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KMID : 0613820050150061013
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Journal of Life Science
2005 Volume.15 No. 6 p.1013 ~ p.1021
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Cloning of α-Amylase Gene from Unculturable Bacterium Using Cow Rumen Metagenome
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Cho Soo-Jeong
Yoon Han-Dae
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Abstract
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The metagenomes of complex microbial communities are rich sources of novel biocatalysts. The gene encoding an extracellular α-amylase from a genomic DNA of cow rumen was cloned in Escherichia coli DH5α and sequenced. The α-amylase (amyA) gene was 1,893 bp in length, encoding a protein of 631 amino acid residues with calculated molecular weight of 70,734 Da. The molecular weight of the enzyme was estimated to be about 71,000 Da by active staining of a SDS-PAGE. The enzyme was 21 to 59% sequence identical with other amyloyltic enzymes. The AmyA was optimally active at pH 6.0 and 40℃. The AmyA had a calculated pI of 5.87. AmyA expressed in E. coli DH5α was enhanced in the presence of Mg^(2+) (20 mM) and Ca^(2+) (30 mM) and inhibited in the presence of Fe^(2+) and Cu^(2+). The origin of amyA gene could not be confirmed by PCR using internal primer of amyA gene from extracted genomic DNA of 49 species rumen culturable bacteria so far. An amyA is supposed to obtained from unculturable rumen bacterium in cow rumen environment.
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