有机磷降解菌生物学特性及降解酶酶学性质分析
发布时间：2018-10-08 20:05
【摘要】：磷是植物生长发育过程中不可或缺的元素,但在田间多以无效的结合态的磷存在。土壤中的微生物不仅能够将无效态的磷转化为游离态的有效磷,还可以降解田间残留的农药。分离土壤中的解磷菌并加以利用,便可以充分利用土壤中的难溶性磷,创造更大的收益和经济效益。本实验从大豆根际土壤中分离纯化出了三种既能降解大豆卵磷脂又能降解乐果的有机磷降解菌Yj1、Yj2和Yj3,并对这三种菌进行了鉴定、生长条件优化、酸性磷酸酶、碱性磷酸酶和有机磷降解酶的酶活性测定以及有机磷降解酶的分离纯化。通过对有机磷降解菌生物学特性及有机磷降解酶酶学性质的研究,一方面可以提高自然界中磷素利用率、减少有机磷农药在环境中的残留,同时为提高有机磷降解率提供基础理论依据。另一方面,也为降解有机农药和生产有机磷降解菌剂奠定基础。经vitek2自动分析系统鉴定,Yj1为灵杆菌同粘质沙雷氏菌(Serratia marcescens)且与Serratia marcescens WW4(CP003959.1)的16s rDNA相似度为99%。正交试验对所需培养基进行优化,得到该菌株的最佳生长条件为甘露糖、蛋白胨和pH=8的组合。Yj1菌株在两种磷源条件下,菌株生长量均很低,但72 h内以大豆卵磷脂为磷源时的菌体生长情况优于乐果。以大豆卵磷脂为磷源时酸性磷酸酶、碱性磷酸酶与有机磷降解酶活性明显高于以乐果为磷源时的酶活,且72 h内碱性磷酸酶活性一直都高于酸性磷酸酶和有机磷降解酶。硫酸铵沉淀法结合阳离子交换层析成功从Yj1菌体中分离纯化了有机磷降解酶,SDS-PAGE结果显示纯化的蛋白为单一条带。且阳离子交换层析的提纯倍数是硫酸氨沉淀的5.303倍,硫酸氨沉淀为粗酶的1.416倍。Yj2鉴定为醋酸钙不动杆菌,与Acinetobacter genomosp.13(FJ694759.1)的16s rDNA相似度为99%。正交试验对所需培养基进行优化,得到该菌株的最佳生长条件为葡萄糖、硫酸铵和pH=8的组合。Yj2菌株在两种磷源条件下,72 h内以乐果为磷源时的菌体生长情况优于大豆卵磷脂。以大豆卵磷脂为磷源时酸性磷酸酶、碱性磷酸酶活性高于以乐果为磷源时的酶活,而有机磷降解酶活性却是以乐果为磷源时高于大豆卵磷脂。且72 h内酸性磷酸酶活性一直都高于碱性磷酸酶和有机磷降解酶。硫酸铵沉淀法结合阳离子交换层析成功从Yj2菌体中分离纯化了有机磷降解酶,SDS-PAGE结果显示纯化的蛋白为单一条带。且阳离子交换层析的提纯倍数是硫酸氨沉淀的10.44倍,硫酸氨沉淀为粗酶的1.327倍。Yj3鉴定为芽孢杆菌(Bacillus),与Bacillus sp.B2101(JX266369.1)的16s rDNA相似度为99%。正交试验对所需培养基进行优化,得到该菌株的最佳生长条件为葡萄糖、蛋白胨和pH=9的组合。Yj3菌株在两种磷源条件下,72 h内以大豆卵磷脂为磷源时的菌体生长情况优于乐果。以大豆卵磷脂为磷源时72 h内酸性磷酸酶、碱性磷酸酶活性高于以乐果为磷源时的酶活,而有机磷降解酶活性却是以乐果为磷源时明显高于大豆卵磷脂。硫酸铵沉淀法结合阳离子交换层析成功从Yj3菌体中分离纯化了有机磷降解酶,SDS-PAGE结果显示纯化的蛋白为单一条带。且阳离子交换层析的提纯倍数是硫酸氨沉淀的7.969倍,硫酸氨沉淀为粗酶的1.527倍。
[Abstract]:Phosphorus is an indispensable element in the growth and development of plants, but there are more phosphorus in the field than in the field. the microorganisms in the soil can not only convert the phosphorus of the inactive state into the effective phosphorus of the denitrification state, but also can degrade the residual pesticide in the field. The phosphorus-solubilizing bacteria in the soil can be separated and utilized, so that the insoluble phosphorus in the soil can be fully utilized, and more benefits and economic benefits can be created. Three kinds of organophosphate degrading bacteria Yj1, Yj2 and Yj3, which could degrade soybean lecithin and degrade methamidophos, were isolated and purified from soybean roots. Enzyme activity determination of alkaline phosphatase and organophosphorus degrading enzyme and separation and purification of organophosphorus degrading enzyme. By studying the biological characteristics of organic phosphorus degrading bacteria and the enzymatic properties of organophosphorus degrading enzymes, on the one hand, the utilization rate of phosphorus in nature can be improved, the residual of organophosphorus pesticide in the environment can be reduced, and the basic theory basis is provided for improving the degradation rate of the organic phosphorus. On the other hand, it lays a foundation for degrading organic pesticides and producing organophosphorus degrading bacteria. The 16s rDNA resemblance of Yj1 to Serratia marcescens and Serratia marcescens WW4 (CP003959. 1) was 99%. The optimal growth conditions of the strain were optimized by orthogonal test, and the optimal growth conditions of the strain were mannose, protein ratio and pH = 8. The growth of Yj1 strain was very low under the conditions of two phosphate sources, but the growth of thalli in 72 hours was better than that of soybean lecithin. The activity of acid phosphatase, alkaline phosphatase and organophosphorus degrading enzyme was significantly higher than that when soybean lecithin was the phosphorus source, and the activity of alkaline phosphatase in 72 hours was higher than that of acid phosphatase and organophosphorus degrading enzyme. The organic phosphorus degrading enzyme was successfully isolated from Yj1 thalli in combination with cation exchange chromatography. SDS-PAGE showed that the purified protein was a single band. and the purification multiple of cation exchange chromatography is 5.303 times that of ammonium sulfate precipitation, and the precipitation of ammonium sulfate is 1.416 times of the crude enzyme. Yj2 was identified as Acinetobacter genomosp.13 (FJ694759. 