Purification and characterization of α-amylase from the infective juveniles of the nematode Heterorhabditis bacteriophora
2004
Mohamed, M.A.
Glycogen content and α-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°C. The change in glycogen content coincided with the change of α-amylase activity during storage. α-Amylase was purified from IJs at zero time of storage by ion exchange chromatography and gel filtration. Ion exchange chromatography resolved α-amylase into three isoenzymes. The major isoenzyme α-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. α-Amylase I showed optimum activity at pH 7.0 and had an optimum temperature of 40°C. The enzyme was unstable at temperatures above 40°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 -amylases in the insect hosts of the parasite.
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