99A-19 |
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J. YUN1, H. Yoon1, S. Park1, S. Kang2, and S. Ryu1. (1) Dept. of Food Science & Technology, Seoul National Univ., Laboratory of Molecular Food Microbiology, School of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul, 151-742, South Korea, (2) Seoul National University, Seoul, South Korea The classical approach for isolating enzymes from environmental samples that includes enrichment of microorganisms in pure culture can analyze only subset of the total microbiota in nature since it has been estimated that less than 1% of microorganisms in nature can be cultivated by standard techniques. Metagenome was isolated from soil samples and metagenomic library was constructed using pUC19 vector to obtain useful microbial enzymes from uncultured soil microorganisms. The library was screened for amylase activity and one clone from approximately 30,000 recombinant Escherichia coli clones had amylase activity. Sequencing of the clone revealed that novel amylolytic enzyme was expressed from a novel gene. The putative amylase gene (amyM) was overexpressed and purified for characterization. AmyM had 42 oC and pH 9.0 as the optimum temperature and pH and Ca2+ stabilized the enzyme activity. The amylase hydrolyzed soluble starch and cyclodextrins to produce high level of maltose and pullulan to panose. The enzyme showed high transglycosylation activity to make ƒÑƒ{1,4-linkages exclusively. Interestingly, AmyM could hydrolyze lactose, which was not reported in any other amylases. These results suggest that AmyM has novel characteristics regarded as an intermediate type of maltogenic amylases, ƒÑ-amylases, and 4-ƒÑ-glucanotransferases.
Session 99A, Biotechnology: General
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