Korean J. Microbiol. Biotechnol. Vol. 39, No. 4, 317–323 (2011) Isolation and Characterization of Starch-hydrolyzing Pseudoalteromonas sp. A-3 from the Coastal Sea Water of Daecheon, Republic of Korea Chi, Won-Jae1, Da Yeon Park1, Sung-Cheol Jeong2, Yong-Keun Chang3, and Soon-Kwang Hong1* 1Department of Biological Science, Myongji University, Yongin, 449-728, Korea 2Division of Forest Disaster Management, Korea Forest Research Institute, Seoul, 130-712, Korea 3Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology, Taejon 305-701, Korea Received : July 12, 2011 / Revised : September 2, 2011 / Accepted : September 5, 2011 Strain A-3, an amylase-producing bacteria, was isolated from coastal seawater near Daecheon in the Republic of Korea. It was seen to possess a single polar flagella and grow well, on ASW-YP agar plates, at temperatures of between 20-37oC. However, it grew more slowly at the temperatures of 15oC and 40oC. Similarly, it was observed to grow abundantly, in an Artificial Sea Water-Yeast extract-Peptone (ASW-YP) liquid medium, in a pH range of 6-9, but not grow at pHs of 4-5 and a pH of 10. Strain A-3 was noted as being close to Pseudoalteromonas phenolica O-BC30T, Pseudoalteromonas luteoviolacea NCIMB1893T, Pseudoalteromo- nas rubra ATCC29570T, and Pseudoalteromonas byunsanensis FR1199T, with 98.30%, 97.86%, 97.78%, and 97.25% similarities respectively, in its 16S rRNA sequence. A phylogenetic tree revealed that strain A-3 and P. phenolica O-BC30T belong to a clade. However, strain A-3 differed from P. phenolica O-BC30T in relation to a number of physiological characteristics. Strain A-3 exhibited no growth above 5% NaCl concentrations, no utilization of D-glucose, D-mannose, D-maltose, or D-melibose, and no lipase (C-14) activity. All of these properties strongly indicate that strain A-3 is distant from P. phenolica O-BC30T and thus led us to name it Pseudoalteromonas sp. A-3. Pseudoalteromonas sp. A-3 produces α-amylase throughout growth. Maximal amylase activities of 144.48 U/mL and 149.20 U/mL were seen at pH 7.0 and 37oC, respectively. Pseudoalter- omonas sp. A-3’s high, stable production of α-amylase in addition to its biochemical features, such as alkali- tolerance, suggest that it is a good candidate for industrial applications. Keywords: α-Amylase, marine bacteria, Pseudoalteromonas, phylogenetic analysis Introduction take up close to 25% of the entire enzyme-related indus- tries. Although α-amylase has been identified and charac- Alpha amylase (endo-1, 4-α-D-glucan glucanohydrolase) terized from various sources such as animals and plants, is an extracellular endoenzyme that produces glucose, microorganisms has been regarded as the most important maltose and maltotriose monomers by randomly cutting α- source for identification of valuable α-amylase for indus- 1, 4 linkage between glucose monomers in straight-chain trial purposes [1, 12]. amylose. Conversion of starch into sugar syrups (glucose, For screening of a novel α-amylase, identification of a maltose, maltotriose, dextrin sugar, fructose syrups, etc.) is new microorganism capable of hydrolyzing starch is pri- one of the critical processes in starch processing industry mary important step. Thus we attempted to isolate new and α-amylase is applicable to such industries as textile, bacterial strains from coastal seawater of Daecheon, locat- paper, brewing, bakery, pharmaceutical and bio-energy ap- ing at western part of Korea, which produce extracellular plications [14, 17, 15]. Industries related with α-amylase α-amylase, and characterize enzyme produced from those strains. Here, we describe isolation and identification of a novel strain belonging to genus Pseudoalteromonas and its *Corresponding author biochemical characteristics. Tel: +82-31-330-6198, Fax: +82-31-335-8249 E-mail: [email protected] 318 CHI et al. Material and Methods Phenotypic and biochemical characteristics Gram staining was performed with gram stain kit (BD, Isolation of bacterial strains with amylase activity USA) according to proposed procedures and observed with Coastal seawater of Daecheon, Korea, was collected and an optical microscope. Physiological characteristics were diluted in series at 10-1-10-5 and 100 µL of diluted solution observed with API Staph and API ZYM strip (Biomérieux, was spread on Artificial Sea Water (ASW) agar plate (6.1 g France) according to the instructions of manufacturer with Tris base, 12.3 g MgSO4, 0.74 g KCl, 0.13 g (NH4)2HPO4, the exception that the bacterial suspension was prepared in 17.5 g NaCl, 0.14 g CaCl2, and 15 g Bacto agar Per L, pH 2% NaCl (w/v). Strain A-3 was inoculated on ASW-YP 7.2) and cultivated at 28oC for 24 h [11]. The strains were liquid medium of pH 4-10 (interval of pH 1) and cultured transferred onto ASW agar plate containing 0.3% (w/v) of at 28oC