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 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.
Krohn
B M
BM
Ceregen Division, A Unit of Monsanto Company, St. Louis, MO 63198, USA.
Barry
G F
GF
Kishore
G M
GM
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics; D005417:Flavobacterium / Q000201:enzymology / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D007517:Isoamylase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D011993:Recombinant Fusion Proteins; D007517:Isoamylase