Open AccessNucleotide sequence of the gene encoding cis ‐biphenyl dihydrodiol dehydrogenase ( bphB ) and the expression of an active recombinant His‐tagged bphB gene product from a PCB degrading bacterium, Pseudomonas putida OU83
Author(s) -
Khan Ashraf A,
Wang RongFu,
Nawaz Mohamed S,
Cerniglia Carl E
Publication year - 1997
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1997.tb12662.x
Subject(s) - pseudomonas putida , nucleic acid sequence , gene , biology , microbiology and biotechnology , ribosomal binding site , biochemistry , start codon , peptide sequence , codon usage bias , escherichia coli , open reading frame , nucleotide , genetics , ribosome , rna , genome
The nucleotide sequence of the bphB gene of Pseudomonas putida strain OU83 was determined. The bphB gene, which encodes cis ‐biphenyl dihydrodiol dehydrogenase (BDDH), was composed of 834 base pairs with an ATG initiation codon and a TGA termination codon. It can encode a polypeptide of 28.91 kDa, containing 277 amino acids. Promoter‐like and ribosome‐binding sequences were identified upstream of the bphB gene. The bphB nucleotide sequence was used to produce His‐tagged BDDH, in Escherichia coli . The His‐tagged BDDH construction, carrying a single 6×His tail on the N‐terminal portion, was active. The molecular mass of the native enzyme was 128 kDa and on SDS‐PAGE analysis the molecular mass was 31 kDa. This enzyme requires NAD + for its activity and its optimum pH is 8.5. Nucleotide and the deduced amino acid sequence analyses revealed a high degree of homology between the bphB gene from Pseudomonas putida OU83 and the bphB genes from P. cepacia LB400 and P. pseudoalcaligenes KF707.