Cloning and Analysis of Genes Encoding synthetic Polymer-degrading Enzymes

1)聚乙二醇（PEG）利用菌的再鉴定利用PEG的细菌通过化学分类学、DNA/DNA同源性等方法进行了再鉴定，确定利用PEG 2/，000共生混合培养物中的PEG 4000利用菌和PEG 20，000代谢菌均为同一新种，分别鉴定为Sphingomonasmacrogoltabidus和Sphin-gomonastervis。2）PCR扩增引物肽段DNA片段我们根据PEG脱氢酶60 kDa亚基的蛋白酶片段的氨基酸序列和PQQ结合位点（PBS）的共有序列合成了多条寡核苷酸引物。将一对与肽片段相对应的寡核苷酸和PBS用作PCR中的反向引物。具有强强度的扩增片段的大小约为0.3kb（pagA ...更多信息 A）和0.5 kb（pagA B）。3）pUC 119和Charomid 9-36基因文库的构建及菌落杂交将细菌总DNA用Sal Ⅰ酶切，将部分酶切片段连接到pUC 119或Charomid 9-36的Sal Ⅰ位点，转化E. coli JM 109。4）EMBL 3和噬菌斑杂交法构建基因文库用Mbo Ⅰ酶切细菌总DNA，将部分酶切片段连接到EMBL 3的Mbo Ⅰ位点，转化E. coli ER 1647。利用寡核苷酸探针进行噬菌斑杂交，未获得阳性克隆。5）PEG的外膜转运及PEG代谢的生物降解性。聚乙二醇（PEG）对203号巨噬细胞进行诱导。在PEG培养的细胞中形成外膜中的孔。另一方面，S. macrogoltabidus No.203组成型形成PEG脱氢酶和孔蛋白，表明该菌株中酶的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