CN109055288B - 一种重组枯草芽孢杆菌及其应用 - Google Patents
一种重组枯草芽孢杆菌及其应用 Download PDFInfo
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- CN109055288B CN109055288B CN201810703969.3A CN201810703969A CN109055288B CN 109055288 B CN109055288 B CN 109055288B CN 201810703969 A CN201810703969 A CN 201810703969A CN 109055288 B CN109055288 B CN 109055288B
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- bacillus subtilis
- polymyxin
- polypeptide
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- recombinant
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Abstract
本发明公开了一种重组枯草芽孢杆菌及其在降解多肽类抗生素中的应用,所述工程菌是将SEQ ID NO.1所示基因片段与线性化pWBUC01载体连接后转入枯草芽孢杆菌获得的。本发明中降解多肽类抗生素的枯草芽孢杆菌工程菌能有效快速地去除水体环境中的多肽类抗生素,与传统化学降解、物理吸附相比较具有安全、高效、节能等优点。本发明枯草芽孢杆菌WB800/pWBUC01‑apr能够有效降解受污染淤泥中的多粘菌素残留,16000U/mL的多粘菌素在8天时间基本降解完全,而含有空载pWBUC01的WB800对多粘菌素降解率仅为38%左右。
Description
(一)技术领域
本发明涉及一种用于多肽类抗生素降解的枯草芽孢杆菌工程菌的构建及其应用。
(二)背景技术
抗生素是一种低微浓度就能干扰或抑制其他生物生理机能的化学物质。现临床常用的抗生素有微生物培养液中提取物以及用化学方法合成或半合成的化合物。它的发现和应用,在人类医疗健康和动植物疾病防治等方面发挥了巨大的作用。但是由于抗生素在生物体内存留时间短、代谢率效低等原因,大部分抗生素都以原药或代谢物形式随粪便排放环境中,造成了环境的严重污染,进而危及到人类健康。多肽类抗生素是具有多肽结构特征的一类抗生素,包括多粘菌素类(多粘菌素B、多粘菌素E)、杆菌肽类(杆菌肽、短杆菌肽)等,对一些真菌或病毒引起的感染有较好的治疗效果。此类抗生素由于能有效预防动物疾病和明显促进动物生长等原因在畜牧养殖业中被广泛应用。但是随着其大量的生产和应用,抗生素残留引发的污染问题更加严峻,环境中耐药菌越来越多。
制药企业硫酸多粘菌素提炼收率在85%~90%的范围内,其中10%~15%的损失包括提取过程操作损失,以及提取不彻底的工艺上的必然损失,比如发酵液的过滤工序,无论如何处理发酵液,过滤菌渣内都会残留一定量的多粘菌素;树脂解析过程中,解析尾液都不可能全部回收;这些生产工业上的硫酸多粘菌素的损失都是造成抗生素环境污染的一部分来源。近些年来,在土壤、水体以及动植物性食品中频繁检测出多肽类抗生素残留。随着国家对环境抗生素残留监测力度的加大,规范使用要求的提高,如何有效去除环境中残留的多粘菌素类抗生素已成为当下亟待解决的严峻问题。
减少抗生素的使用是解决抗生素污染的根本途径,去除抗生素的残留是解决抗生素污染的重要途径。微生物降解是抗生素类药物在土壤或水体环境中迁移、转化和消失的重要途径。相比较于传统的物理、化学方法,生物降解抗生素具有无毒害、无残留、无二次污染和作用时间短等优点,是有效解决环境中抗生素残留的安全、高效、廉价的方法。
目前,国内外近50多年来的文献分析显示当前国内外对于多肽类抗生素的研究工作主要在药物代谢动力学、药效学分析、药物纯化和耐药检测等方面,关于生物降解多肽类抗生素的研究却鲜有报道。
(三)发明内容
本发明目的是克服现有技术的不足,提供一种高效降解多肽类抗生素的工程菌,以及在有效去除环境中多肽类抗生素污染物中的应用,解决多粘菌素E在生产厂和环境中的残留。
本发明采用的技术方案是:
本发明提供一种重组枯草芽孢杆菌,所述工程菌是将SEQ ID NO.1所示基因片段(编码蛋白的氨基酸序列为SEQ ID NO.2所示)与线性化pWBUC01载体连接后转入枯草芽孢杆菌获得的。
进一步,所述枯草芽孢杆菌为枯草芽孢杆菌(Bacillus subtilis)WB800(购于上海北诺生物科技有限公司)。
进一步,所述重组枯草芽孢杆菌为枯草芽孢杆菌(Bacillus subtilis)WB800/pWBUC01-apr,保藏于中国典型培养物保藏中心,保藏日期2017年11月27日,保藏编号为:CCTCC NO:M 2017732,保藏地址为中国武汉,武汉大学,邮编430072。
本发明还提供一种所述的重组枯草芽孢杆菌在降解多肽类抗生素中的应用,所述多肽类抗生素为多粘菌素E或杆菌肽。
进一步,所述应用方法为:将重组枯草芽孢杆菌(枯草芽孢杆菌WB800/pWBUC01-apr)经发酵培养获得的湿菌体用pH为5~10(优选7.5)磷酸缓冲溶液清洗后悬浮配制成OD600为0.4~5.0(优选2.0)的菌悬液;将菌悬液以体积浓度2-16%(优选8%)的接种量接种至含终浓度为5000~15000U/mL(优选10000U/mL)多肽类抗生素的LB液体培养基中,在25~50℃、50~250r/min条件下培养(优选37℃、200r/min),获得多肽类抗生素降解的培养液;所述LB液体培养基组成为:酵母粉1.0~10.0g/L、胰蛋白胨2.0~25.0g/L、氯化钠2.0~25.0g/L,溶剂为去离子水,pH 5~10(优选酵母粉5.0g/L、胰蛋白胨10.0g/L、氯化钠10.0g/L,溶剂为去离子水,pH7.0~7.2)。
进一步,所述湿菌体按如下方法制备:
将重组枯草芽孢杆菌(WB800/pWBUC01-apr)接种至含5~100μg/mL(优选50μg/mL)卡那霉素固体培养基,于25~50℃倒置培养12~72h(优选37℃培养24h)后,挑选单菌落接种在LB液体培养基中,25~50℃、50~250r/min培养12~72h(优选37℃、200r/min培养24h),获得种子液;随后按体积浓度2~16%的接种量接种至LB液体培养基,25~50℃、50~250r/min培养12~72h(优选8%接种量扩大培养48h),取培养结束后的菌液在4000~12000r/min的转速下离心2~20min(优选8000r/min、5min)后,弃上清液,收集湿菌体;LB固体培养基终浓度组成:酵母粉1.0~10.0g/L、胰蛋白胨2.0~25.0g/L、氯化钠2.0~25.0g/L,琼脂15.0~25.0g/L,溶剂为去离子水,pH5~10。
本发明还提供一种所述的重组枯草芽孢杆菌在修复多肽类抗生素污染水体中的应用,所述的应用是将重组枯草芽孢杆菌(WB800/pWBUC01-apr)经发酵培养获得的湿菌体用pH为5~10(优选7.5)的磷酸缓冲溶液清洗后悬浮配制成OD600为0.4~5.0(优选2.0)的菌悬液;将菌悬液以体积浓度2-16%(优选8%)的接种量接种至含多肽类抗生素的水体中,在25~50℃、50~250r/min(优选30℃、200r/min)条件下培养,获得多肽类抗生素降解的培养液,实现污染水体中多肽类抗生素的降解,修复多肽类抗生素污染水体;所述多肽类抗生素污染水体中多肽类抗生素终浓度为5000~20000U/mL(优选160000U/mL),所述多肽类抗生素为多粘菌素E或杆菌肽。
本发明所述多肽类抗生素降解的培养液中多肽类抗生素检测方法为:将培养液离心,取上清液作为待测菌液;以OD600值为1.0的大肠杆菌菌液为指示菌,以pH6.0的灭菌磷酸缓冲液配制的浓度5000U/mL的多肽类抗生素为校正液进行抑菌圈直径测量,4℃条件下静置12h后,转移到37℃培养箱中培养到出现明显的抑菌圈,测定抑菌圈大小,根据多肽类抗生素标准曲线计算待测菌液中多粘菌素含量,获得多肽类抗生素降解率;所述多肽类抗生素标准曲线是在待测菌液检测相同条件下,以不同浓度梯度的多肽类抗生素标准品浓度为横坐标,以抑菌圈直径为纵坐标制成。
所述多肽类抗生素标准曲线按如下方法制备:用pH6.0磷酸缓冲液分别配制2000U/mL、4000U/mL、6000U/mL、8000U/mL和10000U/mL的多肽类抗生素(优选多粘菌素E)标准溶液,同时配制5000U/mL的多肽类抗生素标准溶液作为校正液;以大肠杆菌为指示菌,将灭菌后的LB固体培养基冷却至60℃时,加入体积浓度为5%,OD600值为1.0的大肠杆菌菌液混匀倒平板;待平板冷却后在对角放置四个牛津杯,一组对角牛津杯中加入体积200μL、浓度5000U/mL的多肽类抗生素校正液,另一组对角牛津杯中加入不同浓度梯度的多肽类抗生素标准溶液;4℃条件下静置12h后,转移到37℃培养箱培养到出现明显的抑菌圈,测量记录抑菌圈大小;以多肽类抗生素标准溶液浓度为横坐标,以抑菌圈直径为纵坐标制成多肽类抗生素标准曲线。
