Xiao Liang, Liu Chuan, Xie Chichu, Cai Jun, Liu Dong, Chen Yuehua
Department of Microbiology, Nankai University, Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Tianjin 300071, China.
Wei Sheng Wu Xue Bao. 2010 Jun;50(6):749-54.
To heterogeneously express the chitinase gene of Bacillus licheniformis strain MY75 in E. coli, and to characterize the recombinant chitinase ChiMY.
The extracelluar crude protein from B. licheniformis MY75 was analyzed by zymogram analysis. The partial amino acid sequence of the protein owned chitinase activity was given by time-of-flight mass spectrometry (TOF-MS). Then the corresponding chitinase gene chiMY was cloned and heterogeneously expressed in E. coli. The optimum temperature and pH of the ChiMY, and the effect of various metal ions on chitinase activity were studied. The antifungal activity and the synergistic effect on insecticidal activity were demonstrated by bioassays.
A 55 kDa extracelluar protein produced by B. licheniformis MY75 exhibited chitinase activity in zymogram analysis. The chiMY gene was 1797 bp long and encoded a 599 amino acid protein. The molecular weight of the recombinant protein ChiMY over-expressed in E. coli was 67 kDa. The amino acid sequence of the 55 kDa extracelluar protein was proved identical to the 67 kDa ChiMY by the TOF-MS. The optimum temperature and pH were 50 degrees C and 7.0, respectively. The enzyme activity was improved by Li+, Na+ and Mg2+ and inhibited by Mn2+, Cr3+, Zn2+, and Ag+. Cu2+ and Fe3+ can inactivate the enzyme. The bioassays demonstrated the heterogeneously expressed ChiMY could inhibit the sporangia germination of G. saubinetii and A. niger, and reduce the LC50 (50% lethal concentration) of the crystal protein of Bacillus thuringiensis against S. exigua by approximately 27%.
The B. licheniformis MY75 could produce a 55 kDa chitinase. The corresponding chitinase gene was over-expressed in E. coli. The molecular weight of heterogeneously expressed ChiMY showed significant different to the wild-type chitinase protein. This implicated the the protein processing of chitinase in the B. licheniformis MY75. The ChiMY also owned the antifungal activity and could improve the insecticidal activity of the Bacillus thuringiensis crystal protein against S. exigua. This is the first report about the chitinase from B. licheniformis in China.
在大肠杆菌中异源表达地衣芽孢杆菌MY75菌株的几丁质酶基因，并对重组几丁质酶ChiMY进行特性分析。
用地衣芽孢杆菌MY75的胞外粗蛋白进行酶谱分析。通过飞行时间质谱（TOF-MS）测定具有几丁质酶活性的蛋白的部分氨基酸序列。随后克隆相应的几丁质酶基因chiMY并在大肠杆菌中进行异源表达。研究了ChiMY的最适温度和pH值，以及各种金属离子对几丁质酶活性的影响。通过生物测定证明了其抗真菌活性以及对杀虫活性的协同作用。
地衣芽孢杆菌MY75产生的一种55 kDa胞外蛋白在酶谱分析中表现出几丁质酶活性。chiMY基因长1797 bp，编码一个599个氨基酸的蛋白。在大肠杆菌中过表达的重组蛋白ChiMY的分子量为67 kDa。通过TOF-MS证实55 kDa胞外蛋白的氨基酸序列与67 kDa的ChiMY相同。最适温度和pH值分别为50℃和7.0。Li+、Na+和Mg2+可提高酶活性，而Mn2+、Cr3+、Zn2+和Ag+则抑制酶活性。Cu2+和Fe3+可使酶失活。生物测定表明，异源表达的ChiMY可抑制茄腐镰刀菌和黑曲霉的孢子囊萌发，并使苏云金芽孢杆菌晶体蛋白对甜菜夜蛾的LC50（50%致死浓度）降低约27%。
地衣芽孢杆菌MY75可产生一种55 kDa的几丁质酶。相应的几丁质酶基因在大肠杆菌中过表达。异源表达的ChiMY的分子量与野生型几丁质酶蛋白有显著差异。这暗示了地衣芽孢杆菌MY75中几丁质酶的蛋白加工过程。ChiMY还具有抗真菌活性，并可提高苏云金芽孢杆菌晶体蛋白对甜菜夜蛾的杀虫活性。这是中国关于地衣芽孢杆菌几丁质酶的首次报道。
