CN110038249B - 一种促进除草剂降解的方法 - Google Patents

一种促进除草剂降解的方法 Download PDF

Info

Publication number
CN110038249B
CN110038249B CN201910331736.XA CN201910331736A CN110038249B CN 110038249 B CN110038249 B CN 110038249B CN 201910331736 A CN201910331736 A CN 201910331736A CN 110038249 B CN110038249 B CN 110038249B
Authority
CN
China
Prior art keywords
isoproturon
degradation
laccase
degradation rate
initial concentration
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201910331736.XA
Other languages
English (en)
Other versions
CN110038249A (zh
Inventor
刘芹
宋志波
孔维丽
袁瑞奇
孔维威
崔筱
段亚魁
胡素娟
康源春
张玉亭
韩玉娥
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institute of Plant Nutrition and Resource Environmentof of Henan Academy of Agricultural Sciences
Original Assignee
Institute of Plant Nutrition and Resource Environmentof of Henan Academy of Agricultural Sciences
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institute of Plant Nutrition and Resource Environmentof of Henan Academy of Agricultural Sciences filed Critical Institute of Plant Nutrition and Resource Environmentof of Henan Academy of Agricultural Sciences
Priority to CN201910331736.XA priority Critical patent/CN110038249B/zh
Publication of CN110038249A publication Critical patent/CN110038249A/zh
Application granted granted Critical
Publication of CN110038249B publication Critical patent/CN110038249B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A62—LIFE-SAVING; FIRE-FIGHTING
    • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
    • A62D3/00—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances
    • A62D3/02—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances by biological methods, i.e. processes using enzymes or microorganisms
    • A—HUMAN NECESSITIES
    • A62—LIFE-SAVING; FIRE-FIGHTING
    • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
    • A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
    • A62D2101/04—Pesticides, e.g. insecticides, herbicides, fungicides or nematocides
    • A—HUMAN NECESSITIES
    • A62—LIFE-SAVING; FIRE-FIGHTING
    • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
    • A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
    • A62D2101/20—Organic substances
    • A62D2101/26—Organic substances containing nitrogen or phosphorus
    • A—HUMAN NECESSITIES
    • A62—LIFE-SAVING; FIRE-FIGHTING
    • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
    • A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
    • A62D2101/20—Organic substances
    • A62D2101/28—Organic substances containing oxygen, sulfur, selenium or tellurium, i.e. chalcogen

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Toxicology (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Business, Economics & Management (AREA)
  • Emergency Management (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

Abstract

本发明公开了一种促进除草剂降解的方法，属于生物技术领域，该方法利用本实验纯化出的白灵菇菌渣漆酶降解除草剂异丙隆，采用单因素实验和响应面法研究异丙隆初始浓度、白灵菇菌渣漆酶活力及反应温度、pH值、反应体系体积对异丙隆降解率的影响，确定白灵菇菌渣漆酶降解异丙隆的最佳反应条件为：异丙隆初始浓度10mg/L，白灵菇菌渣漆酶活力300U/mL，反应温度为40℃，pH值为6.4，反应体系体积为5mL，此条件下理论降解率为78.27％。本发明反应条件温和，降解率高，具有很好的应用前景。

