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Cloning and Characterization of an Endoglucanase Gene from Actinomyces sp. Korean Native Goat 40
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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;
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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;
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