The gene encoding an isoamylase with neutral pH optimum (iam) from a Flavobacterium species was cloned using a PCR probe generated from highly conserved regions of amylolytic enzymes. Active isoamylase was expressed from a 4.9-kb Pst I fragment in Escherichia coli, and was detected in the extracellular medium by a plate assay. The iam nucleotide sequence has an open reading frame of 2334 nucleotides (778 amino acids) with a GC content of 69%. Sequence analysis suggests that transcriptional control of the Flavobacterium sp. iam gene is mediated through the product of a malT regulatory gene. The deduced amino acid sequence of iam contained an N-terminal signal peptide of 32 amino acids, and was 61% homologous with Pseudomonas amyloderamosa isoamylase. The mature enzyme, which was engineered for overexpression in E. coli and purified to homogeneity, has a relative molecular mass of 83 kDa, a pH optimum of 6-7, and a highest rate of hydrolysis for glycogen (but did not cleave pullulan). Polyclonal antiserum generated from purified donor isoamylase cross-reacted with crude and purified recombinant isoamylase from E. coli. This is the first report of the cloning, characterization, and sequence of an novel isoamylase that has a neutral pH optimum. A comparison of the sequence of Flavobacterium sp. iam with acidic isoamylase from Pseudomonas sp. identified putative residues which may be associated with the pH for optimal activity of isoamylases.

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The cyclomaltodextrinase gene from Bacillus subtilis high-temperature growth transformant H-17 was cloned on separate PstI, BamHI, and EcoRI fragments into the plasmid vector pUC18, but was expressed in an inactive form in the host, Escherichia coli DH5 alpha. High level constitutive expression of the gene product was also detrimental to the E. coli host, which led to structural instability of the recombinant plasmid. The cyclomaltodextrinase gene was cloned on a 3-kb EcoRI fragment into the plasmid vector pPL708, and the fragment was structurally maintained in the host B. subtilis YB886. The cloned gene product was synthesized in an enzymatically active form in the B. subtilis host; however, expression was at a low level. Subcloning of the 3-kb EcoRI fragment into pUC18 and transformation into E. coli XL1-Blue (F' lacIq) indicated that the cyclomaltodextrinase gene was cloned with its own promoter, since expression of the gene occurred in the absence of IPTG. Subcloning of the cyclomaltodextrinase gene downstream from the Bacillus temperature phage SPO2 promoter of pPL708 may increase expression of this gene.

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