In this work, we have purified the His-tagged oxygenase (ht-oxygenase) component of Rhodococcus globerulus P6 biphenyl dioxygenase. The α or β subunit of P6 oxygenase was exchanged with the corresponding subunit of Pseudomonas sp. strain LB400 or of Comamonas testosteroni B-356 to create new chimeras that were purified ht-proteins and designated ht-αP6βP6, ht-αP6βLB400, ht-αP6βB-356, ht-αLB400βP6, and ht-αB-356βP6. ht-αP6βP6, ht-αP6βLB400, ht-αP6βB-356 were not expressed active in recombinant Escherichia coli cells carrying P6bphA1 and bphA2, P6 bphA1 and LB400bphE, or P6 bphA1 and B-356 bphEbecause the [2Fe-2S] Rieske cluster of P6 oxygenase α subunit was not assembled correctly in these clones. On the other hand ht-αLB400βP6 and ht-αB-356βP6 were produced active inE. coli. Furthermore, active purified ht-αP6βP6, ht-αP6βLB400, ht-αP6βB-356, showing typical spectra for Rieske-type proteins, were obtained from Pseudomonas putidaKT2440 carrying constructions derived from the new shuttle E. coli-Pseudomonas vector pEP31, designed to produce ht-proteins inPseudomonas. Analysis of the substrate selectivity pattern of these purified chimeras toward selected chlorobiphenyls indicate that the catalytic capacity of hybrid enzymes comprised of an α and a β subunit recruited from distinct biphenyl dioxygenases is not determined specifically by either one of the two subunits.
. 1999. Northern blotting analysis with RNA probes derived from amidase and nitrile hydratase genes from Rhodococcus sp. ACV2 revealed that both genes are part of the same operon. RNase protection mapping and sequence analysis indicated that the operon is probably under the control of a sigma 70-like promoter located upstream from the amidase gene. Plasmids were constructed with the cloned genes under tac and lac promoter control. Expression of amdA was demonstrated in Escherichia coli. In another construction, the amdA gene was inserted under the control of the bacteriophage T7 promoter. Large amounts of recombinant amidase (at least 20% of total proteins) in a soluble and active form were obtained with the E. coli -T7 expression system by lowering the growth temperature to 29°C, without IPTG induction. The ratio of amidase activity of strain ACV2 to E. coli was approximately 1:3. Purification of the recombinant amidase was carried out in one chromatographic step, giving an enzyme preparation that could be used directly in a biotechnological process.
The adipamidase of a mutant strain Brevibacterium sp. R312 involved in the degradation of adiponitrile to adipic acid was purified. Its N-terminal amino acid sequence was shown to be identical to Brevibacterium sp. R312 enantio selective amidase and Rhodococcus sp. N-774 amidase.
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