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Plant growth promoting bacteria produce ACC deaminase (EC4.1.99.4) which regulates the biosynthesis of ethylene through cleavage of ACC (immediate precursor of Ethylene) into -ketobutyarate and ammonia. Therefore, it has an important role in plant growth promotion via lowering indigenous ethylene levels especially when the plants are exposed to an environmental stress. Therefore, this study aimed to investigate the cloning, expression, purification and determination of biochemical properties of ACC deaminase from Pseudomonas fluorescense. In this regard, the ACC deaminase encoding gene of Pseudomonas fluoresense FY32 was isolated and cloned in pET28 a (+) and the resultant pET28/acdS construct then was transformed into E. coli BL21(DE3). The expressed enzyme was purified by metal-affinity chromatography on Ni(2+)-TED-Sepharose column and then the optimum conditions and biochemical properties of the purified enzyme was examined. This enzyme showed the highest activity at 28 °C, pH 7 in the presence of 30 mM MgSO4. Also, the significant reduction of ACC deaminase activity was observed in 160 ppm of NaCl. The Km and Vmax of enzyme were calculated to be 9.66 mM and 0.11 nM mg-1 h-1, respectively (determined by the concentration of the produced α-ketobutyrate), which were relatively higher than those previously reported.

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Pertussis toxin (PT), the main virulence factor of Bordetella pertussis is a protein-based AB5-type exotoxin. Methods of pertussis toxin purification are not available exactly because of economic considerations by vaccine companies. The aim of this study was to setup and modify an in-house method for the PT purification based on affinity chromatography to develop acellular pertussis vaccine in future. B. pertussis and CHO cells were provided from Razi Institute (Karaj, Iran). The bacteria were grown in a 300L fermenter (44 h, 35o c, in B2 medium). The fermentation broth was clarified and concentrated by 0.45 µm membrane filter and 10 KDa molecular weight cut-off membrane respectively. Isolation of pertussis toxin was performed based on affinity chromatography by Fetuin Sepharose column. Immune dot blot test showed significant amounts of pertussis toxin qualitatively. The clustering of CHO- cells mono-layer were observed after first hour of applying the purified pertussis toxin and stopped after the twelfth hour. The average amount of extracted PT was 2.53 IU/ml± 0.43. Among the production procedure of whole cell pertussis vaccine, culture broth is discarded, whereas, results showed it was a suitable source for extraction of pertussis toxin. Finally examine other strains and bacterial culture methods to obtain desired pertussis toxin are recommended.