Advanced SearchSearch Tips
Cloning and Characterization of an Endoglucanase Gene from Actinomyces sp. Korean Native Goat 40
facebook(new window)  Pirnt(new window) E-mail(new window) Excel Download
 Title & Authors
Cloning and Characterization of an Endoglucanase Gene from Actinomyces sp. Korean Native Goat 40
Kim, Sung Chan; Kang, Seung Ha; Choi, Eun Young; Hong, Yeon Hee; Bok, Jin Duck; Kim, Jae Yeong; Lee, Sang Suk; Choi, Yun Jaie; Choi, In Soon; Cho, Kwang Keun;
  PDF(new window)
A gene from Actinomyces sp. Korean native goat (KNG) 40 that encodes an endo--1,4-glucanase, EG1, was cloned and expressed in Escherichia coli (E. coli) . Recombinant plasmid DNA from a positive clone with a 3.2 kb insert hydrolyzing carboxyl methyl-cellulose (CMC) was designated as pDS3. The entire nucleotide sequence was determined, and an open-reading frame (ORF) was deduced. The ORF encodes a polypeptide of 684 amino acids. The recombinant EG1 produced in E. coli harboring pDS3 was purified in one step using affinity chromatography on crystalline cellulose and characterized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis/zymogram analysis of the purified enzyme revealed two protein bands of 57.1 and 54.1 kDa. The amino terminal sequences of these two bands matched those of the deduced ones, starting from residue 166 and 208, respectively. Putative signal sequences, a Shine.Dalgarno-type ribosomal binding site, and promoter sequences related to the consensus sequences were deduced. EG1 has a typical tripartite structure of cellulase, a catalytic domain, a serine-rich linker region, and a cellulose-binding domain. The optimal temperature for the activity of the purified enzyme was , but it retained over 90% of maximum activity in a broad temperature range ( to ). The optimal pH for the enzyme activity was 6.0. Kinetic parameters, and of rEG1 were 0.39% CMC and 143 U/mg, respectively.
Korean Native Goat;Actinomyces sp.;Endo--1,4-glucanase;Cellulase;
 Cited by
Areej, A., E. M. Altenaiji, and L. F. Yousef. 2014. Fungal cellulases from mangrove forests - A short review. J. Biochem. Tech. 5:765-774.

Baird, S. D., D. A. Johnson, and V. L. Seligy. 1990. Molecular cloning, expression, and characterization of endo-beta-1,4-glucanase genes from Bacillus polymyxa and Bacillus circulans. J. Bacteriol. 172:1576-1586.

Bedford, M. R. and G. G. Partridge. 2001. Enzymes in Farm Animal Nutrition. CABI publishing. Wallingford, Xofrodshire, UK. 38 p.

Bradford, M. M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72:248-254. crossref(new window)

Chang, L., M. Ding, L. Bao, Y. Chen, J. Zhou, and H. Lu. 2011. Characterization of a bifunctional xylanase/endoglucanase from yak rumen microorganisms. Appl. Microbiol. Biotechnol. 90:1933-1942. crossref(new window)

Clarke. A. J. 1997. Biodegradation of Cellulose: Enzymology and Biotechnology. A Technomic Publishing Company Book, Lancaster, PA, USA. 43 p.

Cho, K. K., S. C. Kim, J. J. Woo, J. D. Bok, and Y. J. Choi. 2000. Molecular cloning and expression of a novel family A endoglucanase gene from Fibrobacter succinogenes S85 in Escherichia coli. Enzyme Microb. Technol. 27:475-481. crossref(new window)

Coughlan, M. P. 1985. The properties of fungal and bacterial cellulases with comment on their production and application. Biotechnol. Genet. Eng. Rev. 3:39-110. crossref(new window)

Culleton, H., V. A. McKie, and R. P. de Vries. 2014. Overexpression, purification and characterisation of homologous $\alpha$-L-arabinofuranosidase and endo-1,4-$\beta$-D-glucanase in Aspergillus vadensis. J. Ind. Microbiol. Biotechnol. 41:1697-1708. crossref(new window)

Forsberg, C. W., J. Gong, L. M. J. Malburg, H. Zhu, A. Iyo, K. J. Cheng, P. J. Krell, and J. P. Phillips. 1993. Preceedings of MIE Bioforum 93: Genetics, Biochemistry and Ecology of Lignocellulose Degradation. Toba, Japan. 125-136.

