CN117089484A - 一种降解农药残留的芽孢粉及其制备方法 - Google Patents
一种降解农药残留的芽孢粉及其制备方法 Download PDFInfo
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Abstract
本发明提供了一种降解农药残留的芽孢粉及其制备方法,本发明首次将交联剂微生物转谷氨酰氨酶用于降解农药生物酶与枯草芽孢表面蛋白实现共价交联,固定化率高达86.91%,该共价交联得到交联孢子可提高交联的降解农药生物酶的活性和酶的热稳定性,且交联的多种降解农药生物酶以适应不同的复杂环境的应用,本发明采用的交联剂无毒无害,避免产生二次污染和化学污染,解决了现有技术的弊端,为解决土壤环境中有机农药环境残留问题提供了生物修复新方法。
Description
技术领域
本发明涉及农药残留清除领域,特别涉及一种降解农药残留的芽孢粉及其制备方法。
背景技术
农药残留是指在农业生产中施用农药后一部分农药直接或间接残存于谷物、蔬菜、果品、畜产品、水产品中以及土壤和水体中的现象。目前去除残留农药的方法主要包括物理去除法、化学去除法和生物酶类清除法,其中物理去除法效果较差,化学清除法容易造成二次污染,生物酶类清除法具有一定清除效果,安全无毒,对环境无污染,是目前主要研究方向。但天然游离酶存在一系列缺陷,例如溶液状态的OPH易降解失活、对温度和PH的变化敏感、催化条件对催化能力影响较大、贮存稳定性差、生产方式烦琐、不利于在自然环境甚至恶劣条件下使用等问题,限制了其实际应用。
酶固定化降解法具有酶稳定性高、可重复利用等优点受到了更广泛的应用。然而,传统的酶固定化工艺有着载体与酶结合位点不固定,具有随机性,存在载体与酶活性位点结合的可能,降低酶的活性,酶利用率低,会限制对有机磷农药的降解能力。
然而,通过枯草芽孢杆菌芽孢表面展示固定化技术可有效解决这一难题,该技术能够将外源蛋白展示在芽孢表面,并保持其相对独立的空间构想和原有的生物活性。借助芽孢优良的抗逆性,可提升外源蛋白的贮存稳定性,并适合在复杂条件中的应用,芽孢表面展示技术还避免了酶的分离纯化步骤,降低了生产成本和可重复利用效果。目前枯草芽孢杆菌芽孢表面展示技术现在逐渐发展为以基因重组和非基因重组2种锚定方式的表面展示策略。其中基因重组表达方式是以芽孢衣壳蛋白如CotB、CotC、CotE、CotG、CotZ和OxdD等作为锚定蛋白,将外源蛋白与芽孢衣壳蛋白进行融合表达展示在芽孢表面,专利《展示有机磷水解酶的枯草芽胞及其制备方法-CN110305824A》中选择CotG作为锚定蛋白,在枯草芽孢杆菌重组孢子上成功展示了有机磷水解酶OPH,增强了其对各种恶劣条件的抗性,重组孢子展现出有机磷水解酶的活性,但该发明存在引入抗性基因、芽孢表达催化活性低、表达外源蛋白单一等问题,其中芽孢展示低阳性率是导致有机磷水解酶活性催化活性低的根本原因。
非基因重组方式的芽孢表面展示是将细菌芽孢与纯化的外源蛋白一起进行培养,由于两者之间存在静电作用、疏水相互作用力结合,该非共价作用力易受环境因素(如表面活性剂、pH等)的影响而致酶蛋白与芽孢分离。而共价交联法是目前一种有效固定化酶、提高酶稳定性的策略。相关文章《The combinationofrecombinantandnon-recombinantBacillussubtilisspore displaytechnologyforpresentationofantigenandadjuvantonsingle spore.》