AN ISOAMYLASE WITH NEUTRAL PH OPTIMUM FROM A FLAVOBACTERIUM SPECIES -CLONING, CHARACTERIZATION AND EXPRESSION OF THE IAM GENE

The gene encoding an isoamylase with neutral pH optimum (iam) from a F lavobacterium species was cloned using a PCR probe generated from high ly conserved regions of amylolytic enzymes. Active isoamylase was expr essed from a 4.9-kb Pst I fragment in Escherichin coli, and was detect ed in the extracellular medium by a plate assay. The iam nucleotide se quence has an open reading frame of 2334 nucleotides (778 amino acids) with a GC content of 69%. Sequence analysis suggests that transcripti onal control of the Flavobacterium sp. iam gene is mediated through th e product of a malT regulatory gene. The deduced amino acid sequence o f iam contained an N-terminal signal peptide of 32 amino acids, and wa s 61% homologous with Pseudomonas amyloderamosa isoamylase. The mature enzyme, which was engineered for overexpression in E. coli and purifi ed to homogeneity, has a relative molecular mass of 83 kDa, a pH optim um of 6-7, and a highest rate of hydrolysis for glycogen (but did not cleave pullulan). Polyclonal antiserum generated from purified donor i soamylase cross-reacted with crude and purified recombinant isoamylase from E. coli. This is the first report of the cloning, characterizati on, and sequence of an novel isoamylase that has a neutral pH optimum. A comparison of the sequence of Flavobacterium sp. iam with acidic is oamylase from Pseudomonas sp. identified putative residues which may b e associated with the pH for optimal activity of isoamylases.