本发明大肠杆菌-枯草芽孢杆菌穿梭质粒pWBUC01含有能够在枯草芽孢杆菌中发挥作用的复制起始位点和卡那霉素抗性基因(Kan),以及能在大肠杆菌中发挥作用的复制起始位点和氨苄青霉素抗性基因(Amp)。
本发明相对于现有的技术,具有如下的优点和效果:
本发明中降解多肽类抗生素的枯草芽孢杆菌工程菌能有效快速地去除水体环境中的多肽类抗生素,与传统化学降解、物理吸附相比较具有安全、高效、节能等优点。本发明枯草芽孢杆菌WB800/pWBUC01-apr能够有效降解受污染淤泥中的多粘菌素残留,16000U/mL的多粘菌素在8天时间基本降解完全,而含有空载pWBUC01的WB800对多粘菌素降解率仅为38%左右。
(四)附图说明
图1是重组穿梭质粒pWGPapr构建示意图。
图2是多粘菌素E效价对数与抑菌圈直径大小关系标准曲线图。
图3是重组芽孢杆菌WB800dc的生长对多粘菌素E降解的影响曲线图。
图4是重组芽孢杆菌WB800dc对污染环境中多粘菌素的降解曲线图。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此:
下述实施例中的方法,如无特殊说明,都为常规方法;所用合成引物均购自擎科生物,大肠杆菌-枯草芽孢杆菌穿梭质粒pWBUC01(专利申请号201710873809.9),地衣芽孢杆菌(Bacillus licheniformis)ATCC 14580以及枯草芽孢杆菌WB800购于上海北诺生物科技有限公司。
实施例1:重组穿梭质粒pWGPapr的构建
1、多肽类抗生素降解酶基因片段papr的PCR扩增。
以地衣芽孢杆菌ATCC 14580基因组为模板,以含有限制性酶切位点的PaprF和PaprR为引物,PCR扩增多肽类抗生素降解酶基因目的片段papr,扩增产物通过1%琼脂糖凝胶电泳检测,PCR产物大小为1539bp(大小为1539bp,包括启动子部分,序列如SEQ ID NO.1所示)。纯化后使用限制性内切酶Xho I和BamH I(大连TaKaRa公司)进行双酶切,纯化回收后备用。
上游引物(PaprF:5’-CCG CTCGAGATCTTTCACCCGTTTCTG-3’,下划线为Xho I酶切位点);
下游引物(PaprR:5’-CGCGGATCCTTATGGAGCGGCAGCTTC-3’,下划线为BamH I酶切位点)。
PCR反应体系(50μL):DNA模板2μL,2×phanta Max Master Mix 25μL,引物PaprF2μL,引物PaprR 2μL,无菌水19μL。
PCR程序:95℃3min;95℃15sec,52℃15sec,72℃60sec,35个循环;72℃5min。
2、线性化pWBUC01载体的获得。
以pWBUC01质粒为模板,以含有限制性酶切位点的WG01F和WG01R为引物,采用反向PCR法扩增除启动子和信号肽以外的载体片段,扩增产物通过1%琼脂糖凝胶电泳检测,PCR扩增产物大小为5975bp,为避免微量的模板质粒影响后续实验,在PCR扩增产物中加入限制性内切酶Dpn I消除模板质粒,然后使用限制性内切酶Xho I和BamH I进行双酶切,纯化回收后备用,记为线性化载体pWG01。
正向引物(WG01F:5’-CCGCTCGAGTTCAGTGCCGACCAAAACCA-3’,下划线为Xho I酶切位点);
反向引物(WG01R:5’-CGCGGATCCTCCGCTCACAATTCCACACA-3’,下划线为BamHI酶切位点)。
PCR反应体系(50μL):质粒模板2μL,2×phanta Max Master Mix 25μL,引物WG01F2μL,引物WG01R 2μL,无菌水19μL。
PCR程序:95℃30sec;95℃15sec,60℃15sec,72℃4min,35个循环;72℃5min。
3、连接和转化
将酶切后的基因片段papr与线性化载体pWG01通过T4DNA连接酶(大连TaKaRa公司)连接,按《分子克隆实验指南》将连接产物转化至100μL大肠杆菌DH5α感受态细胞,在氨苄青霉素抗性100μg/mL的LB平板上对单克隆进行筛选,通过菌落PCR和测序验证后得到含有重组穿梭质粒pWGPapr(图1)的大肠杆菌DH5α阳性转化子。