Description

一种促进除草剂降解的方法
技术领域
本发明属于生物技术领域，涉及一种促进除草剂降解的方法，具体地说，涉及一种促进除草剂异丙隆降解的方法。
背景技术
漆酶(Laccase，EC 1.10.3.2)，全称为对苯二酚：(双)氧氧化还原酶，又名酚酶、多酚氧化酶、漆酚氧化酶，其实质是一种含铜的氧化酶，和植物中的抗坏血酸氧化酶、动物中的血浆铜蓝蛋白同属于蓝色多铜氧化酶的家族。漆酶降解具有特异性、无二次污染及反应条件比较温和等特性，这使得漆酶在农药和染料的降解、造纸业、食品工业、能源和生物检测等方面具有广泛的应用。异丙隆是一种取代脲类选择性内吸传导型除草剂，具有适用作物范围广、除草谱宽且效果好，是我国应用最广、使用量最大的麦田除草剂之一。但是异丙隆的大量使用会造成水体和土壤的污染，并且通过食物链传递将污染范围扩大。利用漆酶降解环境中难降解的污染物-异丙隆，反应条件温和，能够避免出现再次污染，具有很大的应用前景。
发明内容
本发明的目的在于提供一种促进除草剂降解的方法。该方法利用一种新的白灵菇菌渣漆酶能够有效的降解除草剂异丙隆，降低其危害。
其具体技术方案为：
一种促进除草剂降解的方法，包括以下步骤：
步骤1、采用C18反相色谱柱高效液相色谱对异丙隆进行检测，检测波长为241nm；流动相为甲醇∶水＝70∶30V/V；流速0.6mL/min；进样量20μL，异丙隆初始浓度10mg/L；
步骤2、在离心管中加入pH值为6.37-6.4的白灵菇菌渣漆酶和步骤1的异丙隆溶液，白灵菇菌渣漆酶活力300U/mL，震荡均匀后在40℃下水浴反应，反应体系体积为4.95-5mL；
步骤3、将反应后的样品经过微滤处理按步骤1中的条件：C18反相色谱柱，检测波长241nm；流动相为甲醇：水＝70：30V/V；流速0.6mL/min；进样量20μL，进行高效液相色谱检测出反应后样品中残余异丙隆浓度。降解率的计算公式为：异丙隆降解率(％)＝100×(初始浓度-残余浓度)/初始浓度。
进一步，步骤1中，采用C18反相色谱柱为150mm×4.6mm，5μm。
进一步，步骤2中，所述pH值为6.4。
进一步，步骤2中，所述反应体系体积为5mL。
与现有技术相比，本发明的有益效果：
本发明的实验设计科学，漆酶原料是出过菇的白灵菇菌渣简单易得，且除草剂降解条件温和，能够避免出现再次污染，具有很大的应用前景。
漆酶是一类大的含铜多酚氧化酶，具有特异性的氧化还原位点，参与植物生长发育、调节植物对外源有毒物质如除草剂、重金属等环境胁迫响应具有重要的作用。异丙隆是一种内吸传导型的取代脲类除草剂，通过干扰光合作用II的电子传递，抑制光合作用而起到去除杂草的作用。有研究表明土壤中使用异丙隆后，微生物的生物量碳下降，且随着异丙隆浓度的增加，下降趋势愈发明显。持续施用异丙隆会导致小麦叶片失绿变白、枯叶较多，茎秆变细，严重药害时会导致麦苗枯死，从而导致小麦减产。异丙隆是非离子型的除草剂，极性较弱，通过羰基N的两个位点可在土壤中与阳离子进行交换，以配位体形式结合或者通过电荷作用被土壤颗粒表面吸附。异丙隆在土壤中向下渗透会污染地下水，并通过食物链传递会将污染范围扩大，水体和土壤中的异丙隆难降解，毒性大，治理的难度也大。异丙隆还会和Hg、Cd、Cr等重金属形成毒性更大的金属复合物，对环境的污染更大。本发明利用白灵菇菌渣漆酶在24h内对异丙隆的降解率可以达到78.27％，反应条件温和，降解率高，具有很好的应用前景。
附图说明
图1是pH值对异丙隆降解率的影响；
图2是温度对异丙隆降解率的影响；
图3是反应体系体积对异丙隆降解率的影响；
图4是响应面立体分析图，其中，a、b、c分别为体积-温度，温度-pH，体积-pH对异丙隆降解的相互作用。
具体实施方式
下面结合附图和实施例对本发明的技术方案作进一步详细地说明。
一、本发明所用的原料的来源如下：
白灵菇菌渣漆酶(实验室纯化)；甲醇(色谱纯)，上海国药集团化学试剂有限公司；异丙隆(纯度97％)，南通先正达公司。
1220型高效液相色谱仪，美国安捷伦公司；BS-124S型电子天平，德国赛多利斯公司；HH-4型数显恒温水浴锅，国华电器有限公司；PHS-320型多功能酸度计，成都世纪方舟科技有限公司。
