한천 분해효소를 생산하는 해양 미생물 SH-1의 분리 및 특성 분석
Isolation and Characterization of Marine Bacterial Strain SH-1 Producing Agar-Degrading Enzymes
2
조회
0
다운로드
3
인용
0
참고문헌
키워드
초록
A marine bacterial strain producing agar-degrading enzymes was isolated from a mud flat in Jeboo-do (Korea) using a selective artificial sea water (ASW) agar plate containing agar as the sole carbon source. The isolate, designated as SH-1, was gram-negative, aerobic, and motile with single polar flagellum. 16S rRNA gene sequence similarity analysis showed the isolate SH-1 had the highest homology (96.5%) to marine bacterium Neiella marina J221. Cells could grow at 28-37ºC but not at 42ºC, and the agarase activity of the cell culture supernatant was higher when grown at 28ºC than when grown at 37ºC. Cells could grow when concentrations of 1-5% (w/v) NaCl were added to the growth media with the best growth observed at 3% NaCl, and the agardegrading enzyme activity of the cell culture supernatant was best when grown at 3% NaCl-containing growth media under the conditions we examined. The crude enzyme prepared from 48-h culture broth of strain SH-1 exhibited an optimum pH and temperature for agar-degrading activity at 7.0 and 40ºC, respectively. Zymogram analysis of the crude supernatant and cell extract showed that strain SH-1 produced at least 3 agar-degrading enzymes with molecular weights of 15, 35, and 52 KD. Thinlayer chromatography (TLC) analysis also suggested that HS-1 produces β-agarase to degrade agarose to neoagarooligosaccharides.
Abstract
A marine bacterial strain producing agar-degrading enzymes was isolated from a mud flat in Jeboo-do (Korea) using a selective artificial sea water (ASW) agar plate containing agar as the sole carbon source. The isolate, designated as SH-1, was gram-negative, aerobic, and motile with single polar flagellum. 16S rRNA gene sequence similarity analysis showed the isolate SH-1 had the highest homology (96.5%) to marine bacterium Neiella marina J221. Cells could grow at 28-37ºC but not at 42ºC, and the agarase activity of the cell culture supernatant was higher when grown at 28ºC than when grown at 37ºC. Cells could grow when concentrations of 1-5% (w/v) NaCl were added to the growth media with the best growth observed at 3% NaCl, and the agardegrading enzyme activity of the cell culture supernatant was best when grown at 3% NaCl-containing growth media under the conditions we examined. The crude enzyme prepared from 48-h culture broth of strain SH-1 exhibited an optimum pH and temperature for agar-degrading activity at 7.0 and 40ºC, respectively. Zymogram analysis of the crude supernatant and cell extract showed that strain SH-1 produced at least 3 agar-degrading enzymes with molecular weights of 15, 35, and 52 KD. Thinlayer chromatography (TLC) analysis also suggested that HS-1 produces β-agarase to degrade agarose to neoagarooligosaccharides.
참고문헌 (29)
- Araki T, "Purification and characterization of agarases from a marine bacterium, Vibrio sp. PO-303", J. Mar. Biotechnol, 6, pp.260~265 (1998)
- Beer E, "Engineering algae for biohydrogen and biofuel production", Curr. Opin. Biotechnol, 20, pp.264~271 (2009)
- Buck BC, "The offshore ring: A new system design for the open ocean aquaculture of macroalgae", J. Appl. Phycol, 16, pp.355~369 (2004)
- 지원재, "Isolation and Characterization of Starch-hydrolyzing Pseudoalteromonas sp. A-3 from the Coastal Sea Water of Daecheon, Republic of Korea", 한국미생물·생명공학회지, 39(4), pp.317~323 (2011), 한국미생물·생명공학회
- Chi WJ, "Agar degradation by microorganisms and agar-degrading enzymes", Appl. Microbiol. Biotechnol, 94, pp.917~930 (2012)
- Chisti Y, "Biodiesel from microalgae", Biotechnol. Adv, 25, pp.294~306 (2007)
- Chun JS, "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)
- Do JH, "Extraction and purification of agar from Gelidium amansii", J. Korean Fish Soc, 30, pp.423~427 (1997)
- Du ZJ, "Neiella marina gen. nov., sp. nov., isolated from the sea cucumber Apostichopus japonicus", Int. J. Syst. Evol. Microbiol, 63, pp.1597~1601 (2013)
- Duckworth M, "The structure of agar : Part I. Fractionation of a complex mixture of polysaccharides", Carbohydr. Res, 16, pp.189~197 (1971)
- Englard S, "Precipitation techniques", Meth. Enzymol, 182, pp.285~300 (1990)
- Frederiks WM, "Metabolic mapping of proteinase activity with emphasis on in situ zymography of gelatinases:review and protocols", J. Histochem. Cytochem, 52(6), pp.711~722 (2004)
- Fu XT, "Agarase: Review of major sources, categories, purification method, enzyme characteristics and applications", Mar. Drugs, 8, pp.200~218 (2010)
- Kato I, "Antioxidative and antitumorigenic properties of agarooligosaccharide", Bio Industry, 17, pp.13~19 (2000)
- 김정환, "해양 미생물 유래 해조 다당류 분해 효소의 특성 및 산업적 응용", 한국미생물·생명공학회지, 39(3), pp.189~199 (2011), 한국미생물·생명공학회
- Kimura M, "The neutral theory of molecular evolution" (1983), Cambridge Univesity Press
- Kobayashi R, "Neoagarobiose as a novel moisturizer with whitening effect", Biosci. Biotechnol. Biochem, 61, pp.162~163 (1997)
- Laemmli UK, "Cleavage of structural proteins during the assembly of the head of bacteriophage T4", Nature, 227, pp.680~685 (1970)
- Maniatis T, "Molecular cloning:a laboratory manual" (1982), Cold Spring Harbor Laboratory Press
- McCandless E, "The Biology of Seaweeds", pp.559~588 (1981), University of California Press
- Miller GL, "Use of dinitrosalicylic acid reagent for determination of reducing sugar", Anal. Chem, 31, pp.426~428 (1959)
- Ohta Y, "Purification and characterization of a novel α-agarase from a Thalassomonas sp", Curr. Microbiol, 50, pp.212~216 (2005)
- Park DY, "Cloning, expression, and biochemical characterization of a GH16β-agarase AgaH71 from Pseudoalteromonas hodoensis H7", Appl. Biochem. Biotechnol (2014)[DOI]
- 박주이, "Production of DagA, a β-Agarase, by Streptomyces lividans in Glucose Medium or Mixed-Sugar Medium Simulating Microalgae Hydrolysate", Journal of Microbiology and Biotechnology, 24(12), pp.1622~1628 (2014), 한국미생물·생명공학회[DOI]
- Potin P, "Purification and characterization of the alpha-agarase from Alteromonas agarlyticus (Cataldi) comb. Nov., strain GJ1B", Eur. J. Biochem, 214, pp.599~607 (1993)
- Saitou N, "The neighbor-joining method: a new method for reconstructing phylogenetic trees", Mol. Biol. Evol, 4, pp.406~425 (1987)
- Song T, "Isolation and characterization of agar-degrading endophytic bacteria from plants", Curr. Microbiol (2014)[DOI]
- Thompson JD, "CLUSTAL W:improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice", Nucleic Acids Res, 22, pp.4673~4680 (1994)
- Temuujin U, "Overexpression and biochemical characterization of DagA from Streptomyces coelicolor A3(2): an endo-type β-agarase producing neoagarotetraose and neoagarohexaose", Appl. Microbiol. Biotechnol, 92, pp.749~759 (2011)