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Isolation and Characterization of an Agarase-Producing Bacterial Strain, Alteromonas Sp

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Isolation and Characterization of an Agarase-Producing Bacterial Strain, Alteromonas Sp J. Microbiol. Biotechnol. (2012), 22(12), 1621–1628 http://dx.doi.org/10.4014/jmb.1209.08087 First published online September 21, 2012 pISSN 1017-7825 eISSN 1738-8872 Isolation and Characterization of an Agarase-Producing Bacterial Strain, Alteromonas sp. GNUM-1, from the West Sea, Korea Kim, Jonghee1 and Soon-Kwang Hong2* 1Department of Food and Nutrition, Seoil University, Seoul 131-702, Korea 2Division of Bioscience and Bioinformatics, Myongji University, Yongin 449-728, Korea Received: September 3, 2012 / Revised: September 7, 2012 / Accepted: September 8, 2012 The agar-degrading bacterium GNUM-1 was isolated Keywords: Agarase, Alteromonas, agar degradation, Sargassum from the brown algal species Sargassum serratifolium, serratifolium which was obtained from the West Sea of Korea, by using the selective artificial seawater agar plate. The cells were Gram-negative, 0.5-0.6 µm wide and 2.0-2.5 µm long Agar, which is extracted mainly from marine red algae curved rods with a single polar flagellum, forming non- (including Gelidium and Gracilaria spp.), is a mixture of pigmented, circular, smooth colonies. Cells grew at 20oC- o heterogeneous galactans composed mainly of 3,6-anhydro- 37 C, between pH 5.0 and 9.0, and at 1-10% (w/v) NaCl. L-galactose and D-galactose units alternately linked by α- The DNA G+C content of the GNUM-1 strain was 45.5 (1,3) and β-(1,4) linkages [2, 8]. It is a major cell-wall mol%. The 16S rRNA sequence of the GNUM-1 was very component in red algae and has been used in various similar to those of Alteromonas stellipolaris LMG 21861 laboratory and industrial applications, owing to its gelation (99.86% sequence homology) and Alteromonas addita T properties. Therefore, efficient degradation of polysaccharides R10SW13 (99.64% sequence homology), which led us to in the cell wall of seaweeds is a prerequisite for marine assign it to the genus Alteromonas. It showed positive biomass utilization [20]. Many microorganisms that can activities for agarase, amylase, gelatinase, alkaline phosphatase, hydrolyze and metabolize agar as a carbon and energy esterase (C8), lipase (C14), leucine arylamidase, valine source have been identified in seawater, marine sediments, arylamidase, α-chymotrypsin, acid phosphatase, naphthol- and soil. Agarolytic microorganisms commonly produce AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, agarases, which catalyze the hydrolysis of agar. β-glucosidase, catalase, and urease. It can utilize citrate, Agarases that hydrolyze the glycosidic bonds of agarose malic acid, and trisodium citrate. The major fatty acids are generally grouped into 2 types on the basis of their ω were summed feature 3 (21.5%, comprising C16:1 7c/iso- mode of action on agarose; α-agarases cleave the α-1,3 C15:0 2-OH) and C16:0 (15.04%). On the basis of the linkage, whereas β-agarases cleave the β-1,4 linkage of variations in many biochemical characteristics, GNUM-1 agarose [5]. A number of agar-hydrolyzing bacteria have was considered as unique and thus was named Alteromonas been isolated from marine and other environments, and sp. GNUM-1. It produced the highest agarase activity in several agarases have been purified and characterized in modified ASW medium containing 0.4% sucrose, but the past decade from these isolates, including those belonging lower activity in rich media despite superior growth, to the genera Alteromonas [18, 25, 37], Cytophaga [33], implying that agarase production is tightly regulated and Microscilla [40], Pseudoalteromonas [36], Pseudomonas repressed in a rich nutrient condition. The 30 kDa protein [24], and Streptomyces [30, 31]. with agarase activity was identified by zymography, and During screening of agar-hydrolyzing bacteria, the this report serves as the very first account of such a GNUM-1 strain was isolated, using an artificial seawater protein in the genus Alteromonas. (ASW) agar medium, from the marine algae Sargassum serratifolium, obtained from the West Sea of Korea. This *Corresponding author Phone: +82-31-330-6198; Fax: +82-31-335-8249; report describes the characteristics of the GNUM-1 strain E-mail: [email protected] as a species of the genus Alteromonas, and yield improvement 1622 Kim and Hong of agarase production through the optimization of the Tool (BLAST) program [1] and registered as JN578476. The 16S composition of its culture medium. rRNA gene sequences of the related type strains were obtained from the EzTaxon server (http://www.eztaxon.org [7]). Multiple sequence alignment of the most closely related Alteromonas species was carried out using ClustalW [32] and 5'- and 3'-gaps were edited using MATERIALS AND METHODS the BioEdit program [11]. Neighbor-joining (NJ) [27] and maximum Chemicals parsimony methods [15] from the PHYLIP suit