二、本发明所所采取的的实验方法如下：
1、异丙隆测定条件
采用C18反相色谱柱(150mm×4.6mm，5μm)高效液相色谱对异丙隆进行检测，检测波长为241nm；流动相为甲醇∶水＝70∶30(V/V)；流速0.6mL/min；进样量20μL。
2、异丙隆的降解实验
在离心管中分别加入同样体积的一定pH值的白灵菇菌渣漆酶样品和异丙隆溶液，震荡均匀后在一定温度条件下水浴反应，每组实验设置三个重复。对照组中，用热灭活的漆酶样品代替漆酶样品。将反应后的样品经过微滤处理按条件1进行高效液相色谱检测出反应后样品中残余异丙隆浓度。降解率的计算公式为：异丙隆降解率(％)＝100×(初始浓度-残余浓度)/初始浓度。
3、单因素实验
分别对异丙隆初始浓度、白灵菇菌渣漆酶活力及反应时间、温度、pH值及反应体系体积进行单因素实验，研究这5个因素对异丙隆降解率的影响，分析得出影响异丙隆降解的主要因素为反应温度、pH值及反应体系体积(见图1-图3)。根据实验结果和经济成本确定其他因素如异丙隆初始浓度、白灵菇菌渣漆酶活力和反应时间分别为10mg/L、300U/mL和24h。
4、响应面法实验的设计
在单因素实验的基础上，选择A pH值、B温度、C反应体系体积3个因素所确定的水平范围(表1)，利用Design-expert软件进行响应面实验设计并以异丙隆降解率作为响应值，对Box-Benhnken设计实验结果(表2)进行方差分析及二次多相回归拟合(表3)，得到异丙隆降解对pH值、温度及反应体系体积的多元回归方程：异丙隆降解率(％)＝75.27+2.89*A+3.96*B+3.51*C-0.33*A*B+1.13*A*C-1.25*B*C-7.85*A2-1.45*B2-3.85*C2。
表1响应面试验因素水平表
Figure BDA0002037792440000051
表2 Box-Benhnken实验设计及结果
Figure BDA0002037792440000052
Figure BDA0002037792440000061
表3响应面分析试验方差分析结果
Figure BDA0002037792440000062
由表3可知，整体模型的Prob＞F值＜0.001，表明该二次方程模型极显著。模型失拟项表示的是模型预测值与实际值不拟合的概率，该模型失拟项Prob＞F值小于0.0001，说明预测值与实际值不拟合的概率小于0.01％，说明该方程对实验拟合较好。相关系数R2＝0.9654，Radj 2＝0.9209，说明96.54％的数据可以用此方程来解释，该模型相关度较好。并且B、A2对异丙隆的降解率影响极显著，A、C、、C2对异丙隆的降解率影响显著。对异丙隆降解率影响大小的因素顺序依次为：B(温度)＞C(体积)＞A(pH值)。
根据回归分析结果，作出相应曲面图4。可见反应温度在各项因素中起到极显著的影响，其余单因素为显著。在考察的变量水平范围内，白灵菇菌渣漆酶对异丙隆的降解率在一定范围内随反应温度、pH值及反应体系体积增大而增大，当其水平越过一定值后，异丙隆降解率随之降低。
5、最佳工艺条件的预测与检验
利用Design-Expert 8软件对试验模型进行预测，得到最佳的降解条件为：温度为40℃，pH值为6.37，反应体系体积为4.98mL，此条件下预测为78.3908％。结合实际，调整各因素为：温度为40℃，pH值为6.4，反应体系体积为5.0mL，异丙隆初始浓度10mg/L，白灵菇菌渣漆酶活力300U/mL，反应时间24h。在此条件下进行3次平行实验验证，最后得到的异丙隆降解率为78.27％。
6、结论
通过单因素实验和Box-Behnken实验设计以及响应面分析法，得出白灵菇菌渣漆酶对异丙隆的最佳降解条件为：温度为40℃，pH值为6.4，反应体系体积为5.0mL，在此条件下检测的异丙隆降解率为78.27％，反应条件温和，简单易操作且能较高效率的促进除草剂异丙隆的降解。
以上所述，仅为本发明较佳的具体实施方式，本发明的保护范围不限于此，任何熟悉本技术领域的技术人员在本发明披露的技术范围内，可显而易见地得到的技术方案的简单变化或等效替换均落入本发明的保护范围内。