介绍了利用化学交联剂戊二醛将外源蛋白锚定在芽孢表面,可形成蛋白质-蛋白质之间的共价交联的非基因重组表面展示,该方法交联效果好,但戊二醛为化学毒剂,易造成环境二次污染。文章《Covalentimmobilization ofrecombinantorganophosphorushydrolaseonsporesofBacillus subtilis.》中介绍使用1-(3-二甲氨基丙基)-3-乙基碳二亚胺和N-羟基硫代琥珀酰亚胺(EDC-NHS)作为化学交联剂,将有机磷水解酶共价锚定在芽孢表面,使得该酶在不同pH值和温度条件下的活性都显著提高,但EDC-NHS交联剂作为化学有机溶剂在制备过程中残留物较难去除,且交联度较低,制约了其制备的广泛应用。
综上所述,为解决上述现有技术存在的问题,因此寻找安全无毒、交联程度高、交联底物广泛的芽孢交联技术是解决现有技术弊端的关键,是研究降解农药残留固定酶的重要突破点。
技术方案
为解决现有技术的弊端,本发明提供了一种降解农药残留的芽孢粉及其制备方法。
为了实现上述目的,技术方案包括如下:
S1:枯草芽孢杆菌发酵产生芽孢。
S2:对S1制备的芽孢进行纯化。
S3:降解农药生物酶与芽孢发生交联反应。
S4:对S3制备的交联孢子通过真空冷冻干燥机干燥得到交联芽孢粉。
优选的,所述步骤S1所得到的芽孢浓度在8~9×107cfu/ml;
优选的,所述步骤S3交联的降解农药生物酶为有机磷水解酶、羧酸酯酶的一种或两种,酶活均达2万U/g。
优选的,所述步骤S3交联反应所用的交联剂为微生物转谷氨酰氨酶(MTG酶),酶活达120U/g。
优选的,所述步骤S3交联反应体系包括:1mg/ml干重的交联芽孢,1mg/ml的降解农药生物酶,1.2U/ml的MTG酶,混合于20mMPH7.5Tris-HCI缓冲溶液,总体积为20ml。
优选的,所述步骤S3交联反应条件为在25℃水浴锅水浴反应2h。
与现有技术相比,本发明提供了一种降解农药残留的芽孢粉及其制备方法,具备以下有益效果:
(1)本发明首次将交联剂微生物转谷氨酰氨酶用于降解农药生物酶与枯草芽孢表面蛋白实现共价交联,该共价交联得到交联孢子可提高交联的降解农药生物酶的活性和酶的热稳定性,且交联剂本身无毒无害,避免产生二次污染和化学污染,解决了现有技术的弊端。
(2)本发明采用微生物转谷氨酰氨酶交联剂,具有广泛的底物催化活性,可将芽孢表面蛋白交联多种降解农药生物酶,具有不同农药性质的降解效果,以适应不同的复杂环境的应用,解决了现有技术只能交联一种酶的局限性。
(3)与现有技术相比,本发明采用的交联体系和交联条件,使芽孢表面蛋白和降解农药生物酶充分发生交联反应,交联固定化程度高达86.91%,且在90℃孵育60min仍具有70%的酶活力。
(4)本发明制备的具有降解农药残留的芽孢粉制备简单,反应条件温和,在降解农药残留的同时,芽孢复苏后可分泌多种酶和抗生素,能够抑制土壤中病菌、害虫的滋生,为作物根系的健康生长提供良好的环境,是为解决土壤环境中农药残留问题和土传病害问题提供了生物修复新方法。
附图说明
图1为不同实施例固定化酶和游离酶的热稳定性对比图。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
参阅图1,一种降解农药残留的芽孢粉及其制备方法,包括枯草芽孢杆菌发酵产芽孢的制备、芽孢的纯化、农药降解生物酶与芽孢发生交联反应、真空冷冻干燥得到交联芽孢粉。
