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Enterobacter sp. YB-46의 myo-Inositol dehydrogenase 유전자 클로닝과 특성분석

Molecular Cloning and Characterization of myo-Inositol Dehydrogenase from Enterobacter sp. YB-46

  • 박찬영 (우송대학교 바이오식품과학전공) ;
  • 김광규 ((주)디와이내츄럴) ;
  • 윤기홍 (우송대학교 바이오식품과학전공)
  • 투고 : 2018.05.16
  • 심사 : 2018.05.25
  • 발행 : 2018.06.28

초록

myo-Inositol (MI)을 대사하여 다른 물질로 전환하는 미생물을 과수원 토양으로부터 분리하였다. 분리균 YB-46은 유일한 탄소원으로 MI이 첨가된 배지에서 성장하였고 16S rDNA 염기서열에 따라 Enterobacter 속의 균주로 추정되었다. Fosmid pCC1FOS 벡터를 사용하여 제조된 거대 유전체 은행으로부터 MI을 미지의 대사 물질로 전환하는 Escherichia coli 형질전환주를 선발하였다. 이로부터 플라스미드를 분리하고 삽입된 유전자의 일부 염기서열을 결정한 결과 336 아미노 잔기로 구성된 myo-inositol dehytrogenase (IolG)를 암호화하는 iolG 유전자가 발견되었다. 분리균 YB-46의 IolG는 E. aerogenes와 Bacillus subtilis의 IolG와 약 50% 수준의 상동성을 보였다. 카르복실 말단에 hexahistidine이 연결되도록 제조한 His-tagged IoG (HtIolG)의 유전자를 재조합 대장균에서 발현하여 균체 파쇄액으로부터 HtIolG를 정제하였다. 정제된 HtIolG는 $45^{\circ}C$와 pH 10.5에서 최대 활성을 보였고 MI과 D-glucose에 대한 활성이 가장 높았으며 D-chiro-inositol, D-mannitol 및 D-xylose에도 90% 이상의 활성을 보였다. 최적 반응조건에서 MI을 기질로 하여 반응 동력학적 계수를 측정한 결과 $K_m$$V_{max}$가 1.83 mM과 $0.724{\mu}mol/min/mg$로 확인되었다. HtIolG의 활성은 $Zn^{2+}$에 의해 1.7배 증가하였으며, $Co^{2+}$와 SDS에 의해서는 크게 감소하였다.

A bacterial strain capable of metabolizing myo-inositol (MI) and converting to other substances was isolated from soil of orchard. The isolate, named YB-46, was grown on minimal medium supplemented with MI as the sole carbon source and was presumed to belonging to genus Enterobacter according to the 16S rDNA sequence. Escherichia coli transformant converting MI into unknown metabolites was selected from a metagenomic library prepared with fosmid pCC1FOS vector. Plasmid was isolated from the transformant, and the inserted gene was partially sequenced. From the nucleotide sequence, an iolG gene was identified to encode myo-inositol dehydrogenase (IolG) consisting of 336 amino residues. The IolG showed amino acid sequence similarity of about 50% with IolG of Enterobacter aerogenes and Bacillus subtilis. The His-tagged IolG (HtIolG) fused with hexahistidine at C-terminus was produced and purified from cell extract of recombinant E. coli. The purified HtIolG showed maximal activity at $45^{\circ}C$ and pH 10.5 with the highest activity for MI and D-glucose, and more than 90% of maximal activity for D-chiro-inositol, D-mannitol and D-xylose. $K_m$ and $V_{max}$ values of the HtIolG for MI were 1.83 mM and $0.724{\mu}mol/min/mg$ under the optimal reaction condition, respectively. The activity of HtIolG was increased 1.7 folds by $Zn^{2+}$, but was significantly inhibited by $Co^{2+}$ and SDS.

