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KMID : 0545119950050020068
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Journal of Microbiology and Biotechnology
1995 Volume.5 No. 2 p.68 ~ p.73
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Cloning and Expression of pcbC and pcbD Genes Responsible for 2, 3-Dihydroxybiphenyl Degradation from Pseudomonas sp. P20
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Nam, Jung Hyun
Koh, Hee Mock/Kim, Chi Kyung
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Abstract
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Pseudomonas sp. P20 was shown to be capable of degrading biphenyl and 4-chlorobiphenyl (4CB) to produce the corresponding benzoic acids which were not further degraded. But the potential of the strain for biodegradation of 4CB was shown to be excellent. The pcbA, B, C and D genes responsible for the aromatic ring-cleavage of biphenyl and 4CB degradation were cloned from the chromosomal DNA of the strain. In this study, the pcbC and D genes specifying degradation of 2,3-dihydroxybiphenyl (2,3-DHBP) produced from biphenyl by the pcbAB-encoded enzymes were cloned by using pBluescript SK(+) as a vector. From the pCK102 (9.3kb) containing pcbC and D genes, pCK1022 inserted with a EcoRI-HindⅢ DNA fragment (4.1kb) carrying pcbC and D and a pCK1092 inserted with EcoRI-Xbal fragment (1.95 kb) carrying pcbC were constructed. The expression of pcbC and D in E. coli CK102 and pcbC in E. coli CK1092 was examined by gas chromatography and UV-vis spectrophotometry. 2.3-dihydroxybiphenyl was readily degraded to produce meta-cleavage product (MCP) by E. coli CK102 after incubation for 10 min, and then only benzoic acid(BA) was detected in the 24-h old culture. The MCP was detected in E. coli CK1022 containing pcbC and D genes (by the resting cells assay) for up to 3 h after incubation and then diminished completely in 8 h, whereas the MCP accumulated in the E. coli CK1092 culture even after 6 h of incubation. The 2,3-DHBP dioxygenases (product of pcbC gene) produced by E. coli CK1, CK102, CK1023, and CK1092 strains were measured by native PAGE analysis to be about 250 kDa in molecular weight, which were about same as those of Pseudomonas sp. DJ-12, P. pseudoalcaligenes KF707, and P. putida OU83.
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