进一步的,所述制备枯草芽孢杆菌(Bacillussubtilis)购自中国农业微生物菌种保藏管理中心的微生物农药及菌肥的开发及应用菌种,保藏编号为ACCC60429;该枯草芽孢杆菌在恶劣环境下可产生芽孢抗逆休眠体,在适宜环境下,芽孢复苏后可以分泌多种酶和抗生素,常用作生物类型杀菌剂,能分解并抑制土壤中病菌、害虫的滋生,为作物根系的健康生长提供良好的环境。
进一步的,所述的农药降解生物酶为有机磷降解酶、羧酸酯酶的一种或两种。
进一步的,所述的有机磷降解酶(OPH)可催化有机磷化合物水解,包括甲基对硫磷、乙基对硫磷、对氧磷、毒死蜱、库马磷、氰磷、二嗪磷等有机磷化合物,是处理有机磷农药的重要生物酶。
进一步的,所述的羧酸酯酶(CarE)是一种多聚蛋白,属于酯酶家族,主要催化酯、硫酸酯和酰胺的水解,其作为降解农药的有效成分之一;
进一步的,所述农药降解生物酶与芽孢交联反应,交联剂采用的是微生物转谷氨酰氨酶(简称MTG),它是一种酰基转移酶的一种催化蛋白质间酰基转移反应,从而导致蛋白质之间发生共价交联的酶,这种交联提高蛋白质的性质、抵抗蛋白酶水解、提高凝胶能力、热稳定性和持水力等具有显著的影响,且催化底物具有广泛性,交联剂本身无毒无害,不产生二次污染;
进一步的,对交联后的芽孢分别表面展示有机磷水解酶和羧酸酯酶或同时展示该两种酶进行热稳定性验证。
下面将通过实施例进行进一步的说明:
实施例一:
S1:枯草芽孢杆菌芽孢的制备
取甘油活化保存的枯草芽孢杆菌在LB琼脂平板上划线,37℃培养24h,挑取长出的单菌落接种于50ml的LB液体培养基,200rpm,37℃摇瓶培养至OD600达到1.2-1.5,作为一级种子液;按3%的接种量将一级种子液转接至100ml的LB液体培养基,200rpm,37℃摇瓶培养至OD600达到1.5-1.8,作为二级种子液;按3%的接种量转接于5L发酵罐中,初始转速200rpm,风量0.3L/min,温度37℃,待生长对数期12-18h,转速慢慢调节至最大600rpm,风量慢慢调节最大1.0L/min,整个发酵过程罐压一直保持0.1Mpa,溶氧DO控制在20%,发酵至48h将培养温度提高至65℃继续培养30min后结束,得到枯草芽孢杆菌芽孢发酵液,通过显微镜计算孢子数,算出芽孢浓度为8.21×107cfu/ml,生孢率为67.36%。
所述发酵罐培养基:葡萄糖5g/dl、豆饼粉3.6g/dl、玉米粉2g/dl、磷酸氢二钠0.8g/dl、硫酸铵0.4g/dl,氯化铵0.13g/dl,PH6.5,121℃灭菌20min。
流加补料:准备50%的葡萄糖,121℃单独灭菌20min,糖补料方式为6h开始流加补料,控制整个发酵体系糖浓度在0.3-0.5g/dl。准备40%的硫酸铵,121℃单独灭菌20min,硫酸铵补料方式为3h开始添加补料,控制整个发酵体系氨氮浓度控制在0.5-1.0g/dl。
S2:芽孢纯化
将步骤S1制备的枯草芽孢杆菌芽孢发酵液,通过小型陶瓷膜浓缩设备(陶瓷膜孔径0.2um),将芽孢发酵液浓缩5倍,得到芽孢浓缩液,添加2mg/ml溶菌酶溶液37℃孵育30min,将芽孢浓缩液用离心机12000rpm离心去除上清液,再用去离子水重悬洗涤芽孢沉淀,洗涤三次,最后将芽孢重悬于去离子水中,再在烘干箱中通过55℃条件下干燥4h,得到纯化芽孢。
S3:有机磷水解酶与芽孢交联反应
将得到的芽孢、有机磷水解酶(酶活20000U/g)和MTG酶(120U/g)混合于20mMPH7.5Tris-HCI缓冲溶液,反应体系包括:1mg/ml的纯化芽孢,1mg/ml的有机磷水解酶,1.2U/ml的MTG酶,总体积为20ml,在25℃水浴锅水浴反应2h,12000rpm×10min离心得到交联芽孢和上清液,将得到的交联芽孢用去离子水洗涤两次,最后用去离子水重悬得到交联孢子悬液,分别测定交联孢子重悬液和上清液中游离的有机磷水解酶的酶活性,算出有机磷水解酶交联芽孢的固定化率为82.38%,每毫克交联芽孢含有的酶活为16.48U/mg。