1). The similarity of 16S rDNA was 99%. The orthogonal test was used to optimize the medium required to obtain the optimal growth conditions of the strain as a combination of glucose, pH and pH = 8. The growth of Yj2 strain was better than that of soybean lecithin under two phosphate sources. The activity of acid phosphatase and alkaline phosphatase was higher than that of soybean lecithin when soybean lecithin was the source of phosphorus, but the activity of organophosphorus degrading enzyme was higher than that of soybean lecithin. The activity of acid phosphatase in 72 hours was higher than that of alkaline phosphatase and organophosphorus degrading enzyme. The organic phosphorus degrading enzyme was successfully isolated from Yj2 thalli in combination with cation exchange chromatography. SDS-PAGE showed that the purified protein was a single band. and the purification multiple of cation exchange chromatography is 10.44 times that of ammonium sulfate precipitation, and the precipitation of ammonium sulfate is 1.327 times of the crude enzyme. Yj3 was identified as Bacillus (Bacillus sp.), and the 16s rDNA was 99% similar to that of Bacillus sp. B2101 (JX266369. 1). Orthogonal tests were used to optimize the medium required to obtain the optimal growth conditions for the strain as a combination of glucose, proteolytic enzyme and pH = 9. The growth of Yj3 strain under the conditions of two phosphate sources was better than that of soybean lecithin when soybean lecithin was used as the source of phosphorus. The activity of acid phosphatase and alkaline phosphatase in 72 hours was higher than that of soybean lecithin when soybean lecithin was the source of phosphorus. The organic phosphorus degrading enzyme was successfully isolated from Yj3 thalli in combination with cation exchange chromatography. SDS-PAGE showed that the purified protein was a single band. and the purification multiple of cation exchange chromatography is 7.969 times that of ammonium sulfate precipitation, and the precipitation of ammonium sulfate is 1. 527 times of the crude enzyme.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S154.3
本文编号：2258094
[Abstract]:Phosphorus is an indispensable element in the growth and development of plants, but there are more phosphorus in the field than in the field. the microorganisms in the soil can not only convert the phosphorus of the inactive state into the effective phosphorus of the denitrification state, but also can degrade the residual pesticide in the field. The phosphorus-solubilizing bacteria in the soil can be separated and utilized, so that the insoluble phosphorus in the soil can be fully utilized, and more benefits and economic benefits can be created. Three kinds of organophosphate degrading bacteria Yj1, Yj2 and Yj3, which could degrade soybean lecithin and degrade methamidophos, were isolated and purified from soybean roots. Enzyme activity determination of alkaline phosphatase and organophosphorus degrading enzyme and separation and purification of organophosphorus degrading enzyme. By studying the biological characteristics of organic phosphorus degrading bacteria and the enzymatic properties of organophosphorus degrading enzymes, on the one hand, the utilization rate of phosphorus in nature can be improved, the residual of organophosphorus pesticide in the environment can be reduced, and the basic theory basis is provided for improving the degradation rate of the organic phosphorus. On the other hand, it lays a foundation for degrading organic pesticides and producing organophosphorus degrading bacteria. The 16s rDNA resemblance of Yj1 to Serratia marcescens and Serratia marcescens WW4 (CP003959. 