Mohamed Magda A
Molecular Biology Department, National Research Centre, Dokki 12622, Giza, Egypt.
Comp Biochem Physiol B Biochem Mol Biol. 2004 Sep;139(1):1-9. doi: 10.1016/j.cbpc.2004.03.014.
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.
在不同储存时间下，对嗜菌异小杆线虫的感染性幼虫（IJs）中的糖原含量和α-淀粉酶活性进行了测定。在27℃储存40天期间，糖原含量从5.8 ng/IJ降至2.5 ng/IJ。糖原含量的变化与储存期间α-淀粉酶活性的变化一致。在储存零时，通过离子交换色谱和凝胶过滤从IJs中纯化出α-淀粉酶。离子交换色谱将α-淀粉酶分离为三种同工酶。主要的同工酶α-淀粉酶I具有最高的比活性，并被纯化至同质。天然酶和变性酶的分子量估计均为46 - 47 kDa，表明该酶是单体。以淀粉和糖原作为底物时，Km值分别为6.5和9.6 mg/ml。α-淀粉酶I在pH 7.0时表现出最佳活性，最佳温度为40℃。该酶在温度高于40℃时不稳定。酶活性受到EDTA、对氯汞苯甲酸和碘乙酸的强烈抑制，但受到CaCl2和NaCl的增强。对这些结果进行了讨论，并与先前报道的该寄生虫昆虫宿主中的α-淀粉酶进行了比较。
