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

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.

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Purification and characterization of an extracellular glucoamylase from the thermophilic fungus Humicola grisea var. thermoidea

Canadian Journal of Microbiology ◽

10.1139/m93-126 ◽

1993 ◽

Vol 39 (9) ◽

pp. 846-852 ◽

Cited By ~ 41

Author(s):

Luis Ricardo Orsini Tosi ◽

Héctor Francisco Terenzi ◽

Joāo Atílio Jorge

Keyword(s):

Molecular Mass ◽

Half Life ◽

Carbohydrate Content ◽

Thermophilic Fungus ◽

Apparent Molecular Mass ◽

Purification And Characterization ◽

Sds Page ◽

Humicola Grisea ◽

Temperature Optima

Humicola grisea var. thermoidea mycelium grown on maltose as the main source of carbon produced at least two amylases. The major amylolytic component was purified to homogeneity and classified as a glucoamylase. The apparent molecular mass of the purified enzyme was estimated to be 63 000 Da by SDS-PAGE and 65 000 Da by Bio-Gel P-100 filtration. The purified enzyme was a glycoprotein with 1.8% carbohydrate content and pH and temperature optima of 5.0 and 55 °C, respectively. The purified glucoamylase was thermostable at 60 °C with a half-life of 16 min at 65 °C. In the presence of starch the purified enzyme retained 75% of its thermostability at 65 °C, while the addition of maltose failed to protect the activity. The purified enzyme hydrolyzed branched substrates more efficiently than linear substrates. Starch and amylopectin were the best substrates utilized and amylose was hydrolyzed faster than maltopentaose, maltotetraose, and maltotriose. Kinetic experiments suggested that maltose and starch were hydrolyzed at the same catalytic site.Key words: glucoamylase, amylase, Humicola grisea.

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Isolation and partial characterization of protease from Pseudomonas aeruginosa ATCC 27853

Journal of the Serbian Chemical Society ◽

10.2298/jsc100125088i ◽

2010 ◽

Vol 75 (8) ◽

pp. 1041-1052 ◽

Cited By ~ 2

Author(s):

Lidija Izrael-Zivkovic ◽

Gordana Gojgic-Cvijovic ◽

Ivanka Karadzic

Keyword(s):

Pseudomonas Aeruginosa ◽

Molecular Mass ◽

Gel Filtration ◽

Zinc Ion ◽

Metal Chelators ◽

Temperature Optimum ◽

Ph Optimum ◽

Sds Page ◽

Potential Use

Enzymatic characteristics of a protease from medically important, referent strain of Pseudomonas aeruginosa ATCC 27853 were determined. According to SDS PAGE and gel filtration it was estimated that molecular mass of the purified enzyme was about 15 kDa. Other enzymatic properties were found to be: pH optimum 7.1, pH stability between pH 6.5 and pH 10; temperature optimum around 60?C while the enzyme was stable at 60?C for 30 min. The inhibition of the enzyme was observed with the metal chelators such as EDTA and 1,10- phenanthroline, suggesting that the protease is a metalloenzyme. Further more it was determined that enzyme contains one mole of zinc ion per mole of enzyme. The protease is stable in the presence of different organic solvents, which enable potential use for synthesis of peptides.

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