【作者】 张斌；

【导师】 祁庆生；

【作者基本信息】 山东大学 ， 发酵工程， 2009， 硕士

【摘要】 环糊精及其衍生物以其独特的包合性能已被广泛应用到医药、食品、化工、环境等诸多领域,但在农业生产中的应用还处于起始阶段。为解决环糊精糖基转移酶产生菌菌株筛选效率低的难题,本研究建立了一种从土壤中快速筛选环糊精糖基转移酶产生菌菌株的方法。为了进一步探索环糊精在提高农药溶解度及其药效方面的可行性,本试验以农药毒死蜱为实验对象,采用多种方法制备了环糊精β-CD及其衍生物与毒死蜱的固体包合物,对包合物进行了表征,分析了包合物对杀虫药效的影响。本文主要研究结果如下:1.建立了一种从土壤中快速筛选环糊精糖基转移酶产生菌菌株的方法。本研究从土壤中初步筛选出了149个产胞外淀粉水解酶的菌株,利用高效阴离子交换色谱检测出了微生物发酵液中的环糊精成分,从而确定了产生菌菌株;本研究检测了其中11个产生菌菌株,6株主要产CD6,5株主要产CD7。同时,在鉴定的过程中,定量检测了各种环糊精包括大环糊精(CD大于8)的含量。2.研究了环糊精与农药包合物的制备方法,确定了最佳反应条件。本研究分别利用研磨法、超声波法和饱和水溶液法制备了环糊精β-CD、Hp-β-CD和Me-β-CD与农药毒死蜱的包合物,其中饱和水溶液法包合效果最好。使用该方法环糊精β-CD、Hp-β-CD和Me-β-CD与农药毒死蜱的包合率分别为45.8%、52.0%和65.0%。采用正交试验得到β-CD包合毒死蜱的最佳反应条件:投料比为1:1(摩尔比),包合时间为3h,包合温度为60℃。3.利用紫外光谱法定量研究了β-CD及其衍生物对毒死蜱的包结行为。结果表明β-CD及其衍生物与毒死蜱均形成了包结比为1:1的包结物,形成的包合物的稳定常数分别为K=33.33mol/L(β-CD),K=343.8 mol/L(HP-β-CD),K=461.63 mol/L(Me-β-CD)。由包合物稳定常数大小可知β-CD与毒死蜱包合物的稳定性最差,而HP-β-CD、Me-β-CD都能与毒死蜱形成较稳定的包合物。利用ΔG=-RlnK计算出重要的热力学参数ΔG分别为:-8.69KJ/mol(β-CD)、-20.184KJ/mol(HP-β-CD)及-15.21KJ/mol(Me-β-CD),包合反应的吉布斯函数ΔG均为负。4.利用紫外光谱法对液体包合物进行了鉴定。当固定客体毒死蜱浓度时,随着主体β-CD、HP-β-CD、Me-β-CD浓度的增大,整个体系的吸收峰没有明显的位移,但其最大吸收峰处的吸光值逐渐增大。说明β-CD及其衍生物对毒死蜱产生了识别作用,毒死蜱进入CD的空腔,形成了包合物。5.利用红外吸收光谱和差示热分析法分别对β-CD、HP-β-CD和Me-β-CD与毒死蜱固体包合物进行了表征。差示热分析可知,三种包合物仅存在主体分子的脱水峰,毒死蜱原来的吸收峰消失;红外吸收光谱证明三种包合物中毒死蜱的特征吸收峰消失,主要表现为主体分子图谱。结果表明:β-CD、HP-β-CD和Me-β-CD与毒死蜱均形成了1:1的稳定包合物。6.包合物的室内杀虫毒力试验结果为:β-CD、HP-β-CD和Me-β-CD与毒死蜱包合物致死中浓度LC50分别16.1μg/mL、10.8μg/mL和12.3μg/mL。本研究首次利用高效阴离子交换色谱检测了产CGTase菌株发酵液中的环糊精成分,尤其是大环糊精的组成。建立了一种从土壤中快速筛选环糊精糖基转移酶产生菌菌株的新方法,利用该方法还可以检测环糊精糖基转移酶在不同阶段产生环糊精的能力大小。本研究发现通过环糊精与毒死蜱的包合作用可以增加毒死蜱在水中的溶解度,提高其药效。该研究为农药的水基化推广奠定了基础。更多还原

【Abstract】 The Cyclodextrin(CD) and its derivatives have been widely applied in medical industry,food industry,chemical industry,environment and many other areas for its unique inclusion performance.However,the application in agricultural production is still in the initial stage.In order to resolve the problem that the efficiency of screening strain is low in the production process of the CD,this study established a new method which could screen glycosyltransferase(CGTase)-producing strain from the soil quickly and accurately.In order to explore the possibility of the application of the CD in agriculture.The inclusion ofβ-CD and its derivatives and pesticide chlorpyrifos were made by many ways,the inclusion solid complex was characterizated,and the insecticide efficacy experiments was carried out.The results these studies are as follows:1.A method that can quickly select the CGTase-producing bacteria was set up. 149 extracellular amylase-producing strains have been screened from the soil The CD composition was detected using the High Performance Anion Exchange Chromatography(HPAEC) and the CGTase-producing bacteria was determined.In the study,11 CGTase-producing bacteria strains were detected and 6 strains mainly produced the CD6 and 5 strains CD7.Meanwhile,the content of the different CD including the large ring(CD＞8) was determined during the process.2.The preparation methods of the inclusion complex ofβ-CD,HP-β-CD, M-β-CD and the pesticide chlorpyifos were studied,respectively.And the best conditions were determined.The inclusion rate of the saturated aqueous solution was the best among the grinding method,ultrasonic method and saturated aqueous solution.The inclusion rate of the saturated aqueous solution was 45.8%,52%and 65%for the inclusion complex ofβ-CD,HP-β-CD,M-β-CD and the pesticide chlorpyifos,respetively.The best optimal reaction conditions of inclusion complex ofβ-CD and chlorpyifos by orthogonal was:feed ratio of 1:1(mole ratio),inclusion time was 3h,inclusion temperature is 60℃.3.The inclusion behavior ofβ-cyclodextrin and its derivatives with chlorpyrifos were quantitatively studied using UV spectrum.The results showed that the ratio of inclusion was 1:1,the stability constants of inclusion complexes were K=33.33mol/L (β-CD)、K=343.8 mol/L(HP-β-CD)和K=461.63 mol/L(Me-β-CD).It showed that the stability ofβ-CD inclusion complex with chlorpyrifos was the worst,HP-β-CD、 Me-β-CD inclusion complex were stable.The important thermodynamic parameters△G were calculated by△G =-RlnK,that were -8.69KJ/mol(β-CD)、-20.184KJ/mol(HP-β-CD)、-15.21KJ/mol(Me-β-CD).It showed that inclusion reactions were spontaneous process,and their inclusion constant decreases with increasing temperature,indicating that inclusion is an exothermic process.4.The liquid inclusion complexes ofβ-CD,HP-β-CD,M-β-CD and chlorpyrifos were characterized by UV spectroscopy,respectively.When concentrations of object chlorpyrifos was fixed,with increasing of the concentration of the main,the entire system of absorption there was no obvious shift,however, the maximum absorption peak at the absorbance gradually increased.It showed that chlorpyrifos have been recognized byβ-CD and its derivatives.5.The solid inclusion complexes ofβ-CD,HP-β-CD,M-β-CD and chlorpyrifos were characterized by infrared absorption spectroscopy and differential thermal analysis,respectively.DSC showed that three inclusion only appeared dehydration peak of main elements,original absorption peaks of Chlorpyrifos disappeared.Infrared absorption spectra certified the characteristic absorption peaks of Chlorpyrifos disappeared in three kinds of inclusion,mainly as the main molecular map.It confirmed thatβ-cyclodextrin,HP-β-CD and M-β-CD and chlorpyrifos have formed a stable 1:1 inclusion complex.6.The results of indoor insecticide toxicity of inclusion showed that:the median lethal concentration LC50 of inclusion complex ofβ-CD,HP-β-CD,M-β-CD and chlorpyrifos were 16.1μg/mL,10.8μg/mL and 12.3μg/mL,respectively.The composition of the CD especially the large ring CD were firstly detected using the HPAEC.A novel method that can detect the CD-producing ability of the CGTase during the different stages was set up.The inclusion of the CD and chlorpyrifos can increase the solubility of the chlorpyrifos and its efficacy.The reseach was the basis to the extension of the inclusion of the CD and pesticides.更多还原