Cloning and Characterization of an Endoglucanase Gene fromsp. Korean Native Goat 40

Asian-Australasian Journal of Animal Sciences

Seung Ha Kang¹  Sang Suk Lee²  Sung Chan Kim³  In Soon Choi⁴  Eun Young Choi⁴  Jin Duck Bok⁵  Yun Jaie Choi⁵  Jae Yeong Kim⁶  Kwang Keun Cho⁶  Yeon Hee Hong⁶ 

[1] CSIRO Animal, Food and Health Science, Queensland Bioscience Precinct, St. Lucia, QLD 4067, .Australia;Department of Animal Science and Technology, Sunchon National University, Sunchon 540-742, .Korea;Department of Biochemistry, Institute of Cell Differentiation and Aging, College of Medicine, Hallym University, Chuncheon 200-702, .Korea;Department of Biological Science, Silla University, Busan 617-736, .Korea;Institute of Green-Bio Science and Technology, Seoul National University, Pyungchang 25354, .Korea;;

关键词: Korean Native Goat;    sp.;    Endo-β-1;    4-glucanase;    Cellulase;   

DOI  :  10.5713/ajas.15.0616

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown