有機磷降解菌生物學(xué)特性及降解酶酶學(xué)性質(zhì)分析
發(fā)布時間：2018-10-08 20:05
【摘要】：磷是植物生長發(fā)育過程中不可或缺的元素,但在田間多以無效的結(jié)合態(tài)的磷存在。土壤中的微生物不僅能夠?qū)o效態(tài)的磷轉(zhuǎn)化為游離態(tài)的有效磷,還可以降解田間殘留的農(nóng)藥。分離土壤中的解磷菌并加以利用,便可以充分利用土壤中的難溶性磷,創(chuàng)造更大的收益和經(jīng)濟效益。本實驗從大豆根際土壤中分離純化出了三種既能降解大豆卵磷脂又能降解樂果的有機磷降解菌Yj1、Yj2和Yj3,并對這三種菌進行了鑒定、生長條件優(yōu)化、酸性磷酸酶、堿性磷酸酶和有機磷降解酶的酶活性測定以及有機磷降解酶的分離純化。通過對有機磷降解菌生物學(xué)特性及有機磷降解酶酶學(xué)性質(zhì)的研究,一方面可以提高自然界中磷素利用率、減少有機磷農(nóng)藥在環(huán)境中的殘留,同時為提高有機磷降解率提供基礎(chǔ)理論依據(jù)。另一方面,也為降解有機農(nóng)藥和生產(chǎn)有機磷降解菌劑奠定基礎(chǔ)。經(jīng)vitek2自動分析系統(tǒng)鑒定,Yj1為靈桿菌同粘質(zhì)沙雷氏菌(Serratia marcescens)且與Serratia marcescens WW4(CP003959.1)的16s rDNA相似度為99%。正交試驗對所需培養(yǎng)基進行優(yōu)化,得到該菌株的最佳生長條件為甘露糖、蛋白胨和pH=8的組合。Yj1菌株在兩種磷源條件下,菌株生長量均很低,但72 h內(nèi)以大豆卵磷脂為磷源時的菌體生長情況優(yōu)于樂果。以大豆卵磷脂為磷源時酸性磷酸酶、堿性磷酸酶與有機磷降解酶活性明顯高于以樂果為磷源時的酶活,且72 h內(nèi)堿性磷酸酶活性一直都高于酸性磷酸酶和有機磷降解酶。硫酸銨沉淀法結(jié)合陽離子交換層析成功從Yj1菌體中分離純化了有機磷降解酶,SDS-PAGE結(jié)果顯示純化的蛋白為單一條帶。且陽離子交換層析的提純倍數(shù)是硫酸氨沉淀的5.303倍,硫酸氨沉淀為粗酶的1.416倍。Yj2鑒定為醋酸鈣不動桿菌,與Acinetobacter genomosp.13(FJ694759.1)的16s rDNA相似度為99%。正交試驗對所需培養(yǎng)基進行優(yōu)化,得到該菌株的最佳生長條件為葡萄糖、硫酸銨和pH=8的組合。Yj2菌株在兩種磷源條件下,72 h內(nèi)以樂果為磷源時的菌體生長情況優(yōu)于大豆卵磷脂。以大豆卵磷脂為磷源時酸性磷酸酶、堿性磷酸酶活性高于以樂果為磷源時的酶活,而有機磷降解酶活性卻是以樂果為磷源時高于大豆卵磷脂。且72 h內(nèi)酸性磷酸酶活性一直都高于堿性磷酸酶和有機磷降解酶。硫酸銨沉淀法結(jié)合陽離子交換層析成功從Yj2菌體中分離純化了有機磷降解酶,SDS-PAGE結(jié)果顯示純化的蛋白為單一條帶。且陽離子交換層析的提純倍數(shù)是硫酸氨沉淀的10.44倍,硫酸氨沉淀為粗酶的1.327倍。Yj3鑒定為芽孢桿菌(Bacillus),與Bacillus sp.B2101(JX266369.1)的16s rDNA相似度為99%。正交試驗對所需培養(yǎng)基進行優(yōu)化,得到該菌株的最佳生長條件為葡萄糖、蛋白胨和pH=9的組合。Yj3菌株在兩種磷源條件下,72 h內(nèi)以大豆卵磷脂為磷源時的菌體生長情況優(yōu)于樂果。以大豆卵磷脂為磷源時72 h內(nèi)酸性磷酸酶、堿性磷酸酶活性高于以樂果為磷源時的酶活,而有機磷降解酶活性卻是以樂果為磷源時明顯高于大豆卵磷脂。硫酸銨沉淀法結(jié)合陽離子交換層析成功從Yj3菌體中分離純化了有機磷降解酶,SDS-PAGE結(jié)果顯示純化的蛋白為單一條帶。且陽離子交換層析的提純倍數(shù)是硫酸氨沉淀的7.969倍,硫酸氨沉淀為粗酶的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.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S154.3
本文編號：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.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S154.3
【參考文獻】
相關(guān)期刊論文 前5條
1 黃雅;李政一;趙博生;;有機磷農(nóng)藥樂果降解的研究現(xiàn)狀與進展[J];環(huán)境科學(xué)與管理;2009年04期
2 王繼雯;甄靜;謝寶恩;劉瑩瑩;李冠杰;;有機磷降解菌的分離篩選及初步鑒定[J];河南科學(xué);2011年01期
3 王圣惠;閆艷春;徐剛明;李瑩瑩;;一株有機磷農(nóng)藥降解菌的分離、鑒定及降解酶基因的克隆[J];農(nóng)業(yè)環(huán)境科學(xué)學(xué)報;2007年S1期
4 伍寧豐,梁果義,鄧敏捷,姚斌,范云六;有機磷農(nóng)藥降解酶及其基因工程研究進展[J];生物技術(shù)通報;2003年05期
5 王X;王志春;李榮;郜彥彥;李順鵬;;樂果降解菌L3的分離、鑒定及降解性能[J];中國環(huán)境科學(xué);2008年11期
相關(guān)博士學(xué)位論文 前1條
1 賈開志;久效磷降解菌的分離、鑒定及降解酶基因的克隆[D];南京農(nóng)業(yè)大學(xué);2006年
，本文編號：2258094
本文鏈接：http://sikaile.net/kejilunwen/nykj/2258094.html
最近更新
教材專著
