超好熱性細菌Aquifex aeolicus由来のブランチングエンザイム： その性質と高度分岐環状デキストリン合成への応用

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  • Properties of Branching Enzyme from Hyperthermophilic Bacterium, Aquifex aeolicus, and Its Potential for Production of Highly-branched Cyclic Dextrin.

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By sequencing the whole genome of the hyperthermophilic bacterium, Aquifex aeolicus, a gene (Aq722) encoding a protein with high similarity to the branching enzyme (BE, EC 2.4.1.18) has been found. Based on the putative amino acid sequence, a polynucleotide encoding the BE gene was chemically synthesized and the gene was expressed in Escherichia coli. More than 95% of the recombinant enzyme was present within the cells as insoluble but catalytically active aggregates. Heat treatment of the aggregate suspension at 70°C resulted in about 30% solubilization of the BE activity. Enzymatic properties of both soluble and insoluble forms of the BE were analyzed. Both forms exhibited maximum activities at 75°C and pH 7.5-8.0, and were stable at temperatures up to 85°C. The insolubleform BE was less stable than the soluble form in neutral and acidic pH regions; after 30-min incubation at 70°C and pH 7.0, 90 and 50% activities of soluble andinsoluble forms remained, respectively. When amylopectin was used as a substrate, the A. aeolicus BE cyclized the B-chains, which connect the cluster structures of amylopectin. The product (highly-branched cyclic dextrin), with weight-average degree of polymerization (DPw) 1200, has a ring structure with DPw 50 and noncyclic chains with an average unit chain length of 16 connected to the ring.

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