Cloning and Analysis of Genes Encoding synthetic Polymer-degrading Enzymes

1)利用聚乙二醇（PEG）的细菌的再鉴定利用聚乙二醇的细菌通过化学分类技术、DNA/DNA同源性等方法进行了再鉴定。利用peg4000的细菌和利用peg2 / 000的代谢细菌均为同一新种，分别鉴定为Sphingomonas macrogoltabidus和Sphin-gomonas terrae。前一种细菌被用于接下来的实验。我们合成了许多与PEG脱氢酶60kDa亚基蛋白酶产生片段的氨基酸序列和pqq结合位点（PBS）的一致序列相对应的引物。在PCR中使用一对与肽片段和PBS相对应的寡核苷酸作为对立引物。扩增片段大小分别为0.3 kb （pagA…More A）和0.5 kb （pagA B）。这些片段通过southern blot分析与染色体DNA杂交并测序。3)利用pUC119和Charomid 9-36构建基因文库并进行集落杂交将细菌的总DNA用Sal I酶切，部分酶切片段连接在pUC119或Charomid 9-36的Sal I位点上，转导到大肠杆菌JM109中。4)利用EMBL3和斑块杂交构建基因文库用Mbo - 1酶切细菌的总DNA，将部分酶切的片段连接在EMBL3的Mbo - 1位点，转导到大肠杆菌ER1647中。用寡核苷酸探针进行斑块杂交，未获得阳性克隆。5)聚乙二醇通过细菌外膜的转运及利用聚乙二醇的细菌（除S. macrogoltabidus no.）的聚乙二醇代谢活性的生物降解性。PEG诱导203只。聚乙二醇培养的细胞外膜形成孔蛋白。另一方面，S. macrogoltabidus No.203组成性地形成了PEG脱氢酶和孔蛋白，表明该菌株的酶诱导和膜结构发生了突变。少

1) Reidentification of Polyethylene Glycol (PEG)-utilizing BacteriaPEG-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 PCRWe 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 … More 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 hybridizationThe 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 hybridizationThe 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 PEGPEG-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