Publication details
Využití rekombinantního pseudoazurinu jako donoru elektronů nitritreduktasy s následným měřením dvouenzymové kinetiky
| Title in English | Utilization of recombinant pseudoazurin as an electron donor of nitrite reductase and follow-up measuring of dual enzymatic konetics. |
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| Authors | |
| Year of publication | 2002 |
| Type | Article in Proceedings |
| MU Faculty or unit | |
| Citation | |
| Keywords | Pseudoazurin Kinetics Paracoccus enzyme denitrification |
| Description | Denitrification is an anaerobic respiratory process in which a nitrogen oxycompound serves as the electron acceptor. The key step of the whole denitrification pathway is the transformation of nitrite ion to gaseous nitric oxide. In the bacterium Paracoccus denitrificans this reaction is catalysed by nitrite reductase of the cytochrome cd1 type. The enzyme does not interact directly with membrane components but takes electrons from soluble periplasmic electron mediators (cytochrome c550 or pseudoazurin). Pseudoazurin in amount needed for konetics studies was obtained by procedure consisting of a PCR amplification of the pas gene, cloning in pRSET plasmid, overepression in E.coli DH5(and subsequent affinity chromatography (Ni-NTA Sepharose, fast flow, Qiagen)). Since P.denitrificans contains high concentration of nitrite reductase the enzyme could be isolated from wild strain of P.denitrificans. Activities of three different mediator proteins (horse cytochrome c, P. denitrificans cytochrome c550 and pseudoazurin) were compared by measuring the initial velocity of nitrite consumption in reconstituted systems containig membrane vesicles and nitrite reductase. |