利用HPLC测定二硫苏糖醇(DTT)对苏云金芽孢杆菌伴胞晶体降解的影响  

DETECTION ON DECOMPOSITION OF B.T. CRYSTAL FROM BACILLUS THURINGIENSIS BY DITHIOTHREITOL (DTT) USING HPLC

作　　者：朱育菁[1] 刘波[1] C. SENGONCA 

机构地区：[1]福建省农科院生物技术中心农业环保技术研究室,福建福州350003 [2]德国波恩大学植物病理研究所,波恩d53115

出　　处：《武夷科学》2004年第1期1-7,共7页Wuyi Science Journal

基　　金：德国科学基金(DFG)项目PN-74179;国家863计划项目(2002AA244031-2);福建省科技厅重大项目(98-I-8);福建省发展计划委员会项目(2001)

摘　　要：利用 HPLC测定二硫苏糖醇 (DTT)对苏云金芽孢杆菌伴胞晶体降解的影响。结果表明 ,放置时间对DTT溶液的变化影响显著 ,随着 DTT放置时间的延长 ,2个峰的保留时间都随之延长。利用不同浓度的 DTT降解 Bacillusthuringiensis,伴胞晶体时 ,其检测结果都出现保留时间小于 1 5.0 0 min的 4个主峰 ,与 DTT单独添加在 Na2 CO3 - HCl缓冲液 1 h后检测的保留时间大于 2 5.0 0 min的 2个峰明显不同 ,也比伴胞晶体单独添加到 Na2 CO3 - HCl缓冲液 1 h后检测的结果多了 1个新峰 ,并且峰高显著增加 ,这表明 DTT对晶体起到降解作用。峰 1和峰 4的保留时间与 Na2 CO3 - HCl缓冲液所测到的色谱特征峰相近 ,峰 2和峰 3为 Na2 CO3 -HCl缓冲液不具有的。在用不同浓度的 DTT在 37℃下降解 B.t.伴胞晶体 1 h后 ,立即用 HPLC进行检测的结果表明峰 2高于峰 3,峰 2的高度与 DTT的浓度成正相关 ;放置 2 4 h后所有处理中峰 2的高度都下降 ,峰 3的高度都高于峰 2 ,峰 2 (Y1 )和峰 3(Y2 )的峰高都与 DTT的浓度 (x)成指数增长 ,Y1 =1 9.1 33e0 .0 1 4 8x (R2 =0 .970 8)和 Y2 =2 9.0 62 e0 .0 1 68x(R2 =0 .9589) ;上述结论表明在 DTT的作用下 ,伴胞晶体不断地被降解 ,使得峰3不断增高 ,DTT的消耗 ,使得峰 2下降 ;随着 DTT浓度的增大 ,The present paper dealt with the detection on decomposition of B.t. crystal from Bacillus thuringiensis by Dithiothreitol (DTT) using HPLC. The results showed that the preserve time had significant effect on DTT solution due to it was an active reducer. There were 4 peaks with retention time shorted than 15min on the HPLC chromatograph after the decomposition of B.t. crystal treated with DTT, which were significantly different from peaks with retention time longer than 25.00 min detected from DTT added into Na2CO3-HCl buffer solely for 1h and 3 peaks from B.t. crystal added into Na2CO3-HCl buffer solely respectively. It meat that B.t. crystal was decomposed DTT in pH 9.5 Na2CO3-HCl buffer. Comparing with HPLC chromatographs of reactants during decomposing, it was concluded that peak 1 and peak 4 were related to Na2CO3-HCl buffer. Detected immediately after B.t. crystals decomposed with DTT of 20mmol/m, 40mmol/mL and 100mmol/mL under 37℃ for 1 h, peak heights of peak 2 at all treatments were higher than those of peak 3 and increased as DTT's concentration increasing. Preserved for 24h, peak 2 of all treatments decreased and lower than peak 3. The peak heights of peak 2 and peak 3 increased exponentially to the concentration of DTT, the relationships of peak height of peak 2 (Y1) and peak height of peak 3 (Y2) with the concentration of DTT (x) were Y1 = 19.133e0.0148x(R2 = 0.9708)and Y2 = 29.062e0.0168x(R2 = 0.9589). It was concluded that the heightening of peak 3 indicated the formation of B.t. protoxin created by B.t. crystal decomposed with DTT, and the lowering of peak 2 represented consumption of DTT. The results of qualitative analysis by adding DTT into decomposition product showed that the peak height of peak 2 increased 114.59% and peak 3 only increased 55.56% ( the new decompose product by extra DTT) and no significant difference was found at the change of peak 1 peak 4. It was deduced that peak 2 related to DTT, peak 3 related to the protoxin protein from B.t. crystal.

关 键 词：苏云金芽孢杆菌 伴胞晶体降解 二硫苏糖醇 HPLC 

分 类 号：Q939.9[生物学—微生物学]