Volume 6, Issue 1 (2015)                   JMBS 2015, 6(1): 60-70 | Back to browse issues page

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Farajzadeh D. Cloning, expression, purification and determination of biochemical properties of ACC deaminase from Pseudomonas fluorescence. JMBS 2015; 6 (1) :60-70
URL: http://biot.modares.ac.ir/article-22-2698-en.html
1- Azarbaijan Shahid Madani University, Tabriz
Abstract:   (8247 Views)
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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Article Type: Research Paper | Subject: Biotechnology
Received: 2015/03/16 | Accepted: 2015/09/23 | Published: 2015/10/13

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