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 Asp-374, Glu-422, and Asp-497 were essential. The sequences around those residues were highly conserved in isoamylases of different origins and the glycogen operon protein X, GlgX. The comparison of the distance between these essential residues with those of various amylases suggested that the bacterial and plant isoamylase but not GlgX had a longer fourth loop than the other amylases. This longer fourth loop had a possible role in accommodating the long branched chains of native glycogens and starches.

FIG. 1

Primers for site-directed mutagenesis. Shading indicates substituted nucleotides, and boxes indicate the positions of mutations where Asp or Glu is replaced with Ala. New restriction enzyme sites are underlined.

FIG. 2

SDS-PAGE of the purified extracellular (Ex) and periplasmic (Pe) isoamylase produced by E. coli JM109(pIF13). The purified enzymes were subjected to SDS-PAGE and detected with Coomassie Brilliant Blue R-250 (A) and by Western blotting (B). M_r markers: phosphorylase a (97,000), bovine serum albumin (66,000), glyceraldehyde 3-phosphate dehydrogenase (36,000), and soybean trypsin inhibitor (20,100).

FIG. 3

Sequence alignment of isoamylases. FSPIAM, Flavobacterium sp. isoamylase; PSPIAM, Pseudomonas sp. strain JD210 isoamylase (6); ZMAIAM, Z. mays sugary-1 protein (13); ECOGLG, E. coli GlgX; CTRGLG, C. trachomatis GlgX. SSOGLG, S. solfataricus GlgX; SSPGLG, Synechocystis sp. GlgX. The first 93 residues of ZMAIAM were omitted. Black shading indicates 100%, dark gray shading indicates 80%, and light gray shading indicates 60% conservation, according to the Blosum 62 matrix table.

FIG. 3

Sequence alignment of isoamylases. FSPIAM, Flavobacterium sp. isoamylase; PSPIAM, Pseudomonas sp. strain JD210 isoamylase (6); ZMAIAM, Z. mays sugary-1 protein (13); ECOGLG, E. coli GlgX; CTRGLG, C. trachomatis GlgX. SSOGLG, S. solfataricus GlgX; SSPGLG, Synechocystis sp. GlgX. The first 93 residues of ZMAIAM were omitted. Black shading indicates 100%, dark gray shading indicates 80%, and light gray shading indicates 60% conservation, according to the Blosum 62 matrix table.