Isolation and Characterization of Starch-Hydrolyzing Pseudoalteromonas Sp

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 industries. 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 chloramphenicol) 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. Of them, one strain (C14), crystine arylamidase, α-chymotrypsin, α-galactosidase, with outstanding hydrolyzing activity toward starch azure β-galactosidase, β-glucuronidase, β-glucosidase, N-acetyl- was named as strain A-3 and further studied (Fig. 1A). β-glucosaminase, α-mannosidase and α-fucosidase. Fur- Strain A-3 was confirmed to be Gram negative bacteria and thermore, D-trehalose, D-saccharose, and N-acetyl-gluco- formed brown-colored colony which was circular, smooth, samine were used as sources of energy but not D-glucose, and less than 1 mm in diameter after cultivation on ASW- D-fructose, D-mannose, D-maltose, D-lactose, D-mannitol, YP agar plate at 28oC for 2 days. In addition, strain A-3 xylitol, D-raffinose, D-xylose, and methyl-α-D-glucopyr- was found a rod-shaped (0.6 µm thick, 1.4 µm long) anoside (Table 1). bacterium to have single polar flagella according to the observation by transmission electron microscopy (TEM) Phylogenetic analysis of strain A-3 via negative staining (Fig. 1B). It grows at temperatures 20- Strain A-3 showed 16S rRNA gene sequence similarity 37oC, grows more slowly at 15oC and 40oC, and growth is o o Table 1. Phenotypic and chemotaxonomic characteristics of the stagnated at 4-10 C and 45 C, on ASW-YP agar plate. It strain A-3. grows abundantly under pH range between pH 6-9 but not Characteristic 12345 grows at pH 4-5 and pH 10 in ASW-YP liquid medium. Color Brown Brown Purple Red Violet Growth of strain A-3 was hardly observed in ASW liquid Flagella + + + + + medium containing 0 and 5-20% (w/v) NaCl while its Amylase + + + + ND growth and amylase activity were observed in medium Lipase (C14) - + ND + - Growth at: NaCl (%) 1-3 1-5 3-6 2-6 0.5-5 Temperature (oC) 15-40 18-37 10-30 10-37 10-40 pH 6-9 6.5-9.5 >6 6-10 5-10 Utilization: D-Glucose - + + + + D-Fructose - ND - - ND D-Mannose - + + + + D-Maltose - + + - + D-Trehalose + + + + ND D-Melibiose w - - - - D-Saccharose + + - - - P. phenolica T P. luteoviolacea Fig. 1. Phenotype and amylase activity of the strain A-3 iso- Strains; 1, Strain A-3; 2, O-BC30 [7]; 3, NCIMB1893T [4]; 4, P. rubra ATCC29570T [4]; 5, P. byunsanensis lated from seawater. (A) Detection of amylase activity on agar T plate. The strain A-3 was grown on an ASW-YP agar plate con- FR1199 [13]. All utilize D-mannose and N-acetylglucosamine, but taining starch azure. (B) Transmission electron microscopy analy- not D-lactose, mannitol or D-xylose. sis after negative staining. Cells were grown on ASW-YP agar +, positive; -, negative; w, weak positive; ND, not data available/not plate at 28oC for 2 days. detected. 320 CHI et al.

Table 2. Similarity search of the strain A-3 based on 16S rRNA NCIMB2033T, P. piscicida IAM12932T, P. maricaloris gene sequence KMM636T with 98.30%, 97.86%, 97.78%, 97.37%, 97.25%, Accession Similar- Strain 97.00%, 97.00%, 96.78%, 96.77% and 96.71%, respectively no. ity (%) (Table 2). T Pseudoalteromonas phenolica O-BC30 AF332880 98.30 Neighbor-joining (NJ) method [16] and maximum parsi- Pseudoalteromonas luteoviolacea T X82144 97.86 NCIMB1893 mony (MP) method [9] from the PHYLIP suit program [3] Pseudoalteromonas rubra T X82147 97.78 ATCC29570 were used for construction of phylogenetic tree. Bootstrap Pseudoalteromonas byunsanensis T DQ11289 97.25 FR1199 value was calculated with data restructured close to 1,000 Pseudoalteromonas citrea T X82137 97.00 NCIMB1889 times and marked into branching point and evolutionary Pseudoalteromonas aurantia T X82135 97.00 ATCC33046 distance matrix was estimated according to Kimura two- Pseudoalteromonas flavipulchra NCIMB2033T AF297958 96.78 parameter model [8]. Phylogenetic tree showed that strain Pseudoalteromonas piscicida IAM12932T AF297959 96.77 A-3 and genetically closest P. phenolica O-BC30T belonged Pseudoalteromonas maricaloris KMM636T AF144036 96.71 to a clade (100% bootstrap value, 1000 replications), but did not cluster robustly with any recognized species of to type strains, Pseudoalteromonas phenolica O-BC30T, P. genus Pseudoalteromonas (Fig. 2). Based on 16S rRNA luteoviolacea NCIMB1893T, P. rubra ATCC29570T, P. analysis, it was suggested that strain A-3 should be classifi- spongiae AY769918T, P. byunsanensis FR1199T, P. citrea ed as a bacterium belonging to genus Pseudoalteromonas, NCIMB1889T, P. aurantia ATCC33046T, P. flavipulchra for which the name Pseudoalteromonas sp. A-3 is proposed.

