation was confirmed from the blue coloration that xylanases, are known to have various physiological appeared around the colonies after AZCL-xylan in the activities, which include antimicrobial, antioxidative, media was degraded, and agarase activation was con- and anti-inflammatory activities; thus, it is believed that firmed after staining with Lugol’s Iodine solution. Among they can be used in various industries such as food, med- the selected bacterial colonies, those showing activation ical, agriculture, livestock, and cosmetic [3, 4]. of both xylanase and agarase simultaneously, were inoc- The family Altermonadaceae within the class δ- ulated on a new culture plate and incubated for 2 d. proteobacteria are large group of marine, obligate aerobic Finally, one microbial strain was selected through the heterotroph and requiring sodium to grow. They have screening process based on the morphological character- genomes that contain several macromolecule-degradable istics such as shape, color, transparency, and size of the genes and secondary metabolite synthetic gene clusters colony. The selected strain was named strain A28-5. [5]. Catenovulum is a novel genus belonging to the class δ-proteobacteria. Since the identification of the novel Sequencing of 16S rRNA gene sequence of strain A28-5 species, C. agarivorans YM01, which was first reported Strain A28-5 was cultured in marine broth (MB) by Yan et al. [6], a new different species C. maritimus Q1 under shaking conditions for 2 d and the bacteria was belonging to this genus had been reported until now [7]. harvested by centrifugation at 20,000 ×g for 10 min. The aforementioned hydrolytic degradation methods Genomic DNA was extracted from the collected bacterial using enzymes such as agarase and xylanase can selec- sample by using a Genomic DNA extraction kit from tively and specifically hydrolyze polysaccharides to pro- DyneBio Inc. (Korea). The 16S rRNA gene of the strain duce desired oligosaccharides. In particular, the hydrolysis A28-5 was amplified using bacterial universal primers of plant biomass by microbial enzymes has been accepted (27F and 1492R) [13]. The PCR conditions were (i) 30 s as an eco-friendly method that can replace chemical deg- at 94℃, (ii) 30 cycles of 20 s at 98℃, 30 s at 55℃, and radation methods, which produce harmful by-products 1 min at 72℃, and (iii) 10 min at 72℃. PCR was [8]. Many researchers have been trying to isolate new performed in a Thermal Cycler Dice (Takara, Japan) microorganisms that produce more suitable enzymes [9− and the amplified PCR products were analyzed on a 1% 12]. agarose gel in TAE buffer. The DNA fragments were In this study, a new microorganism that produce inserted into the pGEM-T easy vector (Promega, USA) useful enzymes capable of hydrolyzing the cell walls of and base sequence analysis was performed by Genotech marine and terrestrial plants were identified and the Inc. (Daejeon, Korea). Base sequence homology analysis properties of the enzymes were identified to determine was performed on the 16S rRNA gene sequence of strain their use in the manufacture of plant biomass-derived A28-5 with the data from GenBank database using the oligosaccharides. BlastN program of National Center for Biotechnology Information (NCBI). Additionally, homology with type Materials and Methods strains was determined by comparing the 16S rRNA gene sequence of strain A28-5 with that of type strains Isolation of microorganisms from EzTaxon database (http://www.ezbiocloud.net/ Seawater was collected off the coast of Seogwipo-si, eztaxon).