Claims (1)

1.一种促进除草剂降解的方法，其特征在于，包括以下步骤：
步骤1、采用150mm×4.6mm，5μm的C18反相色谱柱高效液相色谱对异丙隆进行检测，检测波长为241nm；流动相为甲醇∶水＝70∶30；流速0.6mL/min；进样量20μL，异丙隆初始浓度10mg/L；
步骤2、在离心管中加入pH值为6.4的白灵菇菌渣漆酶和步骤1的异丙隆溶液，白灵菇菌渣漆酶活力300U/mL，震荡均匀后在40℃下水浴反应24h，反应体系体积为5mL；
步骤3、将反应后的样品经过微滤处理按步骤1中的条件：C18反相色谱柱，检测波长241nm；流动相为甲醇∶水＝70∶30；流速0.6mL/min；进样量20μL，进行高效液相色谱检测出反应后样品中残余异丙隆浓度，降解率的计算公式为：异丙隆降解率＝100×(初始浓度-残余浓度)/初始浓度。
CN201910331736.XA 2019-04-24 2019-04-24 一种促进除草剂降解的方法 Active CN110038249B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910331736.XA CN110038249B (zh) 2019-04-24 2019-04-24 一种促进除草剂降解的方法

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910331736.XA CN110038249B (zh) 2019-04-24 2019-04-24 一种促进除草剂降解的方法

Publications (2)

Publication Number Publication Date
CN110038249A CN110038249A (zh) 2019-07-23
CN110038249B true CN110038249B (zh) 2023-05-16

Family

ID=67278871

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910331736.XA Active CN110038249B (zh) 2019-04-24 2019-04-24 一种促进除草剂降解的方法

Country Status (1)

Country Link
CN (1) CN110038249B (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113558175A (zh) * 2021-08-10 2021-10-29 赞皇县光森中药材有限公司 一种酸枣仁中黄曲霉毒素的降解方法及装置

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101942421B (zh) * 2010-10-09 2011-10-05 江南大学 一种竹黄菌发酵生产漆酶的方法
CN107686169A (zh) * 2017-08-31 2018-02-13 上海交通大学 蛋白质介体‑漆酶体系降解双酚a的方法
CN108132323A (zh) * 2017-12-11 2018-06-08 常州合全药业有限公司 硼酸酯类化合物的非水反相色谱检测方法

Also Published As

Publication number Publication date
CN110038249A (zh) 2019-07-23

Similar Documents

Publication Publication Date Title
Nguyen et al. The effects of soil phosphorus and zinc availability on plant responses to mycorrhizal fungi: a physiological and molecular assessment
Ren et al. Effects of plant growth-promoting bacteria (PGPB) inoculation on the growth, antioxidant activity, Cu uptake, and bacterial community structure of rape (Brassica napus L.) grown in Cu-contaminated agricultural soil
Hmid et al. Olive mill waste biochar: a promising soil amendment for metal immobilization in contaminated soils
Hammed et al. Growth regime and environmental remediation of microalgae
Li et al. Effects of arsenic on seed germination and physiological activities of wheat seedlings
Li et al. The rice aquaporin Lsi1 mediates uptake of methylated arsenic species
Fernandes et al. Effect of sewage sludge on microbial biomass, basal respiration, metabolic quotient and soil enzymatic activity
Ali et al. Phytoremediation of cadmium improved with the high production of endogenous phenolics and free proline contents in Parthenium hysterophorus plant treated exogenously with plant growth regulator and chelating agent
Mukhtar et al. Potential of sunflower (Helianthus annuus L.) for phytoremediation of nickel (Ni) and lead (Pb) contaminated water
Eissa et al. Growth and biochemical changes in quail bush (Atriplex lentiformis (Torr.) S. Wats) under Cd stress
Lin et al. Accumulation of copper by roots, hypocotyls, cotyledons and leaves of sunflower (Helianthus annuus L.)
Li et al. Integrated metabolomic and proteomic approaches dissect the effect of metal-resistant bacteria on maize biomass and copper uptake
Xiao et al. Characterization of cometabolic degradation of p-cresol with phenol as growth substrate by Chlorella vulgaris
Abalos et al. Trade-offs in greenhouse gas emissions across a liming-induced gradient of soil pH: Role of microbial structure and functioning
Puckett et al. Differential expression of genes encoding phosphate transporters contributes to arsenic tolerance and accumulation in shrub willow (Salix spp.)
Lorenc-Plucińska et al. Capabilities of alders (Alnus incana and A. glutinosa) to grow in metal-contaminated soil
Cao et al. Effects of Cu 2+ and Zn 2+ on growth and physiological characteristics of green algae, Cladophora
Schilling et al. Isotope fractionation of selenium during fungal biomethylation by Alternaria alternata
Marino et al. Inhibition of nitrogen fixation in symbiotic Medicago truncatula upon Cd exposure is a local process involving leghaemoglobin
Suzuki et al. The detection of endogenous 2’-deoxymugineic acid in olives (Olea europaea L.) indicates the biosynthesis of mugineic acid family phytosiderophores in non-graminaceous plants
Ma et al. Jasmonic acids facilitate the degradation and detoxification of herbicide isoproturon residues in wheat crops (Triticum aestivum)
CN110038249B (zh) 一种促进除草剂降解的方法
Hatano et al. Molasses melanoidin-like products enhance phytoextraction of lead through three Brassica species
Wang et al. Removal effects of different emergent-aquatic-plant groups on Cu, Zn, and Cd compound pollution from simulated swine wastewater
Zhao et al. Inter-and intra-specific competition of duckweed under multiple heavy metal contaminated water

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant