我国作为农业大国,对农药的大量使用是不可避免的,但是农药的超范围使用、超标及高检出率对于环境的污染与人体健康的威胁日趋严重。
碱性磷酸酶(alkaline phosphatase,ALP)对有机磷农药具有积极的降解作用,因此,本文对鼠李糖乳杆菌(
使用单因素试验和正交试验优化ALP的提取条件;使用对硝基苯酚法测定酶活力;使用分级沉淀和层析法提纯ALP;使用乙酰胆碱酯酶抑制法测定ALP对有机磷农药的降解率。
LGG Z23所产ALP的最优提取条件为:细胞破碎时间15 min,破碎功率450 W,料液比(质量体积比) 1:6,提取液pH 10.0,此条件下酶活力为(4.95±0.26) U/mL,比优化前提高2.11倍;对6种有机磷农药的降解率效果为敌敌畏(95.79%±0.01%)>甲基对硫磷(90.69%±0.03%)>毒死蜱(88.90%±0.02%)>敌百虫(86.07%±0.03%)>马拉硫磷(85.31%±0.02%)>乐果(83.18%±0.03%),其中对敌敌畏和甲基对硫磷的降解效果最好,可达90%以上,并且降解作用差异显著(
本研究为LGG Z23所产ALP的应用研究提供了理论依据和实验数据。
China, one of the world's largest agricultural economy, has seen the large-scale use of pesticides. However, the overuse of pesticides, which has led to high detection rate, has threatened the environment and human health.
Alkaline phosphatase (ALP) can degrade organophosphorus pesticides. Therefore, we optimized the extraction of ALP from
Single factor test and orthogonal test were used to optimize the extraction. The enzyme activity was determined by detecting the amount of
The optimum conditions for extracting ALP from LGG Z23 were as follows: disruption of cells for 15 min at 450 W, material-liquid ratio (mass to volume ratio) of 1:6, and pH 10.0. Under the conditions, the activity of ALP was (4.95±0.26) U/mL, 2.11 times higher than that before optimization. The degradation rate of 6 organophosphorus pesticides was in the order of DDVP (95.79%±0.01%)>methyl parathion (90.69%±0.03%)>chlorpyrifos (88.90%±0.02%)>trichlorfon (86.07%±0.03%)>malathion (85.31%±0.02%)>dimethoate (83.18%±0.03%). Among them, the activity of degrading DDVP and methyl parathion was the highest (over 90%), and the difference was significant (
The result lays a theoretical basis and provides data for the application of ALP from LGG Z23.
目前,有机磷农药是我国常用的农药,由于其毒性大[
碱性磷酸酶(alkaline phosphatase,ALP)是有机磷农药降解酶之一[
LGG Z23为一株产碱性磷酸酶鼠李糖乳杆菌,由本实验室自行分离保存,该菌株在GenBank数据库中的16S rRNA基因登录号为MN582955[
敌敌畏(纯度99.5%)、敌百虫(纯度99.2%)、毒死蜱(纯度98.7%)、马拉硫磷(纯度98.1%)、甲基对硫磷(纯度98.6%)、乐果(纯度98.7%)等有机磷农药标准品,北京坛墨质检科技有限公司。
MRS液体培养基:蛋白胨10.0 g,牛肉膏10.0 g,乙酸钠5.0 g,酵母提取粉5.0 g,葡萄糖20.0 g,磷酸氢二钠2.0 g,柠檬酸二铵2.0 g,七水合硫酸镁0.58 g,四水合硫酸锰0.25 g,吐温-80 1 mL,将其全部用蒸馏水溶解后定容至1 L,调pH值至6.5,1.0×105 Pa灭菌20 min。
脱脂乳培养基:在100 g/L脱脂乳水溶液中加入1 g/L酵母提取粉,0.7×105 Pa高压灭菌7 min。
10 mmol/L的pH 10.5碳酸缓冲溶液:分别配制0.1 mol/L的碳酸钠(Na2CO3)溶液和碳酸氢钠(NaHCO3)溶液,再将两种溶液以9:1的体积比混合稀释,即为10 mmol/L、pH 10.5的碳酸缓冲溶液。
电热恒温培养箱,上海一恒科技有限公司;高速冷冻离心机,Eppendorf公司;超声波细胞破碎仪,Misonix公司。
将LGG Z23用脱脂乳培养基活化一代[
将已活化的菌株Z23接种于MRS液体培养基,37 ℃静置培养36 h[
将洗涤离心后的菌体沉淀,用pH值为10.5的碳酸缓冲溶液按1:10的质量体积比制成菌悬液,在冰浴条件下用超声波细胞破碎仪分别破碎5、10、15、20、25、30 min,功率300 W,4 ℃、12 000 r/min离心10 min后取上清液,用对硝基苯酚法测定酶活力[
将洗涤离心后的菌体沉淀,用pH值为10.5的碳酸缓冲溶液按1:10的质量体积比制成菌悬液,在冰浴条件下,以100、200、300、400、500 W的功率破碎15 min,4 ℃、12 000 r/min离心10 min后取上清液,酶活力测定方法同1.4.1。
将洗涤离心后的菌体沉淀,用pH值为10.5的碳酸缓冲溶液按1:4、1:6、1:8、1:10、1:12、1:14的质量体积比制成菌悬液,在冰浴条件下,以300 W的功率用超声波细胞破碎仪破碎15 min,4 ℃、12 000 r/min离心10 min后取上清液,酶活力测定方法同1.4.1。
将洗涤离心后的菌体沉淀,按1:10的质量体积比制成菌悬液,缓冲溶液pH值分别为6.0、7.0、8.0、9.0、10.0、11.0,在冰浴条件下,以300 W的功率破碎15 min,4 ℃、12 000 r/min离心10 min后取上清液,酶活力测定方法同1.4.1。
以单因素试验结果为基础,以破碎时间、破碎功率、料液比、提取液pH值4个影响因素进行正交试验(
ALP提取条件正交设计及因素水平
Orthogonal design of ALP extraction conditions and factor levels
Level | ||||
1 | 10 | 350 | 1:4 | 9.5 |
2 | 15 | 400 | 1:6 | 10.0 |
3 | 20 | 450 | 1:8 | 10.5 |
经硫酸铵分级沉淀、DEAE-52离子交换层析、Sephadex G-200凝胶过滤层析等步骤纯化[
在试管中加入450 μL不同浓度(0.078、0.156、0.316、0.625、1.25、2.5、5、10 μg/mL)的有机磷农药稀释液,对照管加入50 mmol/L pH 7.5的磷酸缓冲液、25 μL 220 U/g的乙酰胆碱酯酶、25 μL 75 mmo1/L的碘化硫代乙酰胆碱,混匀后在37 ℃水浴20 min,加入50 μL 40 g/mL的十二烷基硫酸钠(SDS)和50 μL 10 mmol/L的二硫代二硝基苯甲酸(DTNB)显色并终止反应,混匀后于412 nm处测定吸光值[
式(1)中,
有机磷农药可以抑制乙酰胆碱酯酶活性,根据1.6中的乙酰胆碱酯酶抑制率与有机磷农药浓度对数值标准曲线,查找与抑制率对应的农药浓度对数值,计算出不同培养时间碱性磷酸酶降解产物中有机磷农药的残留量[
式(2)中,
每组实验重复3次,数值用平均值±SD表示,用SPSS 23.0进行实验数据统计分析,Origin 8.5作图。
在破碎时间5−15 min,LGG Z23所产ALP酶活力显著上升(
不同破碎时间对碱性磷酸酶酶活力的影响
Effect of different crushing times on the enzyme activity of alkaline phosphatase.
在破碎功率100–400 W范围内,LGG Z23所产ALP酶活力显著上升(
不同破碎功率对碱性磷酸酶酶活力的影响
Effect of different crushing power on the enzyme activity of alkaline phosphatase.
从
不同料液比对碱性磷酸酶酶活力的影响
Effect of different material-liquid ratios on the enzyme activity of alkaline phosphatase.
从不同pH值对ALP提取的影响(
不同pH对碱性磷酸酶酶活力的影响
Effect of different pH on the enzyme activity of alkaline phosphatase.
菌株Z23所产ALP最优提取条件正交试验结果(
正交试验结果
Orthogonal test results
Test No. | Factor | Enzyme activity (U/mL) | |||
1 | 1 (10) | 1 (350) | 1 (1:4) | 1 (9.5) | 2.82±0.28 |
2 | 1 | 2 (400) | 2 (1:6) | 2 (10.0) | 3.24±0.13 |
3 | 1 | 3 (450) | 3 (1:8) | 3 (10.5) | 3.08±0.34 |
4 | 2 (15) | 1 | 2 | 3 | 4.03±0.19 |
5 | 2 | 2 | 3 | 1 | 3.75±0.24 |
6 | 2 | 3 | 1 | 2 | 4.86±0.15 |
7 | 3 (20) | 1 | 3 | 2 | 3.82±0.09 |
8 | 3 | 2 | 1 | 3 | 3.58±0.22 |
9 | 3 | 3 | 2 | 1 | 4.28±0.18 |
MV1 | 3.047 | 3.557 | 3.753 | 3.617 | |
MV2 | 4.213 | 3.523 | 3.850 | 3.973 | |
MV3 | 3.893 | 4.073 | 3.550 | 3.563 | |
1.166 | 0.550 | 0.300 | 0.410 |
由6种不同有机磷农药对乙酰胆碱酯酶的抑制率曲线(
不同有机磷农药对乙酰胆碱酯酶的抑制曲线
Inhibition curves of AChE by different organophosphorus pesticides.
