Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1986: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1985: ¥1,400,000 (Direct Cost: ¥1,400,000)
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Research Abstract |
Pullulanase, a starch-debranching enzyme, hydrolyzes (1-6)- <alpha> -glucosidic linkages in pullulan and starch. Pullulanase is used to elucidate the structures of polysaccharides and to produce useful materials such as maltose, amylose, and glucose by debranching starch with and without other amylases. This enzyme is produced by the gram-negative bacterium, Klebsiella aerogenes, and secreted from the cells into the culture broth. We recently cloned the pullulanase gene (pulA). 1. Deletion analysis and the lac gene fusion of the recombinant plasmid showed that the pul coding sequence, with the regulator gene, was located entirely within a 4.2-kb segment of the chromosomal DNA. The K. aerogenes cells carrying the plas-mid over-produced both extracellular and intracellular pullulanase when the cells were induced by maltose. 2. We determined the entire nucleotide sequence of the pul gene. A unique open reading frame of 3,288 bp was found. The precursor enzyme consists of 1096 amino acid res
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idues and contains a hydrophobic N-terminal signal peptide. The molecular weight of precursor and mature proteins are estimated to be 119,334 and 117,258, respectively. 3. A ribosome binding site is located 8 bases upstream of the ATG. Potential -35 and -10 promoter regions were detected at positions 230 (GGATGA)and 260 (TCTAAT). e confirmed that the pulA gene was controlled by malT, which is induced by maltose 4. Cellular localization of pullulanase in E. coli and K. aerogenes carrying the plasmid were examined. In E.coli about 50 and 46% of the pullulanse remained in the membrane and cytoplasmic fractions, respectively. In K. aerogenes about 20% was released in the culture broth, and about 20% each of pullulanase remained in the cytoplasmic fraction and inner and outer membranes. 5. The amino acid sequence showed that a 19-amino acid signal peptide preceded the pullulanase molecule with a glycine residue at the potential cleavage site, where processing of the precursor occured to yield the mature pullulanase. This se quence was followed by a cystein residue, which was modified by palmitate and probably becomes the amino terminus of the mature protein. A leu-leu-ser-gly-cys sequence is the consensus sequence for the bacterial lipoprotein. Less
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