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Molecular Characterization of a ${\beta}$-1,4-Endoglucanase Gene from Bacillus subtilis H12

  • Oh, Jin-Hwan (Department of Bio-Environmental Chemistry, College of Agriculture and Life Science, Chungnam National University) ;
  • Cha, Jeong-Ah (Department of Bio-Environmental Chemistry, College of Agriculture and Life Science, Chungnam National University) ;
  • Yoon, Min-Ho (Department of Bio-Environmental Chemistry, College of Agriculture and Life Science, Chungnam National University)
  • 발행 : 2008.12.31

초록

A ${\beta}$-1,4-endoglucanase gene from Bacillus subtilis H12 was cloned into Escherichia coli JM109 (pBC8) and sequenced. The endoglucanase gene with an insert DNA of 2.5 kb possessed an open reading frame of 1,500 bp encoding a mature protein of 499 amino acids with a calculated molecular mass of 55 kDa. The deduced amino acid sequence showed similarity to those of the known neutral cellulase genes of B. subtilis PAP115 (99.2%) and BSE616 (97.8%), as well as the alkaline gene of Bacillus sp. N4 (55.1%). The endoglucanase activity expressed by E. coli (pBC8) was localized in the periplasmic fraction (80%) and the cytoplasmic fraction (20%). An endoglucanase was purified from the periplasmic fraction by performing gel filtration and anion exchange chromatography. The molecular weight of the purified enzyme was estimated to be 31 kDa by SDS-PAGE, and the maximum activity occurred at pH 7 and $40^{\circ}C$. The enzyme easily hydrolyzed soluble substrates such as carboxymethyl cellulose and barely ${\beta}$-glucan, whereas the sigmacell and xylan, the known insoluble substrates, were not entirely hydrolyzed.

