降解AFB_(1)的克雷伯氏菌分离、鉴定及降解机理初步研究

Isolation,identification of klebsiella and its degradation mechanism of AFB_(1)

为了获得对黄曲霉具有降解作用的菌株,以秸秆、牛粪、土壤、发霉玉米及花生为研究对象,以香豆素为唯一碳源,筛选获得了能够降解黄曲霉毒素B_(1)(AFB_(1))的多个菌株。通过生理生化及16S rDNA基因序列分析确定菌株种类;通过优化获得了对AFB_(1)具有最优降解效果的培养基组成;采用蛋白酶K和SDS处理发酵上清液,初步判断具有降解作用的物质;采用SDS-PAGE电泳分析比较了不同培养基中发酵培养基上清液的蛋白图谱,并选择差异条带进行质谱鉴定;采用不同萃取剂对降解后的产物进行萃取并进行全波长扫描与液质联用分析,确定降解产物的分子量,并对其可能的结构进行推测。结果表明:克雷伯氏菌(Klebsiella sp.)N1在DJN培养基对黄曲霉毒素降解效果最好,37℃、72 h内降解率为74%,上清液中存在降解AFB_(1)的物质,初步推断为酶;差异蛋白条带质谱分析结果显示,对黄曲霉毒素有降解效果的酶可能为Mn-超氧化物歧化酶(SodA)或NADH-醌氧化还原酶(NuoB);降解产物的全波长扫描结果显示其最大吸收峰为310 nm左右;正丁醇作为最优萃取剂,采用LC-MS/MS对降解产物进行初步分析,结果显示其分子量为241.1,推测其呋喃环上的8、9位双键以及12位的甲氧基被破坏。

In order to attain the aflatoxi N1(AFB_(1)) degrading strains,in this study,strains with the capacity of degrading AFB_(1) was obtained from straw,cow dung,soil,moldy corn and peanuts using coumarin as the sole carbon source in the selected medium.The physiological and biochemical analysis combined with16 S rDNA gene sequence analysis were used to verify the species of the strains;the optimal components of the fermentation medium was investigated;the contents with the ability to degrade AFB_(1) was determined using protease K and SDS treatment;the protein profiles were illustrated using SDS-PAGE analysis and the different protein bands were analyzed using LC-MS;the degrading compounds were extracted and full wavelength scanning and liquid-mass spectrometry analysis were employed for further analysis;the possible formulas of the degraded compounds were inferred based on its molecular mass.The results indicated that the strain Klebsiella sp N1 in DJN medium showed the best effect on aflatoxin degradation,with a degradation rate of 74% within 72 hours at 37 ℃ detected by high performance liquid chromatography(HPLC).Meanwhile,the aflatoxin degradation rates of the fermentation supernatant of SC and DPN medium were58% and 36% respectively and the aflatoxin absorbance of bacteria biomass was 20%.After treatment of fermentation supernatant with protease K and SDS,the degradation rate of AFB_(1) was significantly reduced,which were 10.5% and 0%,respectively.These phenomena suggested that AFB_(1) might be degraded by extracellular enzymes.SDS-PAGE electrophoresis illustrated that the protein content in the supernatant of DJN was higher than that in SC and DPN medium.The different protein bands analysis indicated that the protein might be Mn-superoxide dismutase(SodA) or NADH-quinone redox enzyme(NuoB) with molecular mass 23 kDa.The degradation products were extracted using chloroform,ethyl acetate and n-butanol,the full wavelength scanning results showed that the products could be leach by n-butanol.In addition,the full wavelength scanning of the degradation products showed that the maximum absorption peak was at310 nm,and its molecular weight was 241.1 after extraction by n-butanol and analyzed by LC-MS/MS.The results might suggest that 8-and 9-digit double bonds on the furan ring and the 12-digit methoxy group might be destroyed in aflatoxin B_(1) according to the molecular mass of the degraded compounds.