for 3 days to observe effect of initial pH on growth. starch azure (Sigma Chemical Co., USA) as a sole carbon The isolate was inoculated and cultured on ASW-YP agar source and chromogenic substrate with blue color. Hydrolytic plate at 4, 15, 25, 37, 40, and 45oC to determine optimal zone of starch azure by α-amylase produced by microor- growth temperature. For NaCl requirement test, 0, 1, 2, 3, ganism was visible due to the clear zones around cells on 5, 10, 15, and 20% (w/v) NaCl was added in ASW-YP agar plate. The selected colonies were restreaked on ASW- liquid medium devoid of NaCl. YP (ASW supplemented with 0.3% (w/v) of soluble starch, 1.0% (w/v) of yeast extract and 0.3% (w/v) of bacto peptone Antibiotics susceptibility test for faster growth) agar plate and cultured under the same To test the susceptibility toward various antibiotics, strain conditions. When necessary, bacteria were cultured in ASW- A-3 was smeared on ASW-YP agar plate and incubated at YP liquid medium at 28oC with vigorous shaking. All 28oC for 1 h, and then paper disc containing 30 µL of stock reagents used for medium and enzyme activity assay were solution (100 µg/µL) of each antibiotic (thiostrepton, kana- purchased from Sigma Chemical Co. (USA). mycin, neomycin, ampicillin, apramycin, and chloram- phenicol) was laid on the plate. The plate was incubated at Determination of 16S rRNA gene sequence and phy- 28oC for 24 h and clear zone around the paper disc was logenetic analysis observed. The selected bacteria were cultured in ASW-YP liquid medium for 2 days and genomic DNAs were extracted with Alpha-amylase activity and cell growth genomic DNA extraction kit (DyneBio, Korea). Enzyme The enzyme activity (α-amylase) of strain A-3 was mea- and others used to amplify polyermase chain reaction sured in liquid medium. Strain A-3 was cultured in ASW- (PCR) of 16S rRNA gene were from Takara Shuzo (Japan), YP liquid medium containing 0.3% (w/v) soluble starch at and bacterial universal primer (27F and 1525R) used in 28oC for 24 h. One mL of culture broth was sampled at a PCR was synthesized from Genotech (Korea). 16S rRNA regular interval and measured at 600 nm to determine gene sequencing was performed at Genotech Inc. (Korea) growth curve. The sample was centrifuged and its super- using an Applied Biosystems 3730xl DNA Analyzer. 16S natant was collected to measure α-amylase activity. α- rRNA gene sequence of type strains were collected from amylase activity was measured according to the previously EzTaxon server (http://www.eztaxon.org) [2]. Multi-align- described method using 3, 5-dinitrosalicylic acid (DNS). ment between those of related strains was determined by 0.2% (w/v) of soluble starch was added as substrate to using clustal W software [20] and gaps of 5’ and 3’ were reaction solution. One unit (U) of α-amylase was defined edited via BioEdit program [5]. Neighbor-joining (NJ) as the amount of enzyme that produced 1 µmol of glucose method [16] and maximum parsimony (MP) method [9] per min under the assay conditions. Glucose was used as a from the PHYLIP suit program [3] were used for construc- reference reducing sugar for preparing standard curve. tion of phylogenetic tree. Bootstrap value was calculated with data restructured close to 1,000 times and marked into Determination of optimum condition for enzyme branching point and evolutionary distance matrix was esti- activity mated according to Kimura two-parameter model [8]. Sample at 6 h cultivation was used as enzyme solution. To measure optimum pH condition of α-amylase, enzyme STARCH-HYDROLYZING PSEUDOALTEROMONAS sp. A-3 319 reaction was carried out at 28oC under the various pH containing 1-3% (w/v) of NaCl (Table 1). Strain A-3 showed conditions. 20 mM MOPS buffer (pH 6-7), 20 mM Tris-Cl moderate susceptibility to chloramphenicol but resistance buffer (pH 7-9), and 20 mM glycine-NaOH buffer (pH 9- to kanamycin, neomycin, ampicillin, apramycin and thiost- 10) were used, respectively. To measure optimum tempera- repton. ture of α-amylase, the assays were performed at various Physiological characteristics of strain A-3 were observed temperatures, 20, 30, 37, 45, and 50oC, in 20 mM Tris-Cl with API Staph and API ZYM strip (Biomérieux, France) buffer (pH 7.0). according to the instructions of manufacturer with the exception that the bacterial suspension was prepared in 2% Results and Discussion NaCl (w/v). The strain A-3 produced alkaline phosphatase, esterase (C4 and C8), valine arylamidase (weak positive), Phenotypic and biochemical characteristics of the leucine arylamidase, trypsin protease, acid phosphatase strain A-3 (very weak positive), naphtho-AS-BI-phosphorylase, α- Total 15 strains were selected as the candidates that glucosidase (very weak positive), but did not produce lipase hydrolyze starch azure around colony.