| Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1990: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1989: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
1. In Vivo Expression of the Pseudomonas stutzeri Maltotetoraose-forming Amylase Gene (amyP) The amyP gene of P. stutzeri MO-19 was transcribed as a monocistronic mRNA of 20 kb and the transcription start site is located 81 bp upstream from the first nucleotide of the initiation codon. The amyP gene was expressed weakly in Escherichia coli, and transcription started 49 bp downstream of the P. stutzeri MO-19 transcription start site. Synthesis of the amylase in P. stutzeri MO-19 was induced by the addition of maltose and was repressed by the addition of glucose. The induction by maltose was shown to be the result of transcriptional induction of the amyP gene. In contrast, glucose did not repress transcription of amyP, indicating that it controls synthesis of the enzyme, probably at the posttranscriptional level.
2. Purification and Properties of P. stutzeri RNA Polymerase RNA polymerase from P. stutzeri MO-19 was purified to near homogeneity. The subunit components and their molecular weights were analyzed by SDS-PAGE. The enzyme was composed of putative alpha, beta' and sigma subunits with molecular weights of 68,000, 150,000, 155,000 and 40,000, respectively.
3. Characterization of an Isoamylase-hyperproducing Mutant of Pseudomonas amylo-deramosa Strain MI-414 obtained by treating strain SB-15 with NTG produced 10-fold more isoamylase than SB-15. S1 nuclease mapping of isoamylase mRNAs from the strains showed that the level of isoamylase mRNA was higher in MI-414 than in SB-15. The copy number of isoamylase gene (iam was not changed by the mutation. Moreover, the nucleotide sequence of the promoter region of iam, the transcription initiation site, and the expression patterns of iam-mRNAs in SB-15 and MI-414 were approximately 3 and 22 min, respectively. Therefore, hyperproduction of isoamylase in the mutant results, at least partly, from the greater stability of its mRNA.
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