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Phylogenetic analysis the strain for 4 d on MA plate containing NaCl to final For phylogenetic analysis, the 16S rRNA gene concentrations of 0−10% (intervals of 1%). To investigate sequences of type strains provided by EzTaxon database the effect of varying pH on the growth of strain A28-5, were secured and phylogenetic associations with strain solid media ranging from pH 5.5−10.0 (intervals of pH A28-5 were analyzed by creating a phylogenetic tree 0.5) were prepared and the strain was inoculated, fol- using PHYLIP (PHYLogeny Inference Package) pro- lowed by observing the growth by incubating it for gram. The ClustalW program was used for multiple 4 days. Enzyme activation was performed using argi- alignment between the secured base sequences, and the nine dihydrolase, urease, esculin hydrolase (β-glucosi- gaps at the 5′- and 3′-ends were edited by the GeneDoc dase), gelatinase, and β-galactosidase using an API program. The phylogenetic tree was produced by the 20NE kit (Biomérieux, France) according to the manu- neighbor joining (NJ) method [14]. The produced NJ facturer’s instructions. However, as strain A28-5 was phylogenetic tree was verified by making phylogenetic confirmed to be a NaCl-requiring strain, the medium trees using the maximum parsimony (MP) and maxi- was supplemented with NaCl to a final concentration of mum likelihood (ML) methods. 2% and incubated at 37℃ for 2 d, followed by observing the growth. DNA-DNA hybridization To investigate the optimal carbon sources that can be To investigate the possibility of strain A28-5 being a used for cultivating strain A28-5, the strain was first new species, DNA-DNA hybridization was performed on inoculated into artificial sea water broth (ASW; 6.1 g the type strains with high phylogenetic relationship, Tris base, 12.3 g MgSO4, 0.74 g KCl, 0.13 g (NH4)2HPO4, Catenovulum agarivorans YM01 and Gayadomonas joo- 17.5 g NaCl, 0.14 g CaCl2, per liter; pH 7.2) containing biniege G7, at the Korean Culture Center of Microorgan- 0.5% of each carbon source and cultured under shaking isms. The method used for the DNA-DNA hybridization conditions at 200 rpm and 37℃ for 4 days. The tested analysis is as follows: The bacterial strain was cultured carbon sources were glucose, sucrose, N-acetyl-glucos- on MA plate and the genomic DNA was extracted. amine, carboxylmethyl cellulose (CMC), arabinose, malt- Micrococcus luteus KCCM 11326 was used as a negative ose, starch, agar, lactose, xylose, inositol, mannitol, standard bacterial strain. Probe DNA preparation and sorbitol, xylan, galactose, and glycine. hybridization reaction were conducted using a DIG High To investigate the antibiotic sensitivity of strain A28- Prime DNA Labeling and Detection Starter Kit II 5, the bacteria was evenly smeared on MA plate and a (Roche Applied Science, Germany) according to the man- paper disc containing 30 μl of each antibiotic were placed ufacturer’s instructions. Hybridization signals were at the centre of each plate. The antibiotics tested were measured by the Quantity One Program (Bio-Rad, chloramphenicol (25 μg/ml), kanamycin (50 μg/ml), USA), and the values of type strains were calculated by ampicillin (50 μg/ml), apramycin (50 μg/ml), thiostrepton determining the signal of strain A28-5 as 100%. The (50 μg/ml), nalidixic acid (25 μg/ml), neomycin (30 μg/ hybridization value for each bacterial strain was calcu- ml), and tetracycline (10 μg/ml), and a zone of clearance lated by performing three repetitive experiments. appearing due to inhibition of growth was observed by incubating at 37℃ for 1 d. C. agarivorans YM01 was Analysis of morphological and physiological characteris- used as a comparison strain, on which the same analy- tics of strain A28-5 ses were simultaneously performed as described above. Strain A28-5 was stained using a Gram stain kit (BD, USA), followed by observation under a microscope. The Analysis of biochemical characteristics of strain A28-5 cells cultured on the plate for 2 d were also stained by To analyze the biochemical characteristics of strain 1% phosphotungstic acid (PTA) and were observed under A28-5, the bacteria grown on MA plate at 37℃ for 2 d a transmission electron microscope (TEM) (JEM1010, was harvested. Analyses of main quinones, fatty acids, JEOL, Japan) to confirm flagella. and G+C content were conducted at the Korean Culture To investigate the growth characteristics of strain Center of Microorganisms (KCCM, Korea). The method A28-5, growth was observed by culturing and incubating used for each analysis is as follows: Main quinones were

http://dx.doi.org/10.4014/mbl.1703.03003 Catenovulum Jejuensis A28-5 Isolated from Coastal Seawater of Jeju Island 171

