| Budget Amount *help |
¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 2004: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2003: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 2002: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
In this study, biochemical and molecular studies on polyethylene glycol (PEG) and polyvinyl alcohol (PVA) degradation were practiced with PEG- or PVA-utilizing sphingomonads.
The approximately 14-kb DNA fragment including upstream and downstream regions of a gene for PEG dehydrogenase (pegA) was cloned from Sphingomonas macrogoltabidus strain 103 and sequenced. It contained ten putative genes : orf1 (A,B), pegB,C,D,A,E and R and orf2 (A,B) corresponding to transposases (A,B), TonB-dependent receptor, aldehyde dehydrogenase, permease, PEG dehydrogenase (PEGDH), acyl-CoA ligase, AraC-type transcription regulator (in opposite orientation) and transposases (A,B), respectively. More than 99% homology of gene structures (pegB,C,A,E and orf 2) was found in the other PEG-degrader, S.macrogoltabidus strain 203 and S.terrae. Orf 1 of S.terrae had approximately 50% homology with those of S.macrogoltabidus strains 103 and 203 (homology of them are more than 90%, but the direction was reverse.) Inse
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rtion of another transposase between pegD and pegA was found in strain 203. which led to constitutive expression of pegA. Dot-blot hybridization and RT-PCR revealed that five genes in the same orientation transcribed as single operon. The gene, pegD was expressed in E.coli and the recombinant enzyme was characterized as nicotinoprotein PEG-aldehyde dehydrogenase including NADP tightly bound to the enzyme. This was the first finding on nicotinoprotein aldehyde dehydrogenase. On the other hand, reaction mechanism of PEGDH was characterized, based on 3D molecular modeling.
Oxidized PVA-hydrolyzing enzyme (OPH) was purified from Sphingomonas sp.113P3 and characterized. Base on N-terminal and internal amino acid sequences, the gene encoding OPH was cloned and sequenced. The recombinant enzyme was expressed as inclusion body in E.coli. By homology search, OPH had similarity with OPH from Pseudomonas sp.VM15C and polyhydroxybutyrate depolymerases, keeping the consensus sequence for oxyanion hole and catalytic triads. In addition, the gene encoding PVA dehydrogenase (PVADH) and cytochrome C were located downstream. The genes for OPH and PVADH were located in tandem with almost no space, showing the coexpression controlled by the same promotor.
PEG- or PVA-utilizing sphingomonads changed their cell surface structures when grown either on PEG or PVA-medium or on nutrient broth, which seemed to correspond to their degradation abilities. By analyzing genes involved in respective degradation operons and their vicinities we could show how polymers are recognized and lead to the expression of their metabolic genes. TonB-dependent receptor and permease seem to be involved in transport of substrates or metabolites in PEG degradation. This has to be clarified further. Less
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