And Xylanse-Producing Catenovulum Jejuensis A28-5 from Coastal Seawater of Jeju Island, Korea
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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 bacte- rial 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)agarooligosac- charides, 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). June 2017 | Vol. 45 | No. 2 170 Kim et al. 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)