analyzed by reverse phase HPLC using a C-18 column syringe filter (Millipore, USA) and was concentrated and the fatty acids present in the bacteria were methyl- using an Amicon ultra centrifugal filter (Millipore, esterified following the method previously described by USA), and 2 ml of xylanase was obtained. Agarase was Miller and Berger [15]. The G+C content in the genomic prepared similar to xylanase, using an agar-containing DNA was analyzed by the method of Mesbah et al., culture medium. which uses reverse phase HPLC [16]. C. agarivorans YM01 was used as a comparison strain, on which similar Thin layer chromatography (TLC) analysis analyses were performed simultaneously. To analyze the xylan hydrolysates produced by the action of xylanases, secreted by the bacterial strain A28- Degradation of xylan by strain A28-5 5, 100 μl of the prepared xylanase was added to 1× Strain A28-5 was cultured on MA plate containing phosphate-buffered saline (PBS) solution containing 0.15% AZCL-xylan at 37℃ for 2 d. In AZCL-xylan-con- 0.4% birchwood xylan and allowed to react at 37℃ for taining media, xylan degradation was confirmed by the 24 h. At 12 h interval during the enzyme reaction, 20 μl change in color. Additionally, to confirm that strain A28- of reaction solution was sampled and applied to a Silica 5 secretes the xylan-degrading enzyme extracellularly, it gel 60 plate (Merck, USA) using xylobiose and xylo- was inoculated into MB and incubated at 37℃ for 2 d, tetraose (Megazyme, Ireland) as standards. A solution of followed by centrifugation at 20,000 ×g for 30 min and n-butanol: acetic acid:distilled water (2:1:2) was used as the supernatant was collected by discarding the bacte- a developing solvent, and a solution of ethanol:sulfuric rial pellet. The supernatant was filtered using a 0.22-μm acid (9:1) was used as a coloring reagent. After develop- syringe filter, and 10 μl of the supernatant was spotted ment, the coloring reagents were evenly sprayed and on MA plate containing 0.15% AZCL-xylan and was visualized by heating at 120℃. incubated at 37℃ for 12 h, then the degradation of To analyze hydrolysis products of agar by agarase AZCL-xylan was observed. secreted by strain A28-5, 100 μl of the agarase crude enzyme solution was added to 1×PBS buffer containing Degradation of agar by strain A28-5 0.4% agarose and reacted at 45℃ for 24 h. At this time, Strain A28-5 was cultured on MA at 37℃ for 2 d. The the temperature was set as the minimum temperature plate was then stained with Lugol’s Iodine solution (1% at which the agarose did not solidify after melting. The I2, 2% KI), followed by washing excess Lugol’s Iodine analytical method was the same as that of xylanase solution with distilled water. Agar degradability around hydrolysis products. However, purely purified neoagaro- the bacterial colonies was observed due to the property biose, neoagarotetraose, and neoagarohexaose were used that the Lugol’s Iodine solution stains only polysaccha- as standard to compare the size of hydrolysates [17]. rides. The supernatant without bacterial cells was obtained as described in the previous experiment, and Results 10 μl of this supernatant was spotted on MA plate and incubated at 37℃ for 12 h; agar degradation was Identification of strain A28-5 observed by staining with the Lugol's Iodine solution. A marine microbial strain showing highest xylan and MA used for this experiment contained AZCL-xylan so agar degradabilities was finally selected and designated that not only agar, but also xylan, degradability could be as strain A28-5 (Fig. 1A and B). The homology search confirmed simultaneously. based on the 16S rRNA gene sequence showed the following results for strain A28-5−98.94% homology with Preparation of xylanase and agarase C. agarivorans YM01 (GQ262000), 95.78% with G. Strain A28-5 was inoculated into 200 ml MB contain- joonbiniege G7 (AMRX01000001), 95% with Catenovulum ing 0.1% birchwood xylan and incubated at 37℃ for 2 d, maritimus Q1 (NR_146038), 91.02% with Algicola sag- followed by centrifugation at 20,000 ×g for 30 min, and amiensis B-10-31 (AB063324), and 90.95% with Echini- the supernatant was collected by discarding the bacte- monas agarilytica KMM6351 (JX072970). rial pellet. The supernatant was filtered using a 0.22 μm NJ phylogenetic tree was constructed based on the

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Fig. 1. Isolation of strain A28-5. (A) Xylanase activity. Strain A28-5 was cultured on MA containing AZCL-xylan at 37℃ for 2 d. (B) Agarase activity. Strain A28-5 was cultured on MA at 37℃ for 2 d. The plate was stained with Lugol’s iodine solution. (C) DNA- DNA hybridization. The values obtained from self hybridization of strain A28-5 were set at 100%. (1), strain A28-5; (2), Catenovulum agarivorans YM01; (3), Micrococcus luteus KCCM11326 as the negative control. (D) Neighbor joining phylogenetic analysis. Phyloge- netic tree was constructed based on the 16S rRNA gene sequences of strain A28-5 and its genetically related strains. Distances was determined according to the Kimura-two model and bootstrap values (>50%) based on 1,000 replicates are listed as percentages at nodes. Nucleotide sequence accession numbers are given in parentheses. Scale bar, 0.01 substitutions per 100 nucleotides.

16S rRNA gene sequence homology search results. The ble that strain A28-5 is of the same genus and species as phylogenetic analysis showed that strain A28-5 is most C. agarivorans YM01 or has evolved into a different spe- closely related to C. agarivorans YM01 (Fig. 1D). Phylo- cies. The DNA hybridization analysis between strain genetic trees constructed by the Maximum-Parsimony A28-5 and the strains with high 16S rRNA gene (MP) and the Maximum-Likehood (ML) methods showed sequence and phylogenetic associations showed the a similar relationship as that of the NJ phylogenetic tree hybridization value of 61.83% with C. agarivorans YM01 (data not shown). Based on the results of 16S rRNA gene (Fig. 1C). Generally, it is considered to be a new subspe- sequence homology and phylogenetic analysis, it is possi- cies when the DNA-DNA hybridization value is in the

http://dx.doi.org/10.4014/mbl.1703.03003 Catenovulum Jejuensis A28-5 Isolated from Coastal Seawater of Jeju Island 173

range of 70−90%, the same species when the value is and not in the medium without NaCl. This indicates 90% or higher, or a different species when the value 70% that strain A28-5 requires NaCl for growth. pH 7.0−9.0 or lower [18]. According to these criteria, the DNA was observed to be optimal for the growth of the strain, hybridization value of 61.83% classifies strain A28-5 as a whereas the growth was not observed below or above different species in the same genus as that of C. agariv- these pH conditions. orans YM01. G. joonbiniege G7, the strain with the sec- Strain A28-5 grew in the medium containing glucose, ond highest 16S rRNA gene sequence homology after C. sucrose, N-acetyl-glucosamine, arabinose, maltose, starch, agarivorans YM01, showed a DNA hybridization value agar, lactose, xylose, mannitol, xylan, and galactose. of 13.56% (data not shown). As this value is lower than However, the growth was not observed in the medium the cut-off value (i.e., 20%), it may indicate a different containing CMC, inositol, sorbitol, or glycine. This char- genus, it is thought that these two bacterial strains may acteristic was similar to that of the type strain, C. aga- have evolved into different genera. Thus, in the present rivorans YM01. In case of antibiotic sensitivity, C. study, strain A28-5 can be classified as a novel species of agarivorans YM01 showed resistance towards ampicillin the genus Catenovulum from the results of 16S rRNA only, whereas Catenovulum sp. A28-5 showed resistance gene sequence analysis, phylogenetic tree analysis, and towards two antibiotics—ampicillin and thiostreptone DNA-DNA hybridization analysis. whereas it was sensitive towards other antibiotics including chloramphenicol, kanamycin, apramycin, nali- Morphological and physiological characteristics of strain dixic acid, neomycin, and tetracycline, and this sensitiv- A28-5 ity was similar to that of C. agarivorans YM01. For all Strain A28-5 was confirmed to be gram-negative by the above experiments, the growth of the comparison Gram staining. The motility of strain A28-5 was con- strain, C. agarivorans YM01, was observed in similar firmed by observing flagella using Transmission Election conditions. The characteristics of strain A28-5, C. aga- Microscope (TEM) after negative staining (data not rivorans YM01 and C. maritimus Q1 are compared and shown). shown in Table 1. Strain A28-5 can be distinguished Strain A28-5 grew only in the presence of NaCl (1−4%) from the two type strains by several characteristics such

Table 1. Distinguishable characteristics of strain A28-5, Catenovulum agarivorans YM01, Catenovulum maritimus Q1. Strains: 1, A28-5 (this study); 2, Catenovulum agarivorans YM01 (this study); 3, Catenovulum maritimus Q1 (data from Li et al. [7]). The G+C content (mol%), isoprenoid quinine and motility results of Catenovulum agarivorans YM01 are from Yan et al. [6]. Characteristics 1 2 3 Source Sea water, Jeju Island, Korea Sea water, Qingdao, China Porphyra yezoensis Ueda, Weihai, China G+C content (mol%) 43.96 44.8 37.9 Motility Polar flagella Peritrichous flagella Peritrichous flagella Colony color Pale yellow Pale yellow Pale white Growth at: pH 7.0−9.0 7.5−9.5 6.0−8.5 NaCl concentration (%) 1−41−30.5−5.5 Utilization of: Sorbitol − + − Enzyme activity Arginine dihydrolase − + − Nitrate to nitrite − + − Resistant to: Ampicillin (50 μg/ml) + - ND Isoprenoid quinone MK-7 MK-7 Q-8 Symbols : +, positive reaction; −, negative reaction; MK-7, menaquinone-7; Q-8, ubiquinone-8; ND, not determined.

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as the cells morphology, G+C content, enzyme activity same method. In particular, three types of fatty acids and growth condition. (C10:0, C12:0 2-OH, and C12:0 3-OH) were found only in strain A28-5 but not in C. agarivorans YM01, while two Biochemical characteristics of strain A28-5 types of fatty acids (C13:0 and C15:1 ω6c) were found only The G+C content in the genomic DNA of strain A28-5 in C. agarivorans YM01 but not in strain A28-5. Five was analyzed to be 43.96% which is similar to that of the types of fatty acids (C15:0, C18:0, C18:1 ω7c, summed fea- comparison strain, C. agarivorans YM01 (44.8%) (Table ture 3, and summed feature 5) clearly showed differ- 1). Additionally, menaquinone-7 (MK-7) was observed as ences between the two strains. Such differences in fatty the predominant quinone in the strain A28-5, which is acid content and type between the two strains indicate the similar characteristic of the type strain C. agariv- that the two strains have evolved into different species. orans YM01, and it is specifically present in species of Although strain A28-5 and C. maritimus Q1 share the the class δ-proteobacteria (Table 1). same major cellular fatty acids (C16:0, C18:1 ω7c, and The fatty acids present in the cell wall of strain A28-5 summed feature 3), other quantitative differences in the were analyzed and were composed of straight-chain fatty acids indicated that this isolate may represent a fatty acids (50.42%), unsaturated fatty acid (21.95%)], different species from C. maritmus Q1. The cellular fatty branched fatty acid (0.52%), Summed feature (23.94%), acid composition of strain A28-5, C. agarivorans YM01, and unknown (3.16%). The cellular fatty acids of C. aga- and C. maritimus Q1 is shown in Table 2. rivorans YM01, the phylogenetically associated compari- Based on the results of molecular, biological, morpho- son strain, were simultaneously analyzed using the logical, physiological, and biochemical analyses, strain A28-5 is thought to be a novel species of the genus Catenovulum. Therefore, in the present study, strain Table 2. Cellular fatty acid composition of strain A28-5, Catenovulum agarivorans YM01 and Catenovulm maritimus A28-5 was named as Catenovulum jejuensis A28-5. Q1. Strains: 1, A28-5 (this study); 2, Catenovulum agarivorans YM01 (this study); 3, Catenovulum maritimus Q1 (data from Li et Degradation of xylan by Catenovulum jejuensis A28-5 al. [7]). As shown in Fig. 1A, the insoluble AZCL-xylan parti- Fatty acid 1 2 3 cles were solubilized to form a blue coloration around the C10:0 1.79 ND 0.93 bacterial colonies. This blue coloration indicates that C10:0 3-OH 3.07 5.90 7.54 strain A28-5 secretes xylan-degrading enzymes extracel- C12:0 3.49 1.94 3.90 lularly. Agar degradation ability was observed in liquid C12:0 2-OH 1.38 ND ND culture medium containing agar as a carbon source, but C12:0 3-OH 5.02 ND ND xylan degradation was not observed, which means that C13:0 ND 2.08 ND xylan degrading enzyme produced in C. jejuensis A28-5 C14:0 1.31 2.78 2.33 is induced only in the presence of xylan. Therefore, in

C15:1 ω6c ND 1.19 ND order to produce xylan-degrading enzyme using the

C15:0 1.14 10.63 0.65 strain, it is considered that xylan should be prepared

C16:0 21.83 18.77 26.56 and contained in the medium.