Gao, D., Y. Luan, Q. Wang, Q. Liang, and Q. Qi. 2015. Construction of cellulase-utilizing Escherichia coli based on a secretable cellulase. Microb. Cell Fact. 14:159-167. crossref(new window)

Gilkes, N. R., B. Henrissat, D. G. Kildrun, R. C. Miller, and R. A. J. Warren. 1991. Domains in microbial $\beta$-1, 4-glycanases: Sequence conservation, function, and enzyme families. Microbiol. Mol. Biol. Rev. 55:303-315.

Gong, X., R. J. Gruninger, M. Qi, L. Paterson, R. J. Forster, R. M. Teather, and T. A. McAllister. 2012. Cloning and identification of novel hydrolase genes from a dairy cow rumen metagenomic library and characterization of a cellulase gene. BMC Res. Notes. 5:566-576. crossref(new window)

Kuhad, R. C., R. Gupta, and A. Singh 2011. Microbial cellulases and their industrial applications. Enzyme Res. Article ID 280696.

Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685. crossref(new window)

Lee, D. S. and M. Y. Pack. 1988. Use of Bacilli for overproduction of exocellular endo-beta-1,4-glucanase encoded by cloned gene. Enzyme Microb. Technol. 9:594-597.

Lemaire, M. and P. Beguin. 1993. Nucleotide sequence of the celG gene of Clostridium thermocellum and characterization of its product, endoglucanase CelG. J. Bacteriol. 175:3353-3360.

Mackay, R. M., A. Lo, G. Willick, M. Zuker, S. Baird, M. Dove, F. Moranelli, and V. Seligy. 1986. Structure of a Bacillus subtilis endo-beta-1, 4-glucanase gene. Nucl. Acids Res. 14:9159-9170. crossref(new window)

Malburg, L. M. Jr. and C. W. Forsberg. 1993. Fibrobacter succinogenes prossesses at least nine distinct glucanase genes. Can. J. Microbiol. 39:882-891. crossref(new window)

Min, H. K., Y. J. Choi, J. K. Ha, K. K. Cho, Y. M. Kwon, Y. H. Chang, and S. S. Lee. 1994a. Isolation and identification of anaerobic rumen bacterium, Actinomyces sp. 40 and enzymatic properties of ${\beta}$-1,4-glucanase. Asian Australas. J. Anim. Sci. 7:373-382. crossref(new window)

Min, H. K., Y. J. Choi, K. K. Cho, J. K. Ha, and J. H. Woo. 1994b. Cloning of the endoglucanase gene from Actinomyces sp. 40 in Escherichia coli and some properties of the gene products. J. Microbiol. Biotechnol. 4:102-107.

Mittendorf, V. and J. A. Thomson. 1993. Cloning of an endo- (1o>4)-betaglucanase gene, celA, from the rumen bacterium Clostridium sp. ('C.longisporum') and characterization of its product. CelA, in Escherichia coli. J. Gen. Microbiol. 139:3233-3242. crossref(new window)

Miyatake, M. and K. Imada. 1997. A gene encoding endo-1,4-beta-glucanase from Bacillus sp. 22-28. Biosci. Biotechnol. Biochem. 61:362-364. crossref(new window)