1) was 99%. The optimal growth conditions of the strain were optimized by orthogonal test, and the optimal growth conditions of the strain were mannose, protein ratio and pH = 8. The growth of Yj1 strain was very low under the conditions of two phosphate sources, but the growth of thalli in 72 hours was better than that of soybean lecithin. The activity of acid phosphatase, alkaline phosphatase and organophosphorus degrading enzyme was significantly higher than that when soybean lecithin was the phosphorus source, and the activity of alkaline phosphatase in 72 hours was higher than that of acid phosphatase and organophosphorus degrading enzyme. The organic phosphorus degrading enzyme was successfully isolated from Yj1 thalli in combination with cation exchange chromatography. SDS-PAGE showed that the purified protein was a single band. and the purification multiple of cation exchange chromatography is 5.303 times that of ammonium sulfate precipitation, and the precipitation of ammonium sulfate is 1.416 times of the crude enzyme. Yj2 was identified as Acinetobacter genomosp.13 (FJ694759. 1). The similarity of 16S rDNA was 99%. The orthogonal test was used to optimize the medium required to obtain the optimal growth conditions of the strain as a combination of glucose, pH and pH = 8. The growth of Yj2 strain was better than that of soybean lecithin under two phosphate sources. The activity of acid phosphatase and alkaline phosphatase was higher than that of soybean lecithin when soybean lecithin was the source of phosphorus, but the activity of organophosphorus degrading enzyme was higher than that of soybean lecithin. The activity of acid phosphatase in 72 hours was higher than that of alkaline phosphatase and organophosphorus degrading enzyme. The organic phosphorus degrading enzyme was successfully isolated from Yj2 thalli in combination with cation exchange chromatography. SDS-PAGE showed that the purified protein was a single band. and the purification multiple of cation exchange chromatography is 10.44 times that of ammonium sulfate precipitation, and the precipitation of ammonium sulfate is 1.327 times of the crude enzyme. Yj3 was identified as Bacillus (Bacillus sp.), and the 16s rDNA was 99% similar to that of Bacillus sp. B2101 (JX266369. 1). Orthogonal tests were used to optimize the medium required to obtain the optimal growth conditions for the strain as a combination of glucose, proteolytic enzyme and pH = 9. The growth of Yj3 strain under the conditions of two phosphate sources was better than that of soybean lecithin when soybean lecithin was used as the source of phosphorus. The activity of acid phosphatase and alkaline phosphatase in 72 hours was higher than that of soybean lecithin when soybean lecithin was the source of phosphorus. The organic phosphorus degrading enzyme was successfully isolated from Yj3 thalli in combination with cation exchange chromatography. SDS-PAGE showed that the purified protein was a single band. and the purification multiple of cation exchange chromatography is 7.969 times that of ammonium sulfate precipitation, and the precipitation of ammonium sulfate is 1. 527 times of the crude enzyme.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S154.3
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