title: Cloning and Characterization of an Endoglucanase Gene from Actinomyces sp. Korean Native Goat 40.
abstract: A gene from Actinomyces sp. Korean native goat (KNG) 40 that encodes an endo-β-1,4-glucanase, EG1, was cloned and expressed in Escherichia coli (E. coli) DH5α. 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 DH5α 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 55°C, but it retained over 90% of maximum activity in a broad temperature range (40°C to 60°C). The optimal pH for the enzyme activity was 6.0. Kinetic parameters, Km and Vmax of rEG1 were 0.39% CMC and 143 U/mg, respectively.
authors.affiliation: Department of Biochemistry, Institute of Cell Differentiation and Aging, College of Medicine, Hallym University, Chuncheon 200-702, Korea .
authors.forename: Sung Chan
authors.initials: SC
authors.lastname: Kim
authors.affiliation: CSIRO Animal, Food and Health Science, Queensland Bioscience Precinct, St. Lucia, QLD 4067, Australia .
authors.forename: Seung Ha
authors.initials: SH
authors.lastname: Kang
authors.affiliation: Department of Biological Science, Silla University, Busan 617-736, Korea .
authors.forename: Eun Young
authors.initials: EY
authors.lastname: Choi
authors.affiliation: Department of Biochemistry, Institute of Cell Differentiation and Aging, College of Medicine, Hallym University, Chuncheon 200-702, Korea .
authors.forename: Yeon Hee
authors.initials: YH
authors.lastname: Hong
authors.affiliation: Institute of Green-Bio Science and Technology, Seoul National University, Pyungchang 25354, Korea .
authors.forename: Jin Duck
authors.initials: JD
authors.lastname: Bok
authors.affiliation: Department of Biochemistry, Institute of Cell Differentiation and Aging, College of Medicine, Hallym University, Chuncheon 200-702, Korea .
authors.forename: Jae Yeong
authors.initials: JY
authors.lastname: Kim
authors.affiliation: Department of Animal Science and Technology, Sunchon National University, Sunchon 540-742, Korea .
authors.forename: Sang Suk
authors.initials: SS
authors.lastname: Lee
authors.affiliation: Institute of Green-Bio Science and Technology, Seoul National University, Pyungchang 25354, Korea .
authors.forename: Yun Jaie
authors.initials: YJ
authors.lastname: Choi
authors.affiliation: Department of Biological Science, Silla University, Busan 617-736, Korea .
authors.forename: In Soon
authors.initials: IS
authors.lastname: Choi
authors.affiliation: Department of Biochemistry, Institute of Cell Differentiation and Aging, College of Medicine, Hallym University, Chuncheon 200-702, Korea .
authors.forename: Kwang Keun
authors.initials: KK
authors.lastname: Cho
keywords: 4-glucanase; Actinomyces sp.; Cellulase; Endo-β-1; Korean Native Goat