Cloning and Analysis of Genes Encoding synthetic Polymer-degrading Enzymes

1)聚乙二醇化细菌的再鉴定通过化学分类学、DNA/DNA同源性等方法对聚乙二醇化细菌进行了鉴定，确定聚乙二醇化聚乙二醇2/000共生混合培养中涉及的聚乙二醇4000利用菌和聚乙二醇20000代谢菌为同一新种，分别鉴定为神经鞘氨脂单胞菌和丝氨醇-单胞菌。2)聚合酶链式反应扩增引物肽DNA片段我们合成了多个寡核苷酸引物，分别与所确定的聚乙二醇脱氢酶60 kDa亚单位的蛋白水解酶产生的片段的氨基酸序列和PQQ结合位点(PBS)的一致序列相对应。用一对与多肽片段相对应的寡核苷酸和PBS作为反义引物。具有较强强度的扩增片段的大小约为0.3kb(Paga…更多A)和0.5kb(Paga B)。3)pUC119和Charomid 9-36基因文库的构建及菌落杂交。用Sal I酶切细菌总DNA，将部分酶切片段连接到pUC119或Charomid 9-36的Sal I酶切位点，转化大肠杆菌JM109。与PagA的菌落杂交未获得阳性克隆。4)用EMBL3构建基因文库，并与空斑杂交。用Mbo I酶切细菌总DNA，将部分酶切片段连接到EMBL3的Mbo I酶切位点，转化大肠杆菌ER1647。菌斑杂交未获得阳性克隆。5)聚乙二醇经细菌外膜运输及生物降解性研究除203号金黄色葡萄球菌外，其余利用聚乙二醇菌的细菌对聚乙二醇酯的代谢活性均受聚乙二醇诱导。外膜中的孔蛋白在聚乙二醇组培细胞中形成。另一方面，203号链球菌产聚乙二醇脱氢酶和孔蛋白，表明该菌株对酶和膜结构的诱导发生了突变。较少

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