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참고문헌

  1. Baird SD, Johnson DA, and Seligy VL (1990) Molecular cloning, expression, and characterization of endo-$\beta$-1,4-glucanase genes from Bacillus polymyxa and Bacillus circulans. J Bacteriol 172, 1576-1586 https://doi.org/10.1128/jb.172.3.1576-1586.1990
  2. Bischoff K, Liu S, and Hughes SR (2007) Cloning and characterization of a recombinant family 5 endoglucanase from Bacillus licheniformis strain B-41361. Process Biochem 42, 1150-1154 https://doi.org/10.1016/j.procbio.2007.05.001
  3. Cohen SN, Chang ACY, and Hso L (1973) Nonchromosomal antibiotic resistance in bacteria: Genetic transformation of Escherchia coli by R-factor DNA. Proc Natl Acad Sci USA 70, 3240-3244
  4. Fukumori F, Sashihara N, Kudo T, and Horikoshi K (1986) Nucleotide sequences of two cellulase genes from alkalophilic Bacillus sp. strain N-4 and their strong homology. J Bacteriol 168, 479-485 https://doi.org/10.1128/jb.168.2.479-485.1986
  5. Hansen C, Diderichsen B, and Jorgensen PL (1992) celA from Bacillus lautus PL236 encodes a novel cellulosebinding endo-$\beta$-1,4-glucanase. J Bacteriol 174, 3522-3531 https://doi.org/10.1128/jb.174.11.3522-3531.1992
  6. Lima AO, Quecine MC, Fungaro M, Andreote FD, Maccheroni Jr M, Araujo WM, Silva-Filho M, Pizzirani- Kleiner A, and Azevedo JL (2005) Molecular characterization of a $\beta$-1,4-endoglucanase from an endophytic Bacillus pumilus. Appl Microb Biotech 68, 57-65 https://doi.org/10.1007/s00253-004-1740-1
  7. Lynd LR, Weimer PJ, van Zyl WH, and Pretorius IS (2002) Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol Rev 66, 506-577 https://doi.org/10.1128/MMBR.66.3.506-577.2002
  8. Mackay RM, Lo A, Willick G, Zuker M, Baird S, Dove M, Moranelli F, and Seligy V (1986) Structure of a Bacillus subtilis endo-$\beta$-1,4-glucanase gene. Nucleic Acid Res 14, 9159-9170 https://doi.org/10.1093/nar/14.22.9159
  9. Murasima K, Nisimura T, Nakamura Y, Koga J, Moriya T, and Sumida N (2002) Purification and characterization of new endo1,4-beta-glucanases from Rhizopus oryzae. Enzyme Microb Tech 30, 319-326 https://doi.org/10.1016/S0141-0229(01)00513-0
  10. Nossal NG, and Heppel LA (1966) The release of enzymes by osmotic shock from Escherichia coli in exponential phase. J Biol Chem 241, 3055-3062
  11. Ozaki K, Sumitmoto N, and Ito S (1991) Molecular cloning and nucleotide sequence of a gene encoding an endo-$\beta$-1,4-glucanase gene from Bacillus sp. KSM-330. J Gen Microbiol 137, 2299-2305 https://doi.org/10.1099/00221287-137-10-2299
  12. Park SH, Kim HK, and Pack MY (1991) Characterization and structure of the cellulase gene of Bacillus subtilis BSE616. Agr Biol Chem 55, 441-448
  13. Reese MG (2001) Application of a time-delay neural network to promoter annotation in Drosophila melanogaster genome. Comput Chem 26, 51-56 https://doi.org/10.1016/S0097-8485(01)00099-7
  14. Reverbel-Leroy C, Belaich A, Bernadac A, Gaudin C, Belaich JP, and Tardif C (1996) Molecular study and overexpression of the Clostridium cellulolyticum celF cellulose gene in Escherichia coli. Microbiology 142, 1013-1023 https://doi.org/10.1099/00221287-142-4-1013
  15. Saito H and Miura K (1963) Preparation of transforming DNA by phenol treatment. Biochim Biophys Acta 72, 619-629 https://doi.org/10.1016/0006-3002(63)90288-9
  16. Sashihara N, Kudo T, and Horikoshi K (1984) Molecular cloning and expression of cellulase genes of alkalophilic Bacillus sp. strain N-4 in Escherichia coli. J Bacteriol 158, 503-506
  17. Somogyi M (1952) Notes on sugar determination. J Biol Chem 195, 19-23
  18. Teather RM & Wood PJ (1982) Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl Environ Microb 43, 777-780
  19. Wickner W (1979) The Assembly of proteins into biological membranes: the membrane trigger hypothesis. Ann Rev Biochem 48, 23-45 https://doi.org/10.1146/annurev.bi.48.070179.000323
  20. Woodward J (1991) Synergism in cellulase systems. In Enzymatic Hydrolysis of Cellulose, Walker LP and Wilson DB (ed), pp. 67-75. Elsevier Applied Science, New York
  21. Wymelenberg AV, Denman S, Dietrich D, Bassett J, Yu X, Atalla A, Predki P, Rudsander U, Teeri T, and Cullen D (2002) Transcript analysis of genes encoding a family 61endoglucanase and a putative membrane-anchored family 9 glycosyl hydrolase from Phanerochaete chrysosporium. Appl Environ Microb 68, 5765-5768 https://doi.org/10.1128/AEM.68.11.5765-5768.2002
  22. Yoda K, Toyoda A, Mukoyama Y, Nakamura Y, and Minato H (2005) Cloning, sequencing, and expression of a Eubacterium cellulosolvens 5 gene encoding an endoglucanase (Cel5A) with novel carbohydrate-binding modules, and properties of Cel5A. Appl Environ Microb 71, 5787-5793 https://doi.org/10.1128/AEM.71.10.5787-5793.2005
  23. haracterization and action patterns of two $\beta$-1,4-glucanases purified from Cellulomonas uda CS1-1. J Microb Biotech 57, 1834-1839

피인용 문헌

  1. Cloning and Expression of Endoglucanase Gene from Thermophilic Bacteria Bacillus sp. RP1 vol.8, pp.4, 2014, https://doi.org/10.5454/mi.8.4.4