Cloning and Analysis of Genes Encoding synthetic Polymer-degrading Enzymes
Grant-in-Aid for General Scientific Research (C)
|Allocation Type||Single-year Grants|
|Research Institution||KOBE UNIVERSITY OF COMMERCE|
KAWAI Fusako Kobe Univ. Commerce School of Economics and Business Administration, Professor, 商経学部, 教授 (60118007)
SHIMADA Yoshimi Kobe Univ. Commerce School of Economics and Business Administration, Research As, 商経学部, 助手 (80226216)
|Project Period (FY)
1990 – 1992
Completed(Fiscal Year 1992)
|Budget Amount *help
¥2,300,000 (Direct Cost : ¥2,300,000)
Fiscal Year 1992 : ¥400,000 (Direct Cost : ¥400,000)
Fiscal Year 1991 : ¥400,000 (Direct Cost : ¥400,000)
Fiscal Year 1990 : ¥1,500,000 (Direct Cost : ¥1,500,000)
|Keywords||Xenobiotic Polymer / Polyethylene Glycol(PEG) / Bacterial Assimilation / PEG Dehydrogenase / Gene Library / Cloning / Polymerase Chain Reaction / Synthetic Olingonucleotide Probe / ポリエチレングリコ-ル(PEG) / ジ-ンライブラリ- / クロ-ニング / DNAプロ-ブ|
1) Reidentification of Polyethylene Glycol (PEG)-utilizing Bacteria
PEG-utilizing bacteria were reidentified by chemotaxonomical techniques, DNA/DNA homelogy etc. All the PEG 4000-utilizing bacteria and PEG 20,000-metabolizing bacteria involved in PEG 2/,000-utilizing symbiotic mixed cultures were determined to be the same new species which were identified as Sphingomonas macrogoltabidus and Sphin-gomonas terrae, respectively. The former bacterium was used in the following experiments.
2) Amplification of DNA fragments from primer peptides by PCR
We synthesized many oligonucleotide primers which corresponded to the determined amino acid sequences of the proteinase-generated fragments of the 60kDa subunit of PEG dehydrogenase and to the consensus sequence of PQQ-binding site (PBS). A pair of oligonucleotides which corresponded to a peptide fragment and PBS were used as the oppsing primers in the PCR. The size of the amplified fragments having the strong intensities were about 0.3 kb (pagA
A) and 0.5 kb (pagA B). These fragments were hybridized with the chromosomal DNA by southern blot analysis and sequenced.
3) Construction of gene libraries by pUC119 and Charomid 9-36 and colony hybridization
The total DNA of the bacterium was digested with Sal I and the partially digested fragments were ligated in the Sal I site of pUC119 or Charomid 9-36 and transducted into E. coli JM109. No positive clone was obtained by colony hybridization with pagA A.
4) Construction of gene libraries by EMBL3 and plaque hybridization
The total DNA of the bacterium was digested with Mbo I and the partially digested fragments were ligated in the Mbo I site of EMBL3 and transducted into E. coli ER1647. No positive clone was obtained by plaque hybridization with oligo nucleotide probes.
5) Transport of PEG through bacterial outer membranes and biodegradability of PEG
PEG-metabolizing activities of PEG-utilizing bacteria except S. macrogoltabidus no.203 were induced by PEG. Porins in outer membranes were formed in PEG-grown cells. On the other hand, S. macrogoltabidus No.203 formed PEG dehydrogenase and porins constitutively, suggesting that the induction of the enzyme and membrane structures were mutated in this strain. Less
Research Products (11results)