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【目的】本文对苏云金芽胞杆菌科默尔亚种15A3菌株(Bacillus thuringensis subsp.colmeri 15A3,简称Bt 15A3)几丁质酶B(chitinase B,简称ChiB)的特性及其杀虫抑真菌作用作了初步研究。【方法】将大肠杆菌(Escherichia coli)中表达的Bt 15A3菌株的ChiB,经过硫酸铵沉淀、透析、Sephadex G-200凝胶层析,最终得到初步纯化的几丁质酶。利用酶谱方法确定分子量,并且对2种分子量的酶蛋白进行了质谱鉴定。对各种金属离子对ChiB酶活的影响、最适反应温度及pH、温度及pH稳定性等理化特性进行研究,并且测定了ChiB抑制真菌孢子萌发的活性和对甜菜夜蛾和棉铃虫的杀虫增效作用。【结果】ChiB分子量约为70kDa。同时证明检测到的64kDa蛋白为70kDa蛋白C端的降解产物。ChiB最适作用温度为60℃,最适pH值为5,在温度20℃～60℃和pH4～8的范围内均有良好的稳定性。供试金属离子中Fe3+对ChiB的酶活有促进作用,Zn2+,Ag+有抑制作用。ChiB可抑制产黄青霉等真菌孢子的萌发,半抑制浓度(median effective inhibitory concentration,IC50)约为4μg/mL。该酶可以分别降低Bt晶体蛋白对甜菜夜蛾和棉铃虫的半致死浓度(median lethal concentration,LC50)26%和30%。【结论】Bt科默尔亚种的几丁质酶不但具有较稳定的理化性质,而且具有抑制真菌生长及明显的杀虫增效作用。
【Objective】 In this paper, the characteristics of Bacillus thuringiensis subsp. Colmeri 15A3 (Bt 15A3) chitinase B (ChiB) and its fungicidal and fungicidal activity were studied initial research. 【Method】 ChiB of Bt 15A3 strain expressed in Escherichia coli was precipitated with ammonium sulfate, dialyzed and gel-chromatographed on Sephadex G-200 to obtain chitinase. The molecular weight was determined by zymography and mass spectrometry was performed on enzyme proteins of two molecular weights. The effects of various metal ions on the enzyme activity of ChiB, the optimum reaction temperature and the physicochemical properties such as pH, temperature and pH stability were studied. The activity of ChiB for inhibiting spore germination and the killing of beet armyworm and cotton bollworm Bug synergistic effect. 【Result】 The molecular weight of ChiB was about 70kDa. At the same time, the 64 kDa protein detected was a C-terminal degradation product of 70 kDa protein. The optimum temperature of ChiB is 60 ℃, the optimum pH is 5, and it has good stability at the temperature of 20 ℃ ~ 60 ℃ and pH4 ~ 8. Fe3 + in test metal ions can promote the enzyme activity of ChiB, and Zn2 + and Ag + have inhibitory effects. ChiB inhibited the germination of fungal spores such as Penicillium chrysogenum, with a median effective inhibitory concentration (IC50) of about 4 μg / mL. The enzyme reduced the median lethal concentration (LC50) of Bt crystal protein by beet armyworm and cotton bollworm by 26% and 30%, respectively. 【Conclusion】 Chitinase of Bt Commel species not only has more stable physicochemical properties, but also inhibits fungal growth and obvious insecticidal synergism.