S4:制备得到交联芽孢粉
将步骤S3交联后离心得到的交联芽孢,用去离子水洗涤两次,最后用去离子水重悬制备的交联孢子悬液,通过真空冷冻干燥机干燥得到交联芽孢粉,干燥过程为先将交联孢子悬液在-30℃预冷30min,在干燥箱中控制-30℃,真空度达到66Pa以下时,开始逐渐升温加速水分升华,最后干燥得到交联芽孢粉。
所述有机磷降解酶的酶活性测定:在2mL塑料EP管中,依次加入800ul测活缓冲液(包括778μl50mMTris–HCl(pH8.8)、2μl2mMCoCl2和20μl40mmol/L对氧磷甲醇溶液),800ul的交联孢子悬液或残余上清液。混合均匀后将所有EP管在37℃下反应10分钟。离心后取上清液,并使用2100分光光度计检测410nm处的吸光度。依据对硝基酚标准曲线计算交联孢子和上清液游离的有机磷水解酶的酶活力。有机磷水解酶酶活活性被定义为在37℃下每分钟释放1nmol对硝基苯酚的所需的酶量。交联芽孢的酶比活力定义为:每毫克交联芽孢的所含的酶活力单位(U/mg)。
实施例二:
S1:枯草芽孢杆菌芽孢的制备
取甘油活化保存的枯草芽孢杆菌在LB琼脂平板上划线,37℃培养24h,挑取长出的单菌落接种于50ml的LB液体培养基,200rpm,37℃摇瓶培养至OD600达到1.2-1.5,作为一级种子液;按3%的接种量将一级种子液转接至100ml的LB液体培养基,200rpm,37℃摇瓶培养至OD600达到1.5-1.8,作为二级种子液;按3%的接种量转接于5L发酵罐中,初始转速200rpm,风量0.3L/min,温度37℃,待生长对数期12-18h,转速慢慢调节至最大600rpm,风量慢慢调节最大1.0L/min,整个发酵过程罐压一直保持0.1Mpa,溶氧DO控制在20%,发酵至27h将培养温度提高至65℃继续培养30min后结束,得到枯草芽孢杆菌芽孢发酵液,通过显微镜计算孢子数,算出芽孢浓度为8.53×107cfu/ml,生孢率为68.23%。
所述发酵罐培养基:葡萄糖5g/dl、豆饼粉3.6g/dl、玉米粉2g/dl、磷酸氢二钠0.8g/dl、硫酸铵0.4g/dl,氯化铵0.13g/dl,PH6.5,121℃灭菌20min。
流加补料:准备50%的葡萄糖,121℃单独灭菌20min,糖补料方式为6h开始流加补料,控制整个发酵体系糖浓度在0.3-0.5g/dl。准备40%的硫酸铵,121℃单独灭菌20min,硫酸铵补料方式为3h开始添加补料,控制整个发酵体系氨氮浓度控制在0.5-1.0g/dl。
S2:芽孢纯化
将步骤S1制备的枯草芽孢杆菌WB800芽孢发酵液,通过小型陶瓷膜浓缩设备(陶瓷膜孔径0.2um),将芽孢发酵液浓缩5倍,得到芽孢浓缩液,添加2mg/ml溶菌酶溶液37℃孵育30min,将芽孢浓缩液用离心机12000rpm离心去除上清液,再用去离子水重悬洗涤沉淀,洗涤三次,最后将芽孢重悬于去离子水中,再在烘干箱中通过55℃条件下干燥4h,得到纯化芽孢。
S3:羧酸酯酶与芽孢交联反应
将得到的芽孢、羧酸酯酶(酶活20000U/g)和MTG酶(120U/g)混合于20mMPH7.5Tris-HCI缓冲溶液,反应体系包括:1mg/ml的纯化芽孢,1mg/ml的羧酸酯酶,1.2U/ml的MTG酶,总体积20ml,在25℃水浴锅水浴反应2h,12000rpm×10min离心得到交联芽孢和上清液,将得到的交联芽孢用去离子水洗涤两次,最后用去离子水重悬得到交联孢子悬液,分别测定孢子重悬液和上清液中游离的羧酸酯酶的酶活性,算出羧酸酯酶交联芽孢的固定化率为86.91%,算出每毫克交联芽孢含有的酶活为17.38U/mg。