菌株Z23所产ALP对不同种类的有机磷农药均有降解作用(
ALP对不同浓度有机磷农药的降解作用
Degradation of organophosphorus pesticides by ALP at different concentrations
Pesticide types | Concentration of organophosphorus pesticides at different times (μg/mL) | ||||
0 h | 2 h | 4 h | 6 h | 8 h | |
注:不同小写字母表示差异显著( | |||||
DDVP | 1.250 | 0.963±0.012d | 0.535±0.037c | 0.328±0.026b | 0.062±0.019a |
2.500 | 1.726±0.054d | 1.084±0.032c | 0.626±0.085b | 0.107±0.040a | |
5.000 | 3.483±0.022d | 2.356±0.067c | 1.446±0.029b | 0.169±0.051a | |
Trichlorfon | 1.250 | 0.821±0.038d | 0.604±0.053c | 0.355±0.025b | 0.120±0.033a |
2.500 | 1.828±0.014d | 1.386±0.023c | 1.072±0.034b | 0.374±0.019a | |
5.000 | 3.766±0.031d | 2.469±0.024c | 1.603±0.052b | 0.862±0.066a | |
Chlorpyrifos | 1.250 | 0.946±0.016d | 0.788±0.014c | 0.523±0.035b | 0.174±0.042a |
2.500 | 1.857±0.011d | 1.243±0.033c | 0.757±0.028b | 0.268±0.015a | |
5.000 | 3.331±0.072d | 2.526±0.025c | 1.401±0.042b | 0.433±0.058a | |
Methyl parathion | 1.250 | 0.946±0.064d | 0.788±0.044c | 0.523±0.039b | 0.118±0.048a |
2.500 | 1.008±0.005d | 0.764±0.023c | 0.396±0.016b | 0.152±0.009a | |
5.000 | 1.802±0.038d | 1.334±0.065c | 0.708±0.029b | 0.621±0.055a | |
Malathion | 1.250 | 3.379±0.084d | 2.238±0.058c | 1.985±0.047b | 0.196±0.073a |
2.500 | 0.896±0.006d | 0.578±0.011c | 0.322±0.033b | 0.409±0.030a | |
5.000 | 1.786±0.025d | 1.295±0.054c | 0.934±0.071b | 0.601±0.052a | |
Dimethoate | 1.250 | 3.893±0.005d | 2.690±0.025c | 1.626±0.029b | 0.259±0.031a |
2.500 | 0.919±0.002d | 0.664±0.042c | 0.433±0.017b | 0.379±0.060a | |
5.000 | 1.897±0.028d | 1.393±0.045c | 0.827±0.030b | 0.729±0.024a |
从目前形势来看,在未来较长时间内,有机磷农药仍会被大量、广泛使用,而在其降解途径中,利用微生物或其产物来降解有机磷农药的方法会很大程度降低产生二次污染的可能,是消除和降解农药残留的一种安全、高效的方法[
通过单因素试验和正交试验结果确定,破碎时间对菌株Z23所产碱性磷酸酶活性影响最大,其次为破碎功率、提取液pH及料液比,郭宇星等的研究结果也表明超声时间是影响氨肽酶活性的最显著因素[
高熳熳等的研究结果表明,有机磷水解酶对敌敌畏与甲基对硫磷降解率高达90%以上,但对敌百虫的降解率仅为28.10%±2.20%[
综上所述,LGG Z23所产ALP的最优提取条件确定为:破碎时间15 min,破碎功率450 W,料液比1:6 (质量体积比),提取液pH值10.0,此提取条件下酶活力可达(4.95±0.26) U/mL,比优化前提高2.11倍。而且,LGG Z23所产ALP对6种有机磷农药的降解率均达到80%以上,其中对敌敌畏与甲基对硫磷的降解效果最好,降解率均高于90%。本研究结果为LGG Z23所产ALP的应用研究提供了理论依据和实验数据。
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