Fig. 2. Neighbour-joining based on nearly complete 16S rRNA gene sequences showing relationships between strain A-3 and other species of the genus Pseudoalteromonas. Numbers at nodes represent levels of bootstrap value (%) based on analysis of 1,000 replications (only values 50 were shown). STARCH-HYDROLYZING PSEUDOALTEROMONAS sp. A-3 321

Enzymatic characteristics of α-amylase produced from Pseudoalteromonas sp. A-3 The production of α-amylase by Pseudoalteromonas sp. A-3 was measured in liquid medium. When Pseudoaltero- monas sp. A-3 was cultivated in ASW-YP liquid medium, cell growth was sharply increased at 3-6 h and reached stationary phase after 9 h of cultivation (Fig. 3A). Total α- amylase activity was the highest at 6 h cultivation and slowly decreased through cultivation (Fig. 3A). The 6-h cultured broth of Pseudoalteromonas sp. A-3 showed the highest total α-amylase activity at pH 7.0 when reacted at 28oC (Fig. 3B). The relative activities at pH 9 and 10 were over 80% of maximum activity. Thus this strain has a good potential to be an alkali-stable amylase producer in detergent industry. It also showed the maximum activity at 37oC when reacted at pH 7.0 (Fig. 3C). The activities were approximately 80% and 60% at 45oC and 50oC, respectively, compared with maximum activity at 37oC. A similar result was reported for α-amylase from a marine bacterium Pseudoalteromnonas sp. MY-1 [19]. These results suggest that extracellular α-amylase produced by Pseudoalteromonas sp. A-3 can be suitable for industrial applications as a thermo- and alkali-tolerant enzyme. Strain A-3 was screened and selected as α-amylase producer from coastal sea water of Korea, and identified as a bacterium belonging to genus Pseudoalteromoas and thus named Pseudoalteromonas sp. A-3 based on its genetic and physiological characteristics. Although phylogenetic tree based on 16S rRNA gene sequence indicated that strain A- 3 and P. phenolica O-BC30T exhibit the closest phylogenetic relatedness, two strains distinctly showed different biochemical characteristics. Pseudoalteromonas sp. A-3 does not grow when NaCl concentration in the medium exceeds 5% while P. phenolica O-BC30T does [7]. Pseu- Fig. 3. Production and property of α-amylase activity by doalteromonas sp. A-3 was negative to D-glucose, D- Pseudoalteromonas sp. A-3. (A) Time course of amylase produc- mannose, D-maltose and D-melibiose while P. phenolica tion by Pseudoalteromonas sp. A-3. The cells were grown in ASW-YP broth with agitation. The enzymatic activity was mea- O-BC30T was positive. The isolate showed weak positive sured by DNS method. Cell growth (●), amylase activity (■). (B) reaction to D-melibose but negative reaction to P. phenolica Effects of pH. The activities were measured at 37oC. Arrow indi- ▲ O-BC30T. Lipase (C-14) activity was not observed in the cates optimum pH for enzyme activity. 100 mM Citrate buffer ( ), 20 mM MOPS buffer (◆), 20 mM Tris-Cl buffer (●), 20 mM isolate while activity was observed in P. phenolica O- Glycine-NaOH buffer (■). (C) Effects of reaction temperature. BC30T. All those results strongly indicate that Pseudoal- The activities were measured at pH 7.0. Arrows indicate optimal teromonas sp. A-3 and the genetically closest P. phenolica pH and temperature for enzyme activity, respectively. O-BC30T cannot be classified into the same species in genus Pseudoalteromonas. a few groups have studied on α-amylase from Pseudoal- Generally, genus Pseudoalteromonas has been isolated teromonas which is active at low temperature for industrial from the deep sea water with low temperature. Therefore, application to detergent. A cold-adapted α-amylase (ParAmy) 322 CHI et al. from P. arc t ic a GS230 [10] showed its maximum activity division of the genus Alteromonas into two genera, Altero- at 30oC, and still retained 34.5% of maximum activity at monas (emended) and Pseudoalteromonas gen. nov., and 0oC. However, its activity decreased sharply at above 40oC, proposal of twelve new species combinations. Int. J. Syst. Bacteriol. 45: 755-761. which is quite different from that of Pseudoalteromonas sp. 5. Hall, T. A. 1999. BioEdit: a user-friendly biological sequence A-3. The halotolerance of a cold adapted α-amylase from alignment editor and analysis program for Windows 95/98/ the psychrophilic bacterium P. haloplanktis (AHA) was NT. Nucleic. Acids Symp. Ser. 41: 95-98. also reported to have similar cold-stable property to ParAmy 6. Isnansetyo, A. and Y. Kamei. 2009. Anti-methicillin-resis- [18]. tant Staphylococcus aureus (MRSA) activity of MC21-B, an antibacterial compound produced by the marine bacterium One extracellular alpha-amylase from a marine bacterium T Pseudoalteromonas phenolica O-BC30 . Int. J. Antimicrob. Pseudoalteromnonas sp. MY-1 [19] was cloned and express- Agents. 34: 131-135. ed in Escherichia coli. The recombinant amylase revealed 7. Isnansetyo, A. and Y. Kamei. 2003. Pseudoalteromonas phe- maximum activity at pH 7.0 and 40oC, which is very nolica sp. nov., a novel marine bacterium that produces phe- similar to our result, however, Pseudoalteromnonas sp. nolic anti-methicillin-resistant Staphylococcus aureus sub- 53 MY-1 and Pseudoalteromonas sp. A-3 are apart in phylo- stances. Int. J. Syst. Evol. Microbiol. : 583-588. T 8. Kimura, M. 1983. The Neutral Theory of Molecular Evolu- genetic tree. The marine bacterium P. phenolica O-BC30 , tion. Cambridge University Press, Cambridge, UK. the genetically closest to Pseudoalteromonas sp. A-3, was 9. Kluge, A. G and F. S. Farris. 1969. Quantative phyletics and isolated to produce MC21-B, an antibiotic, active against the evolution of anurans. Syst. Zool. 18: 1-32. clinical isolates of methicillin-resistant Staphylococcus aureus 10. Lu, M., S. Wang, Y. Fang, H. Li, S. Liu, and H. Liu. 2010. (MRSA) [6], however, study on enzymes in this strain has Cloning, expression, purification, and characterization of cold-adapted α-amylase from Pseudoalteromonas arctica never been reported. Judging from the close phylogenetic GS230. Protein J. 29: 591-597. relatedness to Pseudoalteromonas sp. A-3, therefore, P. 11. Lyman, J. and R. H. Fleming. 1940. Composition of seawa- T phenolica O-BC30 may produce several extracellular ter. J. Mar. Res. 3: 134-146. enzymes such as α-amylase, which needs further investiga- 12. Pandey, A., P. Nigam, C. R. Soccol, V. T. Soccol, D. Singh, tion. and R. Mohan. 2000. Advances in microbial amylases. Bio- technol. Appl. Biochem. 31: 135–52. 13. Park, Y. D., K. S. Baik, H. Yi, K. S. Bae, and J. Chun. 2005. Acknowledgement Pseudoalteromonas byunsanensis sp. nov., isolated from tidal flat sediment in Korea. Int. J. Syst. Evol. Microbiol. 55: This work was supported by the National Research 2519-2523. 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CLUSTAL W: improving the sensitivity of progressive mul- tion-specific gap penalties and weight matrix choice. Nucleic tiple sequence alignment through sequence weighting, posi- Acids Res. 22: 4673-4680.