S4:制备得到交联芽孢粉
将步骤S3交联后离心得到的交联芽孢,用去离子水洗涤两次,最后用去离子水重悬制备的交联孢子悬液,通过真空冷冻干燥机干燥得到交联芽孢粉,干燥过程为先将交联孢子悬液在-30℃预冷30min,在干燥箱中控制-30℃,真空度达到66Pa以下时,开始逐渐升温加速水分升华,最后干燥得到交联芽孢粉。
所述羧酸酯酶活性测定:检测方法采用羧酸酯酶(CarE)活性测定试剂盒(购自上海酶联生物科技有限公司)检测。具体操作步骤按照羧酸酯酶(CarE)活性测定试剂盒说明书操作。羧酸酯酶酶酶活定义:每毫克样品在37℃反应体系中每分钟催化吸光值增加1定义为1个酶活单位。交联芽孢的羧酸酯酶酶比活力定义为:每毫克交联芽孢的所含的酶活力单位(U/mg)。
实施例三:
S1:枯草芽孢杆菌芽孢的制备
取甘油保存的枯草芽孢杆菌在LB琼脂平板上划线,37℃培养24h,挑取长出的单菌落接种于50ml的LB液体培养基,200rpm,37℃摇瓶培养至OD600达到1.2-1.5,作为一级种子液;按3%的接种量将一级种子液转接至100ml的LB液体培养基,200rpm,37℃摇瓶培养至OD600达到1.5-1.8,作为二级种子液;按3%的接种量转接于5L发酵罐中,初始转速200rpm,风量0.3L/min,温度37℃,待生长对数期12-18h,转速慢慢调节至最大600rpm,风量慢慢调节最大1.0L/min,整个发酵过程罐压一直保持0.1Mpa,溶氧DO控制在20%,发酵至27h将培养温度提高至65℃继续培养30min后结束,得到枯草芽孢杆菌芽孢发酵液,通过显微镜计算孢子数,算出芽孢浓度为8.55×107cfu/ml,生孢率为66.15%。
所述发酵罐培养基:葡萄糖5g/dl、豆饼粉3.6g/dl、玉米粉2g/dl、磷酸氢二钠0.8g/dl、硫酸铵0.4g/dl,氯化铵0.13g/dl,PH6.5,121℃灭菌20min。
S2:芽孢纯化
将步骤S1制备的枯草芽孢杆菌WB800芽孢发酵液,通过小型陶瓷膜浓缩设备(陶瓷膜孔径0.2um),将芽孢发酵液浓缩5倍,得到芽孢浓缩液,添加2mg/ml溶菌酶溶液37℃孵育30min,将芽孢浓缩液用离心机12000rpm离心去除上清液,再用去离子水重悬洗涤沉淀,洗涤三次,最后将芽孢重悬于去离子水中,再在烘干箱中通过55℃条件下干燥4h,得到纯化芽孢。
S3:有机磷水解酶、羧酸酯酶共同与芽孢交联反应
将得到的芽孢、有机磷水解酶(酶活20000U/g)、羧酸酯酶(酶活20000U/g)和MTG酶(120U/g)混合于20mMPH7.5Tris-HCI缓冲溶液,反应体系包括:0.5mg/ml的纯化芽孢,0.5mg/ml的有机磷水解酶,1mg/ml的羧酸酯酶,1.2U/ml的MTG酶,总体积20ml,在25℃水浴锅水浴反应2h,12000rpm×10min离心得到交联芽孢和上清液,将得到的交联芽孢用去离子水洗涤两次,最后用去离子水重悬得到交联孢子悬液,分别测定交联孢子重悬液和上清液游离的有机磷水解酶和羧酸酯酶的酶活性,分别计算出有机磷水解酶和羧酸酯酶交联芽孢的固定化率,算出每毫克交联芽孢含有两种酶的比活力,结果如下表一所示。有机磷水解酶和羧酸酯酶活性测定分别同上述实施例一和实施例二。
表一:有机磷水解酶和羧酸酯酶同时交联芽孢的固定化率和比活力
交联酶 | 芽孢交联固定化率% | 比活力U/mg |