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참고문헌

  1. Iuorno MJ, Jakubowicz DJ, Baillargeon JP, Dillon P, Gunn RD, Allan G, et al. 2002. Effects of D-chiro-inositol in lean women with the polycystic ovary syndrome. Endocr. Pract. 8: 417-423. https://doi.org/10.4158/EP.8.6.417
  2. McLaurin J, Golomb R, Jurewicz A, Antel JP, Fraser PE. 2000. Inositol stereoisomers stabilize an oligomeric aggregate of Alzheimer amyloid ${\beta}$ peptide and inhibit $A{\beta}$-induced toxicity. J. Biol. Chem. 275: 18495-18502. https://doi.org/10.1074/jbc.M906994199
  3. Berman T, Magasanik B. 1966. The pathway of myo-inositol degradation in Aerobacter aerogenes. Dehydrogenation and dehydration. J. Biol. Chem. 241: 800-806.
  4. Yoshida KI, Aoyama D, Ishio I, Shibayama T, Fujita Y. 1997. Organization and transcription of the myo-inositol operon, iol, of Bacillus subtilis. J. Bacteriol. 179: 4591-4598. https://doi.org/10.1128/jb.179.14.4591-4598.1997
  5. Yoshida K, Sanbongi A, Murakami A, Suzuki H, Takenaka S, Takami H. 2012. Three inositol dehydrogenases involved in utilization and interconversion of inositol stereoisomers in a thermophile, Geobacillus kaustophilus HTA426. Microbiology 158: 1942-1952. https://doi.org/10.1099/mic.0.059980-0
  6. Yebra MJ, Zuniga M, Beaufils S, Perez-Martinez G, Deutscher J, Monedero V. 2007. Identification of a gene cluster enabling Lactobacillus casei BL23 to utilize myo-inositol. Appl. Environ. Microbiol. 73: 3850-3858. https://doi.org/10.1128/AEM.00243-07
  7. Poole PS, Blyth A, Reid CJ, Walters K. 1994. myo-Inositol catabolism and catabolite regulation in Rhizobium leguminosarum bv. viciae. Microbiology 140: 2787-2795. https://doi.org/10.1099/00221287-140-10-2787
  8. Kroger C, Fuch TM. 2009. Characterization of the myo-inositol utilization island of Salmonella enterica serovar Typhimurium. J. Bacteriol. 191: 545-554. https://doi.org/10.1128/JB.01253-08
  9. Jiang G, Krishnan AH, Kim YW, Wacek TJ, Krishnan HB. 2001. A functional myo-inositol dehydrogenase gene is required for efficient nitrogen fixation and competitiveness of Sinorhizobium fredii USDA191 to nodulate soybean (Glycine max [L.] Merr.). J. Bacteriol. 183: 2595-2604. https://doi.org/10.1128/JB.183.8.2595-2604.2001
  10. Galbraith MP, Feng SF, Borneman J, Triplett EW, de Bruijn FJ, Rossbach S. 1998. A functional myo-inositol catabolism pathway is essential for rhizopine utilization by Sinorhizobium meliloti. Microbiology 144: 2915-2924. https://doi.org/10.1099/00221287-144-10-2915
  11. Kohler PR, Choong EL, Rossbach S. 2011. The RpiR-like repressor IolR regulates inositol catabolism in Sinorhizobium meliloti. J. Bacteriol. 193: 5155-5163. https://doi.org/10.1128/JB.05371-11
  12. Morinaga T, Ashida H, Yoshida K. 2010. Identification of two scyllo-inositol dehydrogenases in Bacillus subtilis. Microbiology 156: 1538-1546. https://doi.org/10.1099/mic.0.037499-0
  13. Yoshida K, Yamaguchi M, Morinaga T, Kinehara M, Ikeuchi M, Ashida H, et al. 2008. myo-Inositol catabolism in Bacillus subtilis. J. Biol. Chem. 283: 10415-10424. https://doi.org/10.1074/jbc.M708043200
  14. Fujita Y, Shindo K, Miwa Y, Yoshida K. 1991. Bacillus subtilis inositol dehydrogenase-encoding gene (idh): sequence and expression in Escherichia coli. Gene 108: 121-125. https://doi.org/10.1016/0378-1119(91)90496-X
  15. Kohler PR, Zheng JY, Schoffers E, Rossbach S. 2010. Inositol catabolism, a key pathway in Sinorhizobium meliloti for competitive host nodulation. Appl. Environ. Microbiol. 76: 7972-7980. https://doi.org/10.1128/AEM.01972-10
  16. Ramaley R, Fujita Y, Freese E. 1979. Purification and properties of Bacillus subtilis inositol dehydrogenase. J. Biol. Chem. 254: 7684-7690.
  17. Fry J, Wood M, Poole PS. 2001. Investigation of myo-inositol catabolism in Rhizobium leguminosarum bv. viciae and its effect on nodulation competitiveness. Mol. Plant-Microbe Interact. 14: 1016-1025. https://doi.org/10.1094/MPMI.2001.14.8.1016
  18. Kroger C, Srikumar S, Ellwart J, Fuchs TM. 2011. Bistability in myo-inositol utilization by Salmonella enterica serovar Typhimurium. J. Bacteriol. 193: 1427-1435. https://doi.org/10.1128/JB.00043-10
  19. Bertwistle D, Vogt L, Aamudalapalli HB, Palmer DR, Sanders DA. 2014. Purification, crystallization and room-temperature X-ray diffraction of inositol dehydrogenase LcIDH2 from Lactobacillus casei BL23. Acta Crystallogr. F Struct. Biol. Commun. 70: 979-983. https://doi.org/10.1107/S2053230X14011595
  20. Thompson J, Donkersloot JA. 1992. N-(carboxyalkyl) amino acids: occurrence, synthesis, and functions. Annu. Rev. Biochem. 61: 517-557. https://doi.org/10.1146/annurev.bi.61.070192.002505
  21. van Straaten KE, Zheng H, Palmer DR, Sanders DA. 2010. Structural investigation of myo-inositol dehydrogenase from Bacillus subtilis: implications for catalytic mechanism and inositol dehydrogenase subfamily classification. Biochem. J. 432: 237-247. https://doi.org/10.1042/BJ20101079
  22. Criddle WJ, Fry JC, Keaney MM. 1974. myo-Inositol dehydrogenase(s) from Acetomonas oxydans. Optimization of conditions for solubilization of membrane-bound enzyme. Biochem. J. 137: 449-452. https://doi.org/10.1042/bj1370449
  23. Holscher T, Weinert-Sepalage D, Gorisch H. 2007. Identification of membrane-bound quinoprotein inositol dehydrogenase in Gluconobacter oxydans ATCC 621H. Microbiology 153: 499-506. https://doi.org/10.1099/mic.0.2006/002196-0
  24. Fujita Y, Ramaley R, Freese E. 1977. Location and properties of glucose dehydrogenase in sporulating cells and spores of Bacillus subtilis. J. Bacteriol. 132: 282-293.