국문초록

대한민국 대천 해안에서 분리한 전분 분해능을 갖는 Pseudoalteromonas sp. A-3 균주의 특징 및 동정 지원재 1·박다연1·정성철2·장용근3·홍순광1* 1명지대학교 생명과학정보학부 2한국임업과학연구소 3한국과학기술원 생명화학공학과

Amylase를 생산하는 능력을 갖고 있는 A-3 균주가 대한민국 대천 해변가의 바닷물로부터 분리되었다. A-3 균주는 1개의 polar flagella를 갖으며, Artificial Sea Water-Yeast extract-Peptone(ASW-YP) 한천배지 위에서 배양할 경우 20-37oC에서 잘 자라지만, 15oC와 40oC에서는 천천히 자라는 속성을 보였다. 또한 ASW-YP 액체배지를 사용하는 경 우, pH 6-9 범위에서 잘 자라는 반면 pH 4-5, pH 10에서는 전혀 성장하지 못했다. 16S rRNA sequence 분석 결과, A-3 균주는 Pseudoalteromonas phenolica O-BC30T, Pseudoalteromonas luteoviolacea NCIMB1893T, Pseudoal- teromonas rubra ATCC29570T, Pseudoalteromonas byunsanensis FR1199T와 각각 98.3, 97.86, 97.78, 97.25%의 similarity를 보였으며, 이를 기초로 한 phylogenetic tree 분석결과, P. phenolica O-BC30T와 같은 clade를 형성하였 다. 그러나, A-3 균주는 5% 이상의 NaCl 농도에서 전혀 성장하지 않고, D-glucose, D-mannose, D-maltose, D- melibiose를 이용하지 못하며, lipase 활성(C-14)이 없는 등 많은 생리학적 특성이 P. phenolica O-BC30T와는 상당히 달랐다. 이러한 생리학적 차이로부터 우리는 A-3 균주가 P. phenolica O-BC30T와는 다른 종으로 판단하고, 이 균주 를 Pseudoalteromonas sp. A-3로 명명하였다. Pseudoalteromonas sp. A-3는 배양시기 동안 계속해서 안정적으로 α- amylase를 생산했으며, 총 amylase 활성은 pH 7과 37oC에서 최대값을 보였다. 이 amylase 활성은 pH 10까지도 비교 적 안정적이었으며, 이러한 alkali-tolerant amylase는 산업적으로도 유용성이 클 것으로 사료된다.