有机磷水解酶 | 81.26 | 8.13 |
羧酸酯酶 | 82.43 | 8.24 |
平均值 | 81.84 | 8.19 |
结果表明,芽孢同时交联有机磷水解酶和羧酸酯酶后,交联孢子均表现出两种酶的活性,且每种酶平均固定化率达81.84%,每种酶的比活力平均为8.19U/mg。
S4:制备得到交联芽孢粉
将步骤S3交联后离心得到的交联芽孢,用去离子水洗涤两次,最后用去离子水重悬制备的交联孢子悬液,通过真空冷冻干燥机干燥得到交联芽孢粉,干燥过程为先将交联孢子悬液在-30℃预冷30min,在干燥箱中控制-30℃,真空度达到66Pa以下时,开始逐渐升温加速水分升华,最后干燥得到交联芽孢粉。
实施例四:
为了验证上述实施例一、二、三得到交联孢子酶热稳定性,各将2ml交联孢子悬液放置于5mlEP管中,在不同温度下孵育60分钟,并评估残余活性,同时取2ml配置100U/ml浓度的游离形式存在的有机磷水解酶和羧酸酯酶作为对照实验。交联孢子的酶活性以初始酶活性为100%来计算剩余活性(其中实施例三交联孢子酶活以两种酶的酶活相加计算),游离的有机磷水解酶和羧酸酯酶活性以初始酶活性为100%来计算剩余活性。
结果如图1所示,游离的有机磷水解酶和羧酸酯酶随着温度的升高酶活力下降较快,在90℃孵育60分钟酶活力下降到了20%左右,酶活稳定性较差,而实施例一、二、三中得到的交联孢子,均随着温度的升高,酶活力下降较慢,在90℃孵育60分钟还能保持70%以上的酶活力,说明有机磷水解酶和羧酸酯酶可借助芽孢优良的抗逆性,可提升酶蛋白的贮存稳定性,可适合在不同复杂条件中应用。
本发明交联反应制备降解农药残留的芽孢粉制备简单,反应条件温和,在降解多种不同农药残留的同时,芽孢复苏后还可分泌多种酶和抗生素,能分解并抑制土壤中病菌、害虫的滋生,为作物根系的健康生长提供良好的环境,为解决土壤环境中有机磷农药环境残留问题和土传病害问题提供了生物修复新方法。
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由所附权利要求及其等同物限定。
Claims (7)
1.一种降解农药残留的芽孢粉的制备方法,包括以下步骤:
(1)枯草芽孢杆菌发酵产生芽孢;
(2)对制备的芽孢进行纯化;
(3)降解农药生物酶与芽孢发生交联反应。
(4)对制备的交联孢子通过真空冷冻干燥机干燥得到交联芽孢粉。
2.根据权利要求1所述一种降解农药残留的芽孢粉的制备方法,所述步骤(1)所得到的芽孢浓度在8~9×107cfu/ml。
3.根据权利要求1所述一种降解农药残留的芽孢粉的制备方法,所述步骤(3)交联的降解农药生物酶为有机磷水解酶、羧酸酯酶的一种或两种,酶活为2万U/g。
4.根据权利要求1所述一种降解农药残留的芽孢粉的制备方法,所述步骤(3)交联反应所用的交联剂为微生物转谷氨酰氨酶(MTG酶),酶活为120U/g。
5.根据权利要求1所述一种降解农药残留的芽孢粉的制备方法,所述步骤(3)交联反应体系包括:1mg/ml的芽孢,1mg/ml的降解农药生物酶,1.2U/ml的MTG酶,混合于20mMPH7.5Tris-HCI缓冲溶液,反应体积为20ml。
6.根据权利要求1所述一种降解农药残留的芽孢粉的制备方法,所述步骤(3)交联反应条件:在25℃水浴锅水浴反应2h。
7.根据权利要求1所述一种降解农药残留的芽孢粉的制备方法,所述步骤(4)得到的交联芽孢粉,具有有机磷水解酶、羧酸酯酶的一种或两种。
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