CN109971679A - 高效降解菌核净的单胞属菌酶剂的制备方法及应用 - Google Patents
高效降解菌核净的单胞属菌酶剂的制备方法及应用 Download PDFInfo
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Abstract
本发明公开了一种高效降解菌核净的单胞属菌酶剂的制备方法及应用,其以单胞属菌(Brevundimonas naejangsanensis)CCTCC M 2017738为菌种,经菌悬液扩繁和降解酶剂超声破碎提取制备而得单胞属菌降解酶剂。该降解酶剂可应用于高效降解油菜、大白菜、紫菜苔、生菜、韭菜、黄瓜、番茄、辣椒、马铃薯、烟草等植物体中以及土壤和水体中的菌核净、烯菌酮和异菌脲。具有速效性好、环境友好、效果显著、成本低廉和易于推广等优点。
Description
技术领域
本发明涉及生物技术降解农药残留领域,特别是一种高效降解菌核净的单胞属菌酶剂的制备方法及应用。
背景技术
农药在农业病虫害综合治理中发挥着重要作用,但农药残留及其代谢产物已成为严重污染物,对人类健康和生态系统构成威胁。菌核净(Dimetachlone),又称纹枯利,分子式C10H7Cl2NO2,其相对分子质量244.07,化学名为:N-(3,5-二氯苯基)丁二酰亚胺。在我国广泛用于油菜、大白菜、紫菜苔、生菜、韭菜、黄瓜、番茄、辣椒、马铃薯、烟草等作物上菌核病、纹枯病、赤星病等真菌病害的防治。尽管菌核净的杀菌活力前景看好,但由于健康问题,目前在美国和欧洲被禁止使用,但在一些发展中国家仍然广泛使用(如中国)。此外,以往的研究表明0.4mmol/L的菌核净是大鼠急性肾毒性物质,且其对哺乳动物内分泌具有干扰作用,对人体肾脏也有毒害作用。综上所述,农业中菌核净的大量使用会产生菌核净的暴露,这对人体健康和环境安全构成潜在威胁,消除菌核净污染物的生态友好和低成本策略迫切需要建立。
在许多国家,包括立法、农业标准化、农民教育和修复技术等多种措施来解决菌核净残留问题,虽然这些措施有助于去除农药残留,但菌核净均不能有效地去除。生物修复(Bioremediation)是一种利用活体微生物或来自微生物的酶对污染物进行解毒和降解,其具有操作简便、经济实用、环境友好等诸多优点的环境和经济有效的方法。迄今为止,尚无研究工作开展农产品中菌核净的生物降解。
发明内容
本发明针对菌核净降解技术缺乏,旨在提供一种高效降解菌核净的速效性好、环境友好、效果显著和成本低廉的单胞属菌酶剂。
本发明的技术方案如下:
该单胞属菌(Brevundimonas naejangsanensis)菌株的保藏编号为CCTCC M2017739,为现有菌株。本发明的技术方案是以单胞属菌(Brevundimonasnaejangsanensis)CCTCC M 2017739为菌种,经菌悬液扩繁和降解酶剂超声破碎提取制备而得单胞属菌降解酶剂。
其中,所述高效降解菌核净的单胞属菌酶剂制备方法包括如下步骤:
1)菌悬液制备:无菌条件下加入2mL无菌磷酸盐缓冲液于培养48h的菌种培养皿中平行振荡,并用接种环轻轻刮落培养基表面的菌落,即成粗制菌悬液,将其转移到液体发酵培养基(麦芽糖7g/L、酵母膏6g/L、NaCl 3g/L)中在pH为7.5~8,30~35℃,150~200rpm条件下培养48~56h。将取出的发酵液立即在8000~10000r/min(3~4℃)下离心10~15min,收集湿菌体,并用无菌磷酸盐缓冲液冲洗3次。然后将无菌磷酸盐缓冲液与湿菌体混匀,控制菌体数量为≥6×1010cfu/mL。制得的菌悬液3~4℃保存备用。
2)游离酶提取:对步骤1)制得的菌悬液进行超声破碎提取游离酶,超声破碎时间10~15min,功率320W,每10s一个周期(工作5s,间歇5s)。破碎液在8000~10000r/min,3~4℃下离心10~15min,得上清液即制得游离酶。上清液游离酶含量控制在大于等于3.00mg/mL。
本发明的单胞属菌酶剂可应用于高效降解油菜、大白菜、紫菜苔、生菜、韭菜、黄瓜、番茄、辣椒、马铃薯、烟草等植物体中以及土壤和水体中的菌核净。
进一步的,所述单胞属菌酶剂还可应用于高效降解植物、土壤和水体中的烯菌酮和异菌脲。
优选的,本发明的单胞属菌酶剂田间喷雾推荐用量为400~600mg/L。
本发明首次提供了一种高效降解菌核净的单胞属菌酶剂,具有速效性好、环境友好、效果显著和成本低廉等诸多优点。
附图说明
图1展示了游离酶和酶剂对菌核净的降解效果。
具体实施方式
下面通过实施例对本发明进一步具体描述。下述实施例中所涉及配料或材料,如无特殊说明,均为商业途径可获得。相关实验方法中如无特殊说明的均为本技术领域现有常规方法。其中的数值或数值比例,如无标注,均指质量数值或质量比例。
实施例1:
1.酶剂制备:
1)菌悬液制备:无菌条件下加入2mL无菌磷酸盐缓冲液于培养48h的菌种培养皿中平行振荡,并用接种环轻轻刮落培养基表面的菌落,即成粗制菌悬液,将其转移到液体发酵培养基(麦芽糖7g/L、酵母膏6g/L、NaCl 3g/L)中在pH为7.5~8,30~35℃,150~200rpm条件下培养48h。将取出的发酵液立即在10000r/min(3~4℃)下离心10min,收集湿菌体,并用无菌磷酸盐缓冲液冲洗3次。然后将无菌磷酸盐缓冲液与湿菌体混匀,控制菌体数量为≥6×108cfu/mL。制得的菌悬液3~4℃保存备用。
2)游离酶提取:对步骤1)制得的菌悬液进行超声破碎提取游离酶,超声破碎时间15min,功率320W,每10s一个周期(工作5s,间歇5s)。破碎液在10000r/min,3~4℃下离心10min,得上清液即制得游离酶。上清液游离酶含量为3.03mg/mL。
2.田间试验:
田间试验选在油菜田中进行,试验区域油菜分别喷洒有效成分为4.05和8.10kg/ha(比推荐高剂量高2倍和4倍)的40%菌核净可湿性粉剂,共4个处理(两个浓度的农药降解酶剂处理区,未用农药降解酶剂处理的对照区)。在田间试验中,天气晴朗,有时多云,7d内无降雨。菌核净使用24h后,每个区域分别用降解酶剂500mg/L进行处理,对照组用等量的水处理。每隔2h、12h、1、3、5和7d随机采集油菜植株样品。采用气相色谱法测定油菜中残留的菌核净。
3.测定方法:
Agilent6890N气相色谱仪,检测器:带μECD;色谱柱:HP-5石英毛细管柱(30m×0.32mm×0.25μm);进样口温度:260℃;检测器温度:280℃;柱箱温度:程序升温方式,初始温度200℃,保持1min,以10℃/min升至270℃,载气为高纯氮气,流速1.2mL/min;分流比为10:1;进样量:1μL。在上述条件下,以菌核净标准溶液浓度(x)为横坐标,峰面积(y)为纵坐标绘制标准曲线。线性方程为y=991.37x+11.136,相关系数为:R2=0.9998。
4.实施例效果:
实验前,检测显示两个农药剂量在油菜植株中的原始沉积量为3.05和5.98mg/kg。图1显示,相对于自然消解,降解酶剂能快速高效地降解菌核净。降解酶剂在1~3d内能快速降解油菜植株中的菌核净,5d后降解速率逐渐下降,这可能是游离酶快速接触菌核净的缘故致使开始具有快速的降解速率,之后由于酶逐渐失活而使降解速率减慢。3d内,降解酶剂对4.05和8.10kg/ha(比推荐高剂量高2倍和4倍)的40%菌核净可湿性粉剂降解率分别达92.46和86.43%,残留量分别为0.298和1.057mg/kg。7d内,降解酶剂对4.05和8.10kg/ha(比推荐高剂量高2倍和4倍)的40%菌核净可湿性粉剂降解率分别达99.26和90.13%,残留量分别为0.023和0.590mg/kg。表明本发明提供的单胞属菌酶剂能快速高效降解农产品中的菌核净,保障农产品质量安全。
当然,以上只是本发明的具体应用范例,本发明还有其他的实施方式,凡采用等同替换或等效变换形成的技术方案,均落在本发明所要求的保护范围之内。
Claims (4)
1.一种高效降解菌核净的单胞属菌酶剂的制备方法,其特征在于:其以单胞属菌Brevundimonas naejangsanensis为菌种,经菌悬液扩繁和降解酶剂超声破碎提取制备而得单胞属菌降解酶剂。
2.根据权利要求1所述的高效降解菌核净的单胞属菌酶剂的制备方法,其特征在于:包括如下步骤:
1)菌悬液制备:无菌条件下加入无菌磷酸盐缓冲液于菌种培养皿中平行振荡,并用接种环轻轻刮落培养基表面的菌落,即成粗制菌悬液,将其转移到液体发酵培养基中在pH为7.5~8,30~35℃,150~200rpm条件下培养48~56h;将取出的发酵液立即在8000~10000r/min下离心10~15min,收集湿菌体,并用无菌磷酸盐缓冲液冲洗3次;然后将无菌磷酸盐缓冲液与湿菌体混匀,控制菌体数量为≥6×1010cfu/mL;制得的菌悬液3~4℃保存备用。
2)游离酶提取:对步骤1)制得的菌悬液进行超声破碎提取游离酶,超声破碎时间10~15min,功率320W,每10s一个周期;破碎液在8000~10000r/min,3~4℃下离心10~15min,得上清液即制得游离酶;上清液游离酶含量控制在大于等于3.00mg/mL。
3.根据权利要求2所述的的高效降解菌核净的单胞属菌酶剂的制备方法,其特征在于:所述液体发酵培养基由以下原料按比例制得:麦芽糖7g/L、酵母膏6g/L、NaCl 3g/L。
4.一种如权利要求1或2的方法制备的高效降解菌核净的单胞属菌酶剂的应用,其特征在于:所述高效降解菌核净的单胞属菌酶剂应用于高效降解包括油菜、大白菜、紫菜苔、生菜、韭菜、黄瓜、番茄、辣椒、马铃薯、烟草在内的植物体中以及土壤和水体中的菌核净,或者应用于高效降解植物、土壤和水体中的烯菌酮和异菌脲。
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