C17:0 1.15 2.33 0.62 TLC analysis of the reaction solution containing birch-

C18:1 ω9c 3.25 2.01 1.29 wood xylan and xylanase obtained from the broth in

C18:1 ω7c 17.98 4.68 16.72 which C. jejuensis A28-5 was grown, showed that

C18:0 8.43 2.62 2.17 xylanase secreted by C. jejuensis A28-5 degrades xylan Summed featurea: to produce tetramers and hexamers as major degrada- 3 23.32 37.48 30.91 tion products (Fig. 2A). This TLC pattern is consistent 5 0.622.070.37with the hydrolysis characteristics of the common endo- Unknown 11.799 2.72 2.69 ND type xylanase. Therefore, it is possible to produce useful oligosaccharides such as tetramer and hexamer from ND, not detected; Sum 3, C16:1 ω7c/iso-C15:0 2-OH; 5, C18:2 ω6c, 9c/Anteiso-C18:0. xylan using the enzymes of the strain.

http://dx.doi.org/10.4014/mbl.1703.03003 Catenovulum Jejuensis A28-5 Isolated from Coastal Seawater of Jeju Island 175

Discussion

The first important step for a successful biotechnologi- cal application is to isolate a microorganism that pro- duces industrially useful xylanase and agarase and to reveal the characteristics of the enzymes produced by the strain. In particular, oligosaccharide production through polymer degradation by xylanase and agarase has been recently receiving attention, and specifically activated enzymes are required for the production of tar- geted oligosaccharides. In the present study, a marine microorganism that secretes xylanase and agarase Fig. 2. Thin layer chromatography analysis. (A) Degradation extracellularly was isolated and identified as a novel of birchwood xylan. Crude enzyme of strain A28-5 was reacted species of the genus Catenovulum by morphological, ℃ with birchwood xylan at 40 . S1 : xylobiose (X2); S2 : xylo- physiochemical, and genetic analyses, and the degrada- tetraose (X4); 1 and 2 : reactant of crude enzyme and birch- tion mechanisms of the enzymes produced by this strain wood xylan. Arrows indicate tetramer and hexamer, respectively. (B) Degradation of agarose. Crude enzyme of strain A28-5 was for polymers of xylan and agar were revealed. Through reacted with agarose at 40℃. S : neoagarobiose (NG2), neoag- xylan hydrolysis using xylanase prepared from the arotetraose (NG4), neoagarohexaose (NG6); 1 and 2 : reactant liquid medium in which C. jejuensis A28-5 was grown, of crude enzyme and agarose. Arrows indicate (neo)agaro- the tetramers and hexamers of XOSs were obtained, and tetraose and (neo)agarohexaose, respectively. through agarose hydrolysis using agarase, tetramers and hexamers of (neo)agarooligosaccharides were Degradation of agar by Catenovulum jejuensis A28-5 obtained. The strain was incubated at 37℃ for 2 days and Based on enzyme activities in the liquid medium, it is stained with Lugol’s Iodine solution to confirm agar deg- expected that the genome of strain A28-5 contains many radation around the cells (Fig. 1B). Agar degradation genes encoding xylanase and agarase. In fact, in C. aga- was observed in the supernatant from which the cells rivorans YM01, the type strain belonging to the same were removed by incubation in MB liquid medium con- genus as strain A28-5, after being identified as a new taining agar. It was found that C. jejuensis A28-5 agar-degrading Catenovulum genus in 2011, a genome secreted an agarase enzyme outside the cell. In the sequence of 4.87 Mb was determined by Shi et al., and a medium containing xylan as a carbon source, weak agar total of 15 genes encoding agarase were identified in the degradability was observed. This means that the agar- genome [19]. Among these, it was shown that 2 genes degrading enzyme is produced by C. jejuensis A28-5 at encode α-agarase and 13 genes encode β-agarase. Pre- low levels even in the absence of agar. However, in order ceding this, in Alteromonas sp. strain S89, which was to induce the production of more agarase from the identified as an agarase-producing marine microbial strain, agar should be used as a carbon source. As a strain and whose genome sequence was determined, 6 result of TLC analysis of enzyme reaction with agarose agarase coding genes were discovered [20]. Recently, 2 β- using agarase prepared from C. jejuensis A28-5 liquid agarases were discovered in Agarivorans gilvus WH0801 culture, it was possible to know that the agarase [21]. As compared to these microorganisms, C. agariv- secreted by C. jejuensis A28-5 degrades agarose to produce orans YM01 encodes relatively many agarases. When (neo)agarotetraose (major product) and (neo)agarohexaose cultured on an agar plate, the agar degradation zone (minor product) (Fig. 2B). This TLC pattern is consistent around the colonies of C. jejuensis A28-5 was observed to with hydrolysis characteristics of common endo-type be similar to that of C. agarivorans YM01. This indicates agarases. Therefore, it is possible to produce tetramer that the strain can be expected to encode various aga- and hexamer (neo)agaro-oligosaccharides from agar rases similar to C. agarivorans YM01. Currently, we are using the enzyme of the strain. sequencing the entire genome of C. jejuensis A28-5 using

