苏云金芽胞杆菌几丁质酶B特性及其杀虫抑真菌的作用

Chitinase B from Bacillus thuringiensis and its antagonism and insecticidal enhancing potential

【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]We studied the characteristics of chitinase B （ChiB） from Bacillus thuringiensis subsp. colmeri 15A3 （Bt 15A3） and evaluated its antagonism and insecticidal potential. [Methods] ChiB was expressed in Escherichia coli. Expression product was purified by ammonium sulfate precipitation, dialysis and Sephadex G-200 gel filtration chromatography. The molecular mass of ChiB was directly estimated by zymogram after sodium dodecyl sulfate-polyacryamide gel electroresis （SDS-PAGE）. The proteins with different molecular weight were identified by mass spectrum （MS） and analyzed through bio-informatics. We studied some metal ions＇ impacts on chitinase activity; optimal temperature, pH and its stability at different temperature and pH. We also studied the inhibition effects on sporangia germination of fungi and the synergistic effects of ChiB on larvicidal activity. [Results] The molecular mass of ChiB was estimated as 70 kDa by zymogram. The protein with 64 kDa was the product of C-terminal degradation of ChiB in E. coli. Chitinase activity was improved by Fe^ 3＋ and inhibited by Zn^2＋ and Ag^2＋ . The optimal temperature for ChiB was 60℃ and the optimal pH was 5.0. The enzyme was quite stable at temperature below 60℃ and pH between 4.0 and 8.0. ChiB inhibited sporangia germination and the IC50 （50% inhibited concentration） was about 4 μg/mL. Moreover, the bioassays showed that ChiB could reduce the LC 50 （50% lethal concentration） of the crystal protein of Bt. against Spodoptera exigua and Helicoverpa armigera larvae by approximately 26% and 30%. [Conclution] ChiB was not only stable, but also with good antagonism and insecticidal activity.