Biological and biochemical study on parasporal protein produced by bacillus thuringiensis local isolates

In this study, six isolates, named BtAl 10 BtA6. adopted from ten isolates isolated from samples collected 15 different agriculture sites. By using special spore-enhancement method. It was found that these isolates produce very large -sized crystalline protein, which showing differences in shape as examined under phase-contrast microscopy. Total genomic DNA has been isolated. All isolates showing presence of mega plasmids, ranging from 70 to 125 MD, on fractionation by conventional agarose electrophoreses. Small-acid soluble proteins were extracted from all BtA isolates under study: show no significant differences when examined by TLC. and so, it is not applicable as biomarkers on using simple conventional chromatographical methods. Parasporal crystals, from six isolates were isolated using sodium bromide gradient and sucrose gradient, the later was found to be less efficient, while the former provide powerful method for both, isolation of crystals and exclusion proteases effects. It was found that, four isolates: BtA2, BtA3 and BtA6 contain polyhedral inclusions besides the arasporal crystals, while BtAl and BtA5 have no such polyhedral inclusions. Quantitative estimation of parasporal protein production has been examined for the six isolates. Parasporal proteinaseous crystal yields were ranged from 18 to 24%. Total carbohydrate of the parasporal proteins for both intact and solubilized crystals, have been estimated. Intact crystals 0.4 - 0.8% were obtained, while lower levels were detected in solubilized protein, since it ranged from 0.3 - 0.6%. Parasporal proteins have been analyzed by vertical-disc PAGE. According to PAGE profile obtained, BtAl and BtA5 show a major band with molecular weight of about 180 kD. There are a great variation in number and position for most of bands Tow separated enzymes-treatment (trypsin and proteinase K) for parasporal proteins from the six isolates, were curried out with different concentrations 0.003. 0.03. 0.3 and 3 mg/ml final concentration, to obtain activated forms of these proteins. It was found that 0.03 mg/ml final concentration the most effective concentration and used in subsequent experiments. In order to examine the activity of each fragment alone, each activated parasporal protein for both trypsin and proteinase K from each Bacillus ihuringiemis strain have been separated by ion-exchange chromatography using 35x1.5 cm DEAE-Sepharose column. Different solubilization buffers were examined for their efficiency in solublization and possible effects on their hemolytic activity. It was found that 50 mM Na2CO3, ImM EDTA, l0mM B-Mercabtoethanol (pH 10.5) the most efficient one. BtA2 and BtA3 native parasporal protein, have the ability to lyses erythrocyte. with HD50 1.5 and 1.3 respectively, rest isolates showing no such activity. Hemolytic activity have been checked, also in term of HD50, firstly for proteinase K-activated parasporal protein (at different concentration of enzyme) for the six BtA isolates, results indicate that BtA4 and BtA6 get the ability to lyses erythrocytes. BtA2 and BtA3 have an elevated ability to lyses erythrocytes (decreased values ofliDjo), while BtA5 and BtAl have no ability to lyses red cells. Secondly hemolytic activity have
been checked for trypsin-activated parasporal protein (at different concentration of en/.yme) for the six BtA isolates, results revealed that trypsin activation have, in approximation, as the same as proteinas K activation in corresponding enzyme concentration, but at some extent, proteinas K show more activity than trypsin could show. Protein fragments for each proteinase K-activated parasporal proteins have been tested alone from the six BtA isolates. Tests include separated xperiments for both hemolytic activities in term of HD5o and larvicidal activity. results were: protein fraction of BtA 1 and BtA5 were neither hemolytic nor larvicidal. The rest isolates shows different results, some fragment show hemolytic activity beside the larvicidal activity, other showing one of these activities, and lucking the other activity. Last stage of this work, includes dose response, cytopathic study. Four strain: parasporal proteins of BtA2, BtA3, BtA4 and BtA6, have been examined for their toxicity against CLL. Result indicates that all these isolates have strong cytotoxicity with no significant differences between normal lymphocytes and leukemic lymphocytes. BtAl and BtA5 show discriminative cytotoxicity between normal and leukemic lymphocytes, even when at low extent. This may be referring to the ratio of unsaturated fatty acids which included in cell membrane. Proteinase K-treated parasporal proteins and none treated of both normal and CLL lymphocytes have been photographed under phase contrast microscopy. BtA2, BtA3, BtA4 and BtA6 showing massive cell lyses, both normal and CLL. BtAl and BtA5 causing cell ballooning, nucleus fragmentation and finally cell rapture and further examined against Hep2 cells, results obtained with Hep2 show no significant differences from that of CLL.