아가로오스 분해세균인 Pseudoalteromonas sp. GNUM08122 분리 및 동정
Isolation and Identification of Agarose-degrading Bacterium, Pseudoalteromonas sp. GNUM08122.
1
조회
0
다운로드
2
인용
0
참고문헌
키워드
초록
본 연구는 홍조류를 기질로 사용한 바이오에탄올 생산 공정에서 전처리(홍조류 가수분해) 공정의 효율을 높이기 위하여 성능이 우수한 신규 아가레이즈를 발굴하는 데 있다. 남해안에 서식하는 해조류를 채집하여 이로부터 아가레이즈활성을 갖는 3종의 균주들을 순수 분리 하였다. 이들 균주들을 4일간 배양한 후, 황산암모늄 침전과 투석에 의하여 배양액으로부터 조효소를 회수하였다. 세포외 분비 효소를 포함하는 배양 상등액으로부터 얻은 조효소와 세포 내 효소를포함하는 세포 추출물에서 얻은 조효소 모두에서 아가레이즈 활성이 측정되었고, 동일 균주에서 세포외 분비 단백질이 세포내 축적 단백질보다 높은 활성을 나타내었다. 3종의균주 중 GNUM08122 조효소가 단위 단백질 당 아가레이즈 활성은 낮았으나, p-nitrophenyl-α-D-galactopyranoside의α-결합을 끊는 것으로 관찰되어 α-agarase 활성이 있을 것으로 추측되어 균주 동정을 실시하였다. GNUM08122 균주의 16S rRNA 염기서열을 결정하고 계통수 분석을 수행한 결과 Pseudoalteromonas issachenkonii KMM 3549 및Pseudoalteromonas tetraodonis IMA 14160 균주와 99.7%이상 상동성을 보였는데, 이는 GNUM08122가 Pseudoalteromonas 속 균주임을 나타낸다. 균주의 생화학적·생리적특성을 조사하였다. GNUM08122는 40oC, 산성 조건(pH 4)는 물론 약 알칼리(pH 8)에서도 활발히 성장하였다. 높은NaCl(10%, w/w)에서도 세포생장이 저해를 받지 않았고 다양한 탄수화물을 사용하는 것으로 확인되었다.
Abstract
This study’s aim was to isolate microorganisms producing agarase with a high activity, with possible applications in improving the performance of the pretreatment processes for bioethanol production. Marine algaes were collected from the south coast of Korea, from which three kinds of microorganisms were isolated. After a 4-day culture of these strains at 25oC,crude enzymes were obtained from culture supernatant or cell-free extract by ammonium sulfate precipitation and membrane dialysis. Agarase activity was observed in these crude enzymes. Notably higher specific activity was observed in the crude enzyme obtained from the culture supernatant rather than that from the cell-free extract. This indicates that a secreted enzyme has a much greater activity than a cellular enzyme. Crude enzymes from the GNUM08122 strain were inferred to have α-agarase activity because release of p-nitrophenol was observed, possibly due to the cleavage of p-nitrophenyl-α-D-galactopyranoside. The 16S rRNA sequence of GNUM08122 showed a close relationship to Pseudoalteromonas issachenkonii KMM 3549(99.8%) and Pseudoalteromonas tetraodonis IMA 14160 (99.7%), which led us to assign it to the genus Pseudoalteromonas. Biochemical and physiological study revealed that this strain can grow well at 40oC under a wide range of pH (pH 4~8) in high-salt conditions (10% NaCl).
참고문헌 (38)
- Bowman, J, "Bioactive compound synthetic capacity and ecological significance of marine bacterial genus Pseudoalteromonas", Mar. Drugs, 5, pp.220~241 (2007)
- Chun, J, "ExTaxon: a web-based tool for the identification of prokaryotes based on 16S ribosomal RNA gene sequences", Int. J. Syst. Evol. Microbiol, 57, pp.2259~2261 (2007)
- Chen, H. M, "The preparation and bioactivity research of agaro-oligosaccharides", Food Technol. Biotechnol, 43, pp.29~36 (2005)
- Felsenstein, J, "PHYLIP (phylogeny inference package), Version 3.69. Distributed by the author" (2009), Department of Genome Sciences, University of Washington
- Flament, D, "Alpha-agarases define a new family of glycoside hydrolases, distinct from beta-agarase families", Appl. Environ. Microbiol, 73, pp.4691~4694 (2007)
- Fu, X. T, "Purification and characterization of a novel β-agarase, AgaA34, from Agarivorans albus YKW-34", Appl. Microbiol. Biotechnol, 78, pp.265~273 (2008)
- Fu, X. T, "Agarase: review of major sources, categories, purification method, enzyme characteristics and applications", Mar. Drugs, 8, pp.200~218 (2010)
- Ghadi, S. C, "Screening for agarolytic bacteria and development of a novel method for in situ detection of agarase", J. Mar. Biotechnol, 5, pp.194~200 (1997)
- Hatada, Y, "Hyperproduction and application of α–garase to enzymatic enhancement of antioxidant activity of porphyran", J. Agric. Food Chem, 54, pp.9895~9900 (2006)
- Ivanova, E. p, "Ecophysiological variabilities in ectohydrolytic enzyme activities of some Pseudoalteromonas species, P. citrea, P. issachenkonii, and P. nigrifaciens", Curr. Microbiol, 46, pp.6~10 (2003)
- Ivanova, E. P, "Retrieval of the species Alteromonas tetraodonis Simidu et al. 1990 as Pseudoalteromonas tetraodonis comb. Nov. and emendation of description", Int. J. Syst. Evol. Microbiol, 51, pp.1071~1078 (2001)
