Browse > Article
http://dx.doi.org/10.3839/jksabc.2008.052

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)
Publication Information
Applied Biological Chemistry / v.51, no.4, 2008 , pp. 299-304 More about this Journal
Abstract
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.
Keywords
Bacillus subtilis H12; cloning; ${\beta}$-1,4-endoglucanase; purification;
Citations & Related Records

Times Cited By Web Of Science : 0  (Related Records In Web of Science)
연도 인용수 순위
  • Reference
1 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
2 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
3 Somogyi M (1952) Notes on sugar determination. J Biol Chem 195, 19-23
4 Lynd LR, Weimer PJ, van Zyl WH, and Pretorius IS (2002) Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol Rev 66, 506-577   DOI
5 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   DOI
6 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   DOI   ScienceOn
7 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
8 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   DOI   ScienceOn
9 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   DOI
10 Reese MG (2001) Application of a time-delay neural network to promoter annotation in Drosophila melanogaster genome. Comput Chem 26, 51-56   DOI   ScienceOn
11 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   DOI
12 Saito H and Miura K (1963) Preparation of transforming DNA by phenol treatment. Biochim Biophys Acta 72, 619-629   DOI
13 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
14 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   DOI
15 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
16 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   DOI
17 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   DOI
18 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
19 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   DOI   ScienceOn
20 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   DOI   ScienceOn
21 haracterization and action patterns of two $\beta$-1,4-glucanases purified from Cellulomonas uda CS1-1. J Microb Biotech 57, 1834-1839
22 Wickner W (1979) The Assembly of proteins into biological membranes: the membrane trigger hypothesis. Ann Rev Biochem 48, 23-45   DOI   ScienceOn
23 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   DOI