Purification and characterization of phosphotriesterases from Pseudomonas aeruginosa F10B and Clavibacter michiganense subsp. insidiosum SBL11

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Abstract:

A microbial biodegradation of monocrotophos was studied in the present investigation. The monocrotophos-degrading enzyme was purified and characterized from two soil bacterial strains. The cells were disrupted and the membrane-bound fractions were studied for purification and characterization. Solubilization of the membrane-bound fractions released nearly 80% of the bound protein. Phase separation further enriched the enzyme fraction 34–41 times. The enzyme phosphotriesterase (PTE) from both the strains was purified to more than 1000-fold with 13%–16% yield. Purified PTE from Clavibacter michiganense subsp. insidiosum SBL11 is a monomeric enzyme with a molecular mass of 43.5 kDa (pI of 7.5), while PTE from Pseudomonas aeruginosa F10B is a heterodimeric enzyme with a molecular mass of 43 and 41 kDa (pI of 7.9 and 7.35). Both purified enzymes are stable enzymes with peak activity at pH 9.0. The enzyme from strain F10B was more thermostable (half-life = 7.3 h) than that from SBL11 (half-life = 6.4 h at 50 °C), while both showed the same temperature optimum of 37 °C. Inhibitors like dithiothreitol and EDTA inhibited the purified enzyme, while p-chloromercuribenzoic acid and indoleacetic acid had a very little effect.Key words: biodegradation, monocrotophos, phosphotriesterase, Pseudomonas aeruginosa F10B, Clavibacter michiganense subsp. insidiosum SBL11.

Une biodégradation microbienne du monocrotophos fut étudiée dans l'enquête présente. L'enzyme dégradant le monocrotophos fut purifiée et caractérisée à partir de deux souches bactériennes du sol. Les cellules furent désintégrées et les infractions associées aux membranes furent étudiées pour la purification et la caractérisation. La solubilisation des fractions associées aux membranes ont libéré presque 80 % des protéines liées. La séparation de phase a par la suite enrichi d'un facteur de 34 à 41 la fraction des enzymes. L'enzyme phosphotriestérase (PTE) des deux souches fut purifiée jusqu'à plus de 1000 fois avec un rendement de 13 % à 16 %. La PTE purifiée de Clavibacter michiganense subsp. insidiosum SBL11 est une enzyme monomérique d'une masse moléculaire de 43,5 kDa (pI de 7,5) alors que la PTE de Pseudomonas aeruginosa F10B est une enzyme hétérodimérique d'une masse moléculaire de 43 et 41 kDa (pI de 7,9 et 7,35). Les deux enzymes stables purifiées avaient une activité maximale à un pH de 9,0. L'enzyme de la souche F10B était davantage thermostable (T1/2 = 7,3 h) que celle de la souche SBL11 (T1/2 = 6,4 h à 50 °C) alors que les deux démontraient la même température optimale de 37 °C. Des inhibiteurs tels que le dithioth réitol et l'EDTA ont inhibé l'enzyme purifiée alors que le p-chloromercuribenzoic acid et le indoleacetic acid n'ont eu que peu d'effet.Mots clés : biodégradation, monocrotophos, phosphotriestérase, Pseudomonas aeruginosa F10B, Clavibacter michiganense subsp. insidiosum SBL11.[Traduit par la Rédaction]

Document Type: Research Article

Publication date: February 1, 2006

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  • Published since 1954, this monthly journal contains new research in the field of microbiology including applied microbiology and biotechnology; microbial structure and function; fungi and other eucaryotic protists; infection and immunity; microbial ecology; physiology, metabolism and enzymology; and virology, genetics, and molecular biology. It also publishes review articles and notes on an occasional basis, contributed by recognized scientists worldwide.
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