期刊
BIOTECHNOLOGY LETTERS
卷 29, 期 10, 页码 1529-1535出版社
SPRINGER
DOI: 10.1007/s10529-007-9421-7
关键词
Gentisate 1; 2-dioxygenase; purification; recombinant; Silicibacter pomeroyi
A 1,125-bp long ORF encoding a novel gentisate 1,2-dioxygenase with two-domain bicupins was cloned from Silicibacter pomeroyi DSS-3 and expressed in Escherichia coli. The resulting product was purified to homogeneity and partially characterized. Non-reductive SDS-PAGE and gel filtration showed that the active recombinant gentisate 1,2-dioxygenase had an estimated molecular mass of 132 kDa, and reductive SDS-PAGE indicated an approximate size of 45 kDa. The enzyme thus appears to be a homotrimeric protein. This is in contrast to the homotetrameric or dimeric protein of the gentisate 1,2-dioxygenases that have been characterized thus far. The K-m and K-cat/K-m for gentisate were 12 mu M and 653 x 10(4) M-1 s(-1); the pI was 4.6-4.8. It was optimally active at 40 degrees C and pH 8.0.
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