We determined the entire nucleotide sequence of the Klebsiella aerogenes W70 pullulanase gene (pulA) contained on a 4.2-kilobase-pair fragment of plasmid pPB174. The amino acid composition deduced from an open reading frame of 3,288 base pairs agreed closely with that determined for the intracellular pullalanase. The precursor enzyme consisted of 1,096 amino acid residues and contained a hydrophobic N-terminal signal peptide and the consensus sequence for the bacterial prelipoprotein signal peptide cleavage site.Pullulanase (EC 3.2.1.41, pullulan 6-glucanohydrolase), a starch-debranching enzyme, hydrolyzes (1-6)-a-glucosidic linkages in pullulan and starch to form maltotriose. This enzyme is produced by the gram-negative bacterium Kleb-0 1.0 genes W70 (19). We sequenced the entire pullulanase gene (pulA) and showed that the precursor enzyme contained N-terminal signal peptide. The precursor and mature proteins deduced from the DNA sequence showed a high degree

No abstract

Klebsiella pullulanase is a lipoprotein synthesized as a precursor with a signal peptide, which is processed by lipoprotein signal peptidase. To clarify the role of lipid modification of pullulanase, we purified lipid-modified wild-type and the unmodified (mutant) pullulanases and compared their properties. The Km and Vmax values of both pullulanases for pullulan were the same. The optimal pH and temperature, the stabilities over pH and temperature ranges, the specificity of substrates, and the patterns of inhibition of the lipid-modified and unmodified pullulanases were also the same. However, we found that the wild-type pullulanase formed trimers whereas the unmodified enzyme did not, and that the migrations of the two enzymes on sodium dodecyl sulphate/electrophoresis were different when the samples were applied on the gel without heating. The results presented in this paper and in previous work show that the correct processing and translocation of pullulanase in K. aerogenes require modification of lipid. However, the enzymatic properties and physical stabilities of pullulanase were not affected by the lipid modification.