- Ivanova, E. P, "Pseudoalteromonas issachenkonii sp. nov., a bacterium that degrades the thallus of the brown alga Fucus evanescens", Int. J. Syst. Evol. Microbiol, 52, pp.229~234 (2002)
- Kazlowski, B, "Separation and quantification of neoagaro- and agaro-oligosaccharide products generated from agarose digestion by β-agarase and HCl in liquid chromatography systems", Carbohydr. Res, 343, pp.2443~2450 (2008)
- 김유나, "Pseudoalteromonas sp. 배양액으로부터 아가레이즈 분리를 위한 음이온교환 크로마토그래피 최적화", Korean Chemical Engineering Research(HWAHAK KONGHAK), 49(6), pp.840~845 (2011), 한국화학공학회
- Kimura, M., "The neutral theory of molecular evolution" (1983), Cambridge University Press
- Kirimura, K, "Purification and characterization of a novel β-agarase from an alkalophilic bacterium, Alteromonas sp. E-1", J. Biosci. Bioeng, 87, pp.436~441 (1999)
- Komagata, K, "Lipid and cell-wall analysis in bacterial systematic", Methods Microbiol, 19, pp.161~207 (1987)
- Lakshmikanth, M, "Purification and characterization of β-agarase from agar-liquefying soil bacterium, Acinetobacter sp., AG LSL-1", Proc. Biochem, 44, pp.999~1003 (2009)
- Lakshmikanth, M, "Extracellular β-agarase LSL-1 producing neoagarobiose from a newly isolated agar-liquefying soil bacterium, Acinetobacter sp., AG LSL-1. World", J. Microbiol. Biotechnol, 22, pp.1087~1094 (2006)
- 이성목, "Saccharomyces cerevisiae에 의한 Agar로부터 바이오 에탄올 생산", 공업화학, 20(3), pp.290~295 (2009), 한국공업화학회
- Mesbah, M, "Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography", Int. J. Syst. Bacteriol, 39, pp.159~167 (1989)
- Michel, G, "Bioconversion of red seaweed galactans: a focus on bacterial agarase and carrageenases", Appl. Microbiol. Biotechnol, 71, pp.23~33 (2006)
- Miller, L, "Bacterial identification by gas chromatography of whole cell fatty acid. Hewlett-Packard Application Note", pp.228~241 (1985), Hwelett-Packard Co
- Morrice, L. M, "β-agarase I and II from Pseudomonas atlantica purification and some properties", Eur. J. Biochem, 135, pp.553~558 (1983)
- Morrice, L. M, "β-agarase I and II from Pseudomonas atlantica substrate specificities", Eur. J. Biochem, 137, pp.149~154 (1983)
- Oh, C. H, "Molecular cloning, characterization and enzymatic properties of a novel beta-agarase from a marine isolate Psudoalteromonas sp. AG52", Brazilian J. Microbiol, 41, pp.876~889 (2010)
- Ohta, Y, "Purification and characterization of a novel α-agarase from a Thalassomonas sp", Curr. Microbiol, 50, pp.212~216 (2005)
- Potin, P, "Purification and characterization of the α-agarase from Alteromonas agarlyticus (cataldi) comb. Nov., strain GJ1B", Eur. J. Biochem, 214, pp.559~607 (1993)
- Saitou, N, "The neighbor-joining method: a new method for reconstructing phylogenetic trees", Mol. Biol. Evol, 4, pp.406~425 (1987)
- Sasser, M, "Identification of bacteria by gas chromatography of cellular fatty acids" (1990), MIDI Inc
- Schroeder, D. M, "Investigation of the role of a β(1-4) agarase produced by Pseudomonas gracilis B9 in eliciting disease symptoms in the red alga Gracilaria gracilis", Microbiology, 149, pp.2919~2929 (2003)
- Somogyi, M, "Notes on sugar determination", J. Biol. Chem, 195, pp.19~23 (1952)
- Suzuki, H, "Purificationand characterization of an extracellular α-neoagarooligosaccharide hydrolase from Bacillus sp. MK03", J. Biosci. Bioeng, 93, pp.456~463 (2002)
- Suzuki, H, "Purification and characterization of an extracellular β-agarase from Bacillus sp. MK03", J. Biosci. Bioeng, 95, pp.328~334 (2003)
- Vera, J, "Identification of a marine agarolytic Pseudoalteromonas isolate and characterization of its extracellular agarase", Appl. Environ. Microbiol, 64, pp.4378~4383 (1998)
- Wang, J, "Characterization of a novel β-agarase from marine Alteromonas sp. SY37-12 and its degrading products", Appl. Microbiol. Biotechnol, 71, pp.833~839 (2006)
- Wang, X, "Two-stage hydrolysis of invasive algal feedstock for ethanol fermentation", J. Integr. Plant Biol, 53, pp.246~252 (2011)
- Zhang, W. W, "Cloning, characterization, and molecular application of a beta-agarase gene from Vibrio sp. Strain V134", Appl. Environ. Microbiol, 73, pp.2825~2831 (2007)