Expression of the isoamylase gene of Flavobacterium odoratum KU in Escherichia coli and identification of essential residues of the enzyme by site-directed mutagenesis.

ABSTRACT: The isoamylase gene from Flavobacterium odoratum KU was cloned into and expressed in Escherichia coli JM109. The promoter of the gene was successful in E. coli, and the enzyme produced was excreted into the culture medium, depending on the amount of the enzyme expressed. The enzyme found in the culture medium showed almost the same M(r), heat-inactivating constant, and N-terminal sequence as those of the enzyme accumulated in the periplasmic space. This result indicated that the enzyme accumulated in an active form at the periplasm was transported out of the cell. The primary sequence of the enzyme, which was deduced from its nucleotide sequence, showed that the mature enzyme consisted of 741 amino acid residues. By changing five possible residues to Ala independently, it was found that As

SUBMITTER: Abe J 

PROVIDER: S-EPMC99755 | biostudies-literature | 1999 Sep

REPOSITORIES: biostudies-literature