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the PacBio sequencing technology. Once the genome is ter. Int. J. Syst. Evol. Microbiol. 61: 2866-2873. sequenced, we will be able to compare C. jejuensis A28-5 7. Li DQ, Zhou YX, Liu T, Chen GJ, Du ZJ. 2015. Catenovulum mariti- with type strains at the genetic level. In addition, by mus sp. nov., a novel agarolytic gammaproteobacterium isolated from the marine alga Porphyra yezoensis Ueda (AST58-103), and securing the xylanase and agarase genes, it is expected emended description of the genus Catenovulum. Antonie Van that the mass production of these enzymes will be possi- Leeuwenhoek. 108: 427-434. ble via genetic engineering techniques. 8. Biely P. 1985. Microbial xylanolytic systems. Trends Biotechnol. 11: In the present study, a novel species of the genus 286-290. Catenovulum, which secretes xylanase and agarase, was 9. Chi WJ, Park JS, Kwak MJ, Kim JF, Chang YK, Hong SK. 2013. Isola- identified from the coastal seawater of Jeju Island, tion and characterization of a novel agar-degrading marine bacterium, Gayadomonas joobiniege gen, nov, sp. nov., from the Korea, and its characteristics were revealed. Addition- Southern Sea, Korea. J. Microbiol. Biotechnol. 23: 1509-1518. ally, the hydrolytic properties of the enzymes produced 10. Kim JH, Choi BH, Jo M, Kim SC, Lee PC. 2014. Flavobacterium by the strain were analyzed by TLC. The present study faecale sp. nov., an agarase-producing species isolated from provides important information on the enzymes that can stools of Antarctic penguins. Int. J. Syst. Evol. Microbiol. 64: 2884- produce various oligosaccharides by decomposing plant 2890. cell wall components. 11. Amel BD, Nawel B, Khelifa B, Mohammed G, Manon J, Salima KG, et al. 2016. Characterization of a purified thermostable xylanase from Caldicoprobacter algeriensis sp. nov. strain TH7C1(T). Carbo- GenBanck accession number and strain deposit hydr. 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국문초록

제주 연안해수로부터 한천 분해 효소 및 자일란 분해 효소를 생산하는 Catenovulum jejuensis A28-5의 동정 및 특성 규명 김다솜, 정가람, 배창환, 여주홍, 지원재* 국립생물자원관 생물자원활용부 유용자원분석과

Strain A28-5는 대한민국 제주도 연안의 해수샘플로부터 고체배지내 xylan과 agar를 분해하는 균주로 분리되었다. Strain A28- 5는 그람 음성균으로 한 개의 polar flagella로 운동성을 갖는 Na+ 이온 요구성 균주로 분석되었다. 또한 ampilcillin과 thiostreptone 등의 항생제에 내성을 보였다. Genome 내 G+C content는 43.96%이고, Menaquinone-7 (MK-7)을 predominant quinone으로 함유하고 있었다. Strain A28-5의 세포벽을 구성하는 주요 지방산은 C16:1 ω7c/iso-C15:0 2-OH (23.32%), C16:0(21.83%), C18:1 ω7c (17.98%)였다. strain A28-5의 16S rRNA gene sequence는 Catenovulum agarivorans YM01와 가장 높은 상동성(98.94%)을 보 였으며, Neighbor-Joining phylogenetic tree 제작을 통해서 Catenovulum agarivorans YM01와 가장 높은 근연관계를 보이는 것을 증명하였다. Catenovulum agarivorans YM01과의 DNA-DNA hybridization 분석을 통하여 A28-5을 Catenovulum 속의 신종으로 분류하여Catenovulum jejuensis A28-5로 명명하였다. 이 균주의 액체배양으로부터 준비된 두 종류의 조효소를 이용한 xylan 또는 agarose 와의 효소반응액을 Thin layer chromatography로 분석하여 각각 tetramer와 hexamer의 xylooligosaccharides 와 (neo)agarooligosacchardes가 생산되는 것을 확인하였다.

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