|Budget Amount *help
¥2,400,000 (Direct Cost : ¥2,400,000)
Fiscal Year 1991 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1990 : ¥1,600,000 (Direct Cost : ¥1,600,000)
1. Determination of N-terminal amino acid sequences of the enzymes and preparation of the antibodies against the enzymes. Three novel enzymes involved in biosynthesis of a polyamine, norspermidine, in Vibrio alginolyticus were purified to determine their N-terminal amino acid sequences. Ten to 20 amino acid residues of each enzyme were successfully determined. The antibodies raised against these enzymes were specific for corresponding enzymes.
2. Cloning and expression of the gene encoding carboxynorspermidine decarboxylase(CANS DC). Sixteen positive clones were selected from the gene library by the immunological method, and one clone haboring a recombinant plasmid, designated pCDC14, showed the enzyme activity 3-fold higher than that in the V. alginolyticus crude cell extract. Western blot analysis of a crude cell extract of E. coli HB101 haboring pCDC14 demonstrated a protein band with an M_r of 43500, which was comparable with CANS DC from V. alginolyticus. Insertion of this fragment
(4 kbp)into pUC18 in a reverse orientation resulted in no change in enzyme activity, suggesting that the whole gene including the promoter region is cloned. Subcloning of the deleted plasmids localized the gene in a fragment of 2 kbp in length(pCDC14-1).
3. Nucleotide sequence of the gene encoding CANS DC. The nested deletion plasmids were prepared from pCDC14-1 to determine the nucleotide sequence of this gene. The region upstream from the gene was characterized by a putative promoter consensus region(-10, -35), a ribosome-binding site and ATG start codon. The amino acid sequence deduced from the nucleotide sequence beginning at ATG completely corresponded to the N-terminal amino acid sequence already determined. Moreover, the termination codon TAA followed by a sequence for a structure resembling a p -independent transcription termination ws also identified.
4. We have found that, like V. alginolyticus, Acinetobacter calcoaceticus possesses L-2, 4diaminobuty-rate decarboxylase(DABA DC)catalyzing the production of diaminopropane, which is present in this bactrium in a large amount. It was found that the purified enzyme was different in subunit structure, N-terminal amino acid sequence and immunoreactivity from the DABA DC of V. alginolyticus. Less