Nguyen, N. H., L. Maruset, T. Uengwetwanit, W. Mhuantong, P. Harnpicharnchai, V. Champreda, S. Tanapongpipat, K. Jirajaroenrat, S. K. Rakshit, L. Eurwilaichitr, and S. Pongpattanakitshote. 2012. Identification and characterization of a cellulase-encoding gene from the buffalo rumen metagenomic library. Biosci. Biotechnol. Biochem. 76:1075-1084. crossref(new window)

Ohara, H., J. Noguchi, S. Karita, T. Kimura, K. Sakka, and K. Ohmiya. 2000. Sequence of egV and properties of EgV, a Ruminococcus albus endoglucanase containing a dockerin domain. Biosci. Biotechnol. Biochem. 64:80-88. crossref(new window)

Park, K. M., H. T. Shin, and K. H. Kang. 1993. Isolation and identification of rumen bacteria from Korean native goat. I. Isolation and identification of Gram positive bacteria. Kor. J. Dairy Sci. 15:165-177.

Perlman, D. and H. O. Halvorson. 1983. A putative signal peptidase recognition site and sequence in eukaryotic and prokaryotic signal peptides. J. Mol. Biol. 167:391-409. crossref(new window)

Poole, D. M., G. P. Hazlewood, J. I. Laurie, P. J. Barker, and H. J. Gilbert. 1990. Nucleotide sequence of the Ruminococcus albus SY3 endoglucanase genes celA and celB. Mol. Gen. Genet. 223:217-223.

Rashamuse, K. J., D. F. Visser, F. Hennessy, J. Kemp, M. P. Rouxvan der Merwe, J. Badenhorst, T. Ronneburg, R. Francis-Pope, and D. Brady. 2013. Characterisation of two bifunctional cellulase-xylanase enzymes isolated from a bovine rumen metagenome library. Curr. Microbiol. 66:145-151. crossref(new window)

Sahu, N. P., D. N. Kamra, and S. S. Paul. 2004. Effect of cellulose degrading bacteria isolated from wild and domestic ruminants on in vitro dry matter digestibility of feed and enzyme production. Asian Australas. J. Anim. Sci. 17:199-202. crossref(new window)

Saito, H. and K. Miura. 1963. Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim. Biophys. Acta. Specialized Section on Nucleic Acids and Related Subjects 72:619-629.

Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: A laboratory manual, 2nd Ed. Cold Spring Harbour Laboratory Press. Cold Spring Harbor, NY, USA.

Sanger, R., S. Niclien, and A. R. Coulson. 1997. DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. 74:5463-5467.

Seo, J. K., T. S. Park, I. H. Kwon, M. Y. Piao, C. H. Lee, and J. K. Ha. 2013. Characterization of cellulolytic and xylanolytic enzymes of Bacillus licheniformis JK7 isolated from the rumen of a native Korean goat. Asian Australas. J. Anim. Sci. 26:50-58. crossref(new window)

Shine, J. and L. Dalgano. 1975. Determinant of cistron specificity in bacterial ribosomes. Naure 254:34-38.

Somogyi, M. 1952. Notes on sugar determination. J. Biol. Chem. 195:19-23.

Teather, R. M. and P. J. Wood. 1982. Use of Congo red - polysaccharide interactions in enumeration and characterization of celluloytic bacteria from the bovine rumen. Appl. Environ. Microbiol. 43:777-780.

Von Heijne, G. 1985. Signal sequences: The limits of variation. J. Mol. Biol. 184:99-105. crossref(new window)

Yan, S. and G. Wu. 2014. Signal peptide of cellulase. Appl. Microbiol. Biotechnol. 98:5329-5362. crossref(new window)

Yuan, S. F., T. H. Wu, H. L. Lee, H. Y. Hsieh, W. L. Lin, B. Yang, C. K. Chang, Q. Li, J. Gao, C. H. Huang, M. C. Ho, R. T. Guo and P. H. Liang. 2015. Biochemical characterization and structural analysis of a bifunctional cellulase/xylanase from Clostridium thermocellum. J. Biol. Chem. 290:5739-5748. crossref(new window)