program [9] were used to construct phylogenetic trees. Bootstrap analysis was used to Agar and agarose were purchased from Amresco Inc., USA and evaluate the tree topology of the NJ data, performing 1,000 replicates Takara Shuzo Inc., Japan, respectively. All other chemicals were and marked into branching points. The evolutionary distance matrix purchased from Sigma Chemical Co., USA. was estimated using the Kimura’s 2-parameter model [13]. Isolation of Agarase-Producing Microorganisms Phenotypic and Biochemical Characteristics The agarase-producing strain was isolated from S. serratifolium collected in Asan Bay, which is located near the West Sea in Korea. Gram staining was performed using the standard reaction and was S. serratifolium was dissected into 5 mm sections and placed in confirmed by using the KOH test [19]. Colony morphology was observed on ASW-YP after incubation at 28oC for 3 days. Growth bottles containing 250 ml of sterile seawater. The solution was then o o vortexed thoroughly and the supernatant was spread on an artificial at different temperatures (between 4 C and 40 C), pH range (between - seawater (ASW) agar plate [4] containing 6.1 g Tris base (pH 7.2), pH 4 and 10 at intervals of 1 pH unit), and NaCl concentrations [0 15% (w/v)] were determined after 3 days of incubation at 28oC. To 12.3 g MgSO4, 0.74 g KCl, 0.13 g (NH4)2HPO4, 17.5 g NaCl, 0.14 g o test for antibacterial activity, Escherichia coli and Bacillus subtilis CaCl2, and 15 g agar per liter. After incubation at 28 C for 2 weeks, Lugol’s iodine solution (25 g of iodine and 50 g of potassium iodine were used as the reference strains for Gram-negative and Gram- in 1,000 ml of distilled water) was poured onto the plate to detect positive bacteria, respectively. After culturing for 3 days, each indicator strain was overlaid on the surface of the colony. Protein agarase activity. Each colony selected as the candidate for agarase o producer was streaked on a modified ASW (ASW-YP) plate hydrolysis was determined after 3 days of incubation at 28 C on supplemented with 0.3% of bacto-peptone and 0.02% of yeast ASW-YP plate supplemented with 1% gelatin as a substrate. extract, and the plate was incubated at 28oC for several days. A Productions of cellulase, xylanase, and amylase were tested by single colony was then picked and transferred again onto a new adding 0.3% cellulose azure, 0.3% xylan azure, and 0.3% starch ASW-YP plate. The same procedure was repeated for colony isolation azure by using the same method as that used for the analysis of several times until a pure culture was obtained. gelatin hydrolysis. Biochemical characteristics were observed using the API 20NE and API ZYM kits (bioMérieux, France) according Determination of Agarase Activity to the manufacturer’s instructions. Antibiotics susceptibility was All experiments after sampling were performed at 4oC unless otherwise determined by the paper disk-diffusion method, in which a paper µ mentioned. Agarase activity was indirectly measured by the release disk was immersed in 30 l of each stock solution. of the reducing sugar equivalent using the dinitrosalicylic acid µ Chemotaxonomic Characteristics (DNS) method [22]. A 100- l volume of the sample was mixed o with 3.9 ml of reaction buffer (20 mM Tris-Cl, pH 8.0) containing The isolate was cultured on ASW-YP agar at 28 C for 48 h. Major 0.2% of agarose and incubated at 40oC. After incubation for 30 min, respiratory quinone was analyzed by reverse-phase HPLC as DNS was added to the reaction solution. The reaction samples were described elsewhere [16]. Cellular fatty acids were extracted heated at 100oC for 5 min and then cooled to room temperature, according to the standard protocol of the Microbial Identification measuring the amount of reducing sugar released by agarase using a System (MIDI) and identified by gas chromatography using the spectrophotometer (Genesys 8; Spectronic Unicam Inc., France) at a Microbial Identification software package [28]. DNA G+C content wavelength of 540 nm. Because the culture broth has its own was determined by reverse-phase HPLC as described elsewhere [21]. characteristic color and absorbance at 540 nm, the OD540 (optical density at 600 nm) of the reaction solution before incubation was subtracted from that after incubation. Specific agarase activity was Medium Optimization for Agarase Production calculated as the observed agarase activity per unit cell density To establish the optimal culture media that supports the highest (OD540/OD600) using the described culture conditions. agarase production and cell growth of microbial strains, the ASW- YP medium was modified, especially in carbon source. Five kinds 16S rRNA Sequencing and Construction of Phylogenetic Tree of carbon source (agar, starch, sucrose, glucose, and maltose) were To identify the phylogenetic position of the agarase-producing added to the original ASW-YP medium at various final concentrations strain, 16S rRNA sequencing was performed at Genotech Inc.
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