A purification procedure for the soluble cytochrome oxidase and some other respiratory proteins from Pseudomonas aeruginosa

Abstract

The production of the soluble cytochrome oxidase/nitrite reductase in the bacterium Pseudomonas aeruginosa is favoured by anaerobic conditions and the presence of KNO3(20g/l) in the culture medium. Of three methods commonly used for the disruption of bacterial suspensions (ultrasonication, liquid-shear homogenization and glass-bead grinding), sonication proved the most efficient in releasing the Pseudomonas cytochrome oxidase. A polarographic assay of Pseudomonas cytochrome oxidase activity with sodium ascorbate as substrate and NNN'N'-tetramethyl-p-phenylenediamine dihydrochloride as electron mediator is described. A purification procedure was developed which can be used on the small scale (40-litre cultures) or the large scale (400-litre cultures) and provides high yields of three respiratory-chain proteins, Pseudomonas cytochrome oxidase, cytochrome c551 and azurin, in a pure state. A typical preparation of 250g of Ps.aeruginosa cell paste yielded 180mg of Pseudomonas cytochrome oxidase, 81 mg of Pseudomonas cytochrome c551 and 275mg of Pseudomonas azurin.

Purification of Azurin from Pseudomonas Aeuroginosa

“…The azurin protein optimization and quantification was studied in UV spectrometer (Perkin Elmer, Massachusetts, USA) at 595 nm by Bradford's method. The azurin synthesized from P. aeruginosa MTCC2453 is significantly higher than other strains [11,12].…”

“…The green-brown crude supernatant was stored. Resuspended the precipitate in same buffer, stirred it vigorously and centrifuged as before and the supernatant were stored with the previous extracts [11,12].…”

“…These inoculums was used to seed the bulk 500ml x 4 sterile medium in 2000 liter conical flasks (separate conical flask used for all four strains) which was also kept at stirred mode (100/rev) for 21 hours at 37 0 C [11].…”

Nuclear‐Magnetic‐Resonance Studies of Pseudomonas aeruginosa Cytochrome c‐551

“…It was purified according to the method of Ambler [15], as described (with modification) by Melling [16]. Buffer solutions were 0.05 M in acetic acid, the pH being adjusted by addition of ammonia solution.…”

Can metal replacement increase the antitumor activity of azurin? (in vitro study)

“…Apo-Az (without cofactor ) was prepared by a cyanide treatment of the holo form according to the procedure described elsewhere by dialysis against 0.1 M KCN in 20 mM tris-HCl, pH=8, for 14 days at room temperature [16]. After cyanide treatment, the protein was dissolved in 10 mM tris-base buffer at pH=8.…”