Purification and characterization of alpha-amylase from the infective juveniles of the nematode Heterorhabditis bacteriophora.
Glycogen content and alpha-amylase activity were estimated in the infective juveniles (IJs) of Heterorhabditis bacteriophora at different times of storage. The glycogen content declined from 5.8 to 2.5 ng/IJ during storage for 40 days at 27 degrees C. The change in glycogen content coincided with the change of alpha-amylase activity during storage. alpha-Amylase was purified from IJs at zero time of storage by ion exchange chromatography and gel filtration. Ion exchange chromatography resolved alpha-amylase into three isoenzymes. The major isoenzyme alpha-amylase I had the highest specific activity and was purified to homogeneity. A molecular mass of 46-47 kDa was estimated for both the native and denatured enzyme, suggesting that the enzyme is monomeric. The Km values were 6.5 and 9.6 mg/ml using starch and glycogen as substrates, respectively. alpha-Amylase I showed optimum activity at pH 7.0 and had an optimum temperature of 40 degrees C. The enzyme was unstable at temperatures above 40 degrees C. The enzyme activity was severely inhibited by EDTA, p-CMB and iodoacetic acid, but potentiated by CaCl2 and NaCl. These results are discussed and compared with previously reported alpha-amylases in the insect hosts of the parasite.
Mohamed
Magda A
MA
Molecular Biology Department, National Research Centre, Dokki 12622, Giza, Egypt. magdasoroor@email.com
D000367:Age Factors; D000818:Animals; D004795:Enzyme Stability; D006003:Glycogen / Q000032:analysis; D007700:Kinetics; D008970:Molecular Weight; D017172:Rhabditoidea / Q000201:enzymology* / Q000254:growth & development*; D013696:Temperature; D000516:alpha-Amylases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D006003:Glycogen; D000516:alpha-Amylases