Purification and characterization of carbon-phosphorus bond-cleavage enzyme from glyphosate degrading Pseudomonas putida T5.
An inducible, carbon-phosphorus bond-cleavage enzyme was purified from cells of Pseudomonas putida T5 grown on N-phosphonomethyl glycine. The native enzyme had a molecular mass of approximately 70 kD and upon sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), yielded a homogeneous protein band with an apparent molecular mass of about 70 kD. Activity of purified enzyme was increased by 627-fold compared to the crude extract and showed pH and temperature optima of approximately 7 and 30°C, respectively. The purified enzyme had an apparent Km and Vmax of 3.7 mM and 6.8 mM/min, respectively, for its sole substrate N-phosphonomethyl glycine. The enzyme was inhibited by phenylmethylsulfonyl fluoride (PMSF), indicating the presence of serine at the active site. The enzyme was not inhibited by SDS, suggesting the absence of disulfide linkage in the enzyme. The enzyme was found to be inhibited by most of the metals studied except Mg(2+). Detergents studied also inhibited glyphosate acting as a carbon-phosphorus bond-cleavage enzyme. Thus initial characterization of the purified enzyme suggested that it could be used as a potential candidate for glyphosate bioremediation.
a Fermentation Technology and Bioengineering Department , Central Food Technological Research Institute (CSIR) , Mysore , Karnataka , India.
A Arul
AA
Selvi
H K
HK
Manonmani
D020134:Catalytic Domain; D002850:Chromatography, Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D005998:Glycine / Q000031:analogs & derivatives* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008190:Lyases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D008670:Metals / Q000494:pharmacology; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D016958:Pseudomonas putida / Q000201:enzymology*; D012694:Serine / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D000097797:Glyphosate
D004791:Enzyme Inhibitors; D005998:Glycine; D008190:Lyases; D008670:Metals; D010664:Phenylmethylsulfonyl Fluoride; D012694:Serine; D000097797:Glyphosate; C051328:carbon-phosphorus lyase
N-phosphonomethyl glycine; Pseudomonas putida; bioremediation; carbon-phosphorus bond cleavage enzyme; glyphosate