Expression, purification and characterization of meta-cleavage enzyme carbabb from Novosphiongobium sp. KA1
Keywords:
meta-cleavage enzyme, extradiol dioxygenase, carbazole degradationAbstract
The meta-cleavage enzyme carbabb of carbazole-degrader Novosphiongobium sp. KA1 were cloned, expressed and purified to homogeneity in Escherichia coli strain. The enzyme was cloned with 6x histidine residues attached at the c-terminal of large subunit carbb for purification using affinity chromatography methodprior to gel filtration chromatography. The carbabb, a two-subunit meta-cleavage enzyme, approximately 30 kda for carbb dan 10 kda for carba, was found to be α2β2-heterotetrameric (mr 80,000), showed highest activity at ph 8.5 and temperature 30°c. Carbabb showed highest catalytic activity towards 2,3-dihydroxybiphenyl with kcat/km 4.1 m-1s-1, and overall higher catalytic activities towards biphenyl-type substrates in comparison to catechol-type substrates.Based on the similarities, this meta-cleavage enzyme fromnovosphiongobium sp. Ka1 would also be a good candidate for protein crystallization and structural studies apart from carbabb from strain P. resinovorans strain ca10.References
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