This 1989 study reports the isolation and genetic characterization of an isoamylase-producing Pseudomonas strain (SMP1) from Italian soil. Isoamylases are industrially important enzymes that break α-1,6 glucosidic bonds in starch to produce amylose and maltose. The researchers purified the enzyme and found it nearly identical to a previously characterized isoamylase from Pseudomonas amyloderamosa SB15 isolated in Japan. Both enzymes had similar molecular weights (~90 kDa), optimal pH (3-4), temperature stability, and were inducible by maltose. Using molecular cloning techniques, the isoamylase gene was inserted into a plasmid vector and successfully expressed in E. coli. The complete nucleotide sequence of the 3.3 kb chromosomal fragment was determined, revealing a 776-amino-acid coding sequence with a 26-amino-acid signal peptide for secretion. Sequence analysis identified putative promoter regions resembling maltose-regulated promoters found in other Gram-negative bacteria, suggesting conserved regulatory mechanisms. Comparisons with other starch-degrading enzymes revealed two conserved protein domains shared between isoamylase, pullulanase, and α-amylases, though isoamylase lacked a third domain present in α-amylases.

Key findings

• The isoamylase gene from Pseudomonas sp. SMP1 was successfully cloned and sequenced, with the complete 3337 bp insert containing a 776-amino-acid coding sequence.
• The SMP1 isoamylase is biochemically and structurally nearly identical to the previously characterized P. amyloderamosa SB15 enzyme, differing mainly in source organism.
• Sequence analysis revealed putative promoter regions resembling maltose-regulated promoters, suggesting conserved gene regulation mechanisms across Gram-negative bacteria.
• Comparison with other amylolytic enzymes identified two conserved domains shared between isoamylase and pullulanase, but lacking the third domain found in α-amylases that participates in 1,4-glycosidic bond hydrolysis.
• The isoamylase gene includes a 26-amino-acid signal peptide for protein secretion and produces enzyme activity when expressed in E. coli with its own Pseudomonas promoter.