Isolation Of Enzyme Genes Cloning of Enzyme Genes

The DCase genes from Agrobacterium sp. KNK712 and Pseudomonas sp. KNK003A were isolated and their DNA sequences analyzed as follows (12). Chromosome DNA was partially digested with Sau3AI, and the fractionated DNA

fragment of 4-9 kb was inserted into pUC18 and then transformed into E. coli JM109. Recombinant colonies were collected, inoculated into enrichment culture medium containing A-carbamoyl-D-amino acids or 5-substituted hydantoins, and then incubated at 37 °C. After repeating the enrichment cultures, the recombinant clones were isolated. Plasmid DNAs were prepared from these clones and analyzed with several restriction endonucleases. The DCase gene of Agrobacterium sp. KNK712 was located in a 1.8-kb SalI-EcoRI fragment, and the gene of Pseudomonas sp. KNK003A was found in a 1.8-kb SphI-AccI fragment. These fragments were inserted into pUC19, the resultant plasmids being designated as pAD108 and pPD304, respectively. The restriction maps are shown in Figure 1. The recombinant strains containing these plas-mids showed increased DCase expression compared with the native strains.

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