Function of cell surface and degradation of xenobiotic polymers in Sphingomonas species

鞘氨醇单胞菌细胞表面的功能和外源聚合物的降解

基本信息

  • 批准号:
    14560068
  • 负责人:
  • 金额:
    $ 1.73万
  • 依托单位:
  • 依托单位国家:
    日本
  • 项目类别:
    Grant-in-Aid for Scientific Research (C)
  • 财政年份:
    2002
  • 资助国家:
    日本
  • 起止时间:
    2002 至 2004
  • 项目状态:
    已结题

项目摘要

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 … More 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
在本研究中,利用聚乙二醇或聚乙烯醇的鞘单胞菌对聚乙二醇(PEG)和聚乙烯醇(PVA)进行了生化和分子研究。从Sphingomonas macrogoltabidus菌株103中克隆了一个PEG脱氢酶(PEG dehydrogenase, pegA)基因的上游和下游区域,全长约14kb,并对其进行了测序。orf1 (A,B), pegB,C,D,A,E, R和orf2 (A,B)分别对应转座酶(A,B), tonb依赖性受体,醛脱氢酶,渗透酶,PEG脱氢酶(PEGDH),酰基辅酶A连接酶,arac型转录调节因子(方向相反)和转座酶(A,B)。另一种降解peg的菌株S.macrogoltabidus菌株203与S.terrae的基因结构(pegB、C、A、E和orf2)同源性超过99%。S.terrae的Orf 1与S.macrogoltabidus菌株103和203的同源性约为50%(同源性均在90%以上,但方向相反)。在菌株203中发现了pegD和pegA之间的另一个转座酶的更多部分。导致pegA的本构表达。斑点杂交和RT-PCR结果显示,5个定向相同的基因转录为单操纵子。该基因pegD在大肠杆菌中表达,重组酶被鉴定为烟碱蛋白peg -醛脱氢酶,NADP与该酶紧密结合。这是对烟碱蛋白醛脱氢酶的首次发现。另一方面,利用三维分子模型对PEGDH的反应机理进行了表征。从鞘氨单胞菌(Sphingomonas sp.113P3)中纯化得到氧化pva水解酶(OPH),并对其进行了表征。根据n端和内部氨基酸序列,克隆了编码OPH的基因并进行了测序。重组酶在大肠杆菌中以包涵体的形式表达。通过同源性搜索,OPH与Pseudomonas sp.VM15C和聚羟基丁酸解聚合酶的OPH具有相似性,保持了氧阴离子空穴和催化三联体的一致序列。此外,编码PVA脱氢酶(PVADH)和细胞色素C的基因位于下游。OPH和PVADH基因串联在一起,几乎没有空间,显示出由同一启动子控制的共表达。利用聚乙二醇或聚乙烯醇的鞘单胞菌在聚乙二醇或聚乙烯醇培养基或营养液中生长时,细胞表面结构发生了变化,这似乎与它们的降解能力相对应。通过分析参与降解操纵子及其附近的基因,我们可以展示聚合物是如何被识别并导致其代谢基因的表达的。tonb依赖性受体和渗透酶似乎参与了PEG降解过程中底物或代谢物的运输。这一点必须进一步澄清。少

项目成果

期刊论文数量(144)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
微生物利用の大展開(今中忠行監修)
微生物利用的巨大发展(今中忠之监修)
  • DOI:
  • 发表时间:
    2002
  • 期刊:
  • 影响因子:
    0
  • 作者:
    E.Ueta;E.Suzuki;T.Kurata;河合富佐子
  • 通讯作者:
    河合富佐子
T.Ohta et al.: "Structural analysis of polyethylene glycol dehydrogenase"J.Biol.Chem.またはEur.J.Biochem. (予定).
T.Ohta 等人:“聚乙二醇脱氢酶的结构分析”J.Biol.Chem 或 Eur.J.Biochem(计划)。
  • DOI:
  • 发表时间:
  • 期刊:
  • 影响因子:
    0
  • 作者:
  • 通讯作者:
Expression nicotinoprotein aldehyde dehydrogenase of Sphingomonas macrogoltabidus strain 103 involved in PEG degradation.
Sphingomonas Macrogoltabidus 菌株 103 的表达烟碱蛋白醛脱氢酶参与 PEG 降解。
Regulation of genes relevant to microbial degradation of xenobiotic polymers
外源聚合物微生物降解相关基因的调控
Cloning of polyvinyl alcohol dehydrogenase gene of Sphingomonas sp.113P3 and characterization of the enzyme.
鞘氨醇单胞菌113P3聚乙烯醇脱氢酶基因的克隆及酶的表征。
{{ item.title }}
{{ item.translation_title }}
  • DOI:
    {{ item.doi }}
  • 发表时间:
    {{ item.publish_year }}
  • 期刊:
  • 影响因子:
    {{ item.factor }}
  • 作者:
    {{ item.authors }}
  • 通讯作者:
    {{ item.author }}

数据更新时间:{{ journalArticles.updateTime }}

{{ item.title }}
  • 作者:
    {{ item.author }}

数据更新时间:{{ monograph.updateTime }}

{{ item.title }}
  • 作者:
    {{ item.author }}

数据更新时间:{{ sciAawards.updateTime }}

{{ item.title }}
  • 作者:
    {{ item.author }}

数据更新时间:{{ conferencePapers.updateTime }}

{{ item.title }}
  • 作者:
    {{ item.author }}

数据更新时间:{{ patent.updateTime }}

KAWAI Fusako其他文献

KAWAI Fusako的其他文献

{{ item.title }}
{{ item.translation_title }}
  • DOI:
    {{ item.doi }}
  • 发表时间:
    {{ item.publish_year }}
  • 期刊:
  • 影响因子:
    {{ item.factor }}
  • 作者:
    {{ item.authors }}
  • 通讯作者:
    {{ item.author }}

{{ truncateString('KAWAI Fusako', 18)}}的其他基金

Improvement of acidic soils by aluminium (Al)-resistant fungi and molecular mechanism of Al-resistance
抗铝真菌改良酸性土壤及其抗铝分子机制
  • 批准号:
    17580065
  • 财政年份:
    2005
  • 资助金额:
    $ 1.73万
  • 项目类别:
    Grant-in-Aid for Scientific Research (C)
Cloning and Analysis of Genes Encoding synthetic Polymer-degrading Enzymes
编码合成聚合物降解酶的基因的克隆和分析
  • 批准号:
    02806023
  • 财政年份:
    1990
  • 资助金额:
    $ 1.73万
  • 项目类别:
    Grant-in-Aid for General Scientific Research (C)
{{ showInfoDetail.title }}

作者:{{ showInfoDetail.author }}

知道了