实施例2:含有重组穿梭质粒pWGPapr的重组枯草芽孢杆菌的构建。
利用质粒抽提试剂盒(生工生物工程(上海)股份有限公司)从实施例1构建的含有重组穿梭质粒pWGPapr的大肠杆菌DH5α阳性转化子中抽提重组穿梭质粒pWGPapr,采用电转化方法,在电压21kV/cm,时间常数=5ms的条件下,将重组穿梭载体pWGPapr转化枯草芽孢杆菌WB800菌株,在50μg/mL卡那霉素抗性LB平板上进行筛选,通过菌落PCR和测序验证后得到含重组穿梭质粒pWGPapr的枯草芽孢杆菌(Bacillus subtilis)WB800/pWBUC01-apr,即为降解多肽类抗生素的枯草芽孢杆菌WB800dc,该菌株于2017年11月27日保藏于中国典型培养物保藏中心,保藏编号为:CCTCCNO:M 2017732,保藏地址为湖北省武汉市洪山区八一路珞珈山。
为了使得本发明上述实施例2中的降解多肽类抗生素的工程菌WB800dc能得到更好的应用,本发明还在上述实施例2的基础上提供了实施例3,实施例3中以多肽类抗生素中的代表性物质多粘菌素E作为探究对象,提供了一种用于多肽类抗生素降解的降解工程菌WB800dc生物菌剂的应用。
实施例3:枯草芽孢杆菌WB800dc对多肽类抗生素多粘菌素E降解能力的初步研究。
1、种子菌悬液的制备:
LB液体培养基:酵母粉5.0g/L、胰蛋白胨10.0g/L、氯化钠10.0g/L,溶剂为去离子水,pH 7.0~7.2,装液50mL/250mL三角瓶,121℃灭菌20min。
LB固体培养基:酵母粉5.0g/L、胰蛋白胨10.0g/L、氯化钠10.0g/L,琼脂20.0g/L,溶剂为去离子水,pH 7.0~7.2,装液100mL/250mL三角瓶,121℃灭菌20min。
将枯草芽孢杆菌WB800dc菌体划线接种在含50μg/mL卡那霉素的LB固体培养基上活化,于37℃倒置培养24h后,挑选单菌落接种在LB液体培养基中,37℃、200r/min培养24h进一步活化;随后按照体积浓度8%的接种量接种至LB液体培养基进行扩大培养48h,取培养结束后的菌液在8000r/min离心5min后,弃上清液,沉淀菌体用pH为7.5的磷酸缓冲溶液清洗1~3次,配置成OD600为2.0的菌悬液。
2、枯草芽孢杆菌WB800dc对多粘菌素E降解能力的定量分析:
由于多粘菌素E是对菌体抑制能力很强的抗生素,若以其作为唯一底物培养则很不利于菌体生长,因此不宜用营养贫乏的培养基。在LB液体培养基中加入终浓度为10000U/mL的多粘菌素E,按照体积浓度8%的接种量接种OD600为2.0的菌悬液,对照组为接入等体积的磷酸缓冲液,保持恒温振荡培养箱的温度和振荡速率分别为37℃、200r/min,每隔6h取样测定多粘菌素E的残留浓度。
采用标准曲线法(又称剂量法)测定多粘菌素E残留浓度:
将待测菌液在4℃、8000r/min离心10min,弃菌体沉淀,保存发酵上清液。以大肠杆菌DH5α为指示菌,将灭菌后的固体培养基冷却至60℃时,加入体积浓度为5%,OD600值为1.0的大肠杆菌菌液混匀倒平板;待平板冷却后在对角放置四个牛津杯,一组对角牛津杯中加入体积100μL、浓度5000U/mL的多粘菌素E校正液(pH6.0的灭菌磷酸缓冲液配制),另一组对角牛津杯中加入相同体积的发酵上清液。把加好溶液的平板放置4℃条件下静置12h后,转移到37℃培养箱中培养直到出现明显的抑菌圈;用游标卡尺测定抑菌圈大小,根据标准曲线计算多粘菌素E残留量。结果如图3所示,随着实验组中菌株生物量的增加,多粘菌素E发生了快速降解,表明所述枯草芽孢杆菌WB800dc有高效的多粘菌素E降解能力,而对照组(接入等体积的磷酸缓冲液)未见多粘菌素E浓度的明显变化。
测定多粘菌素E标准曲线的制作:
(1)根据多粘菌素E标样的效价精确称量,用pH6.0的灭菌磷酸缓冲液分别配制2000U/mL、4000U/mL、6000U/mL、8000U/mL和10000U/mL的梯度标准溶液,同时配制5000U/mL的多粘菌素E标准溶液作为校正液。
(2)以大肠杆菌DH5α为指示菌,将灭菌后的LB固体培养基冷却至60℃时,加入体积浓度为5%,OD600值为1.0的大肠杆菌菌液混匀倒平板;待平板冷却后在对角放置四个牛津杯,一组对角牛津杯中加入体积200μL、浓度5000U/mL的多粘菌素E校正液,另一组对角牛津杯中加入不同浓度梯度的多粘菌素E标准溶液,每个梯度做三组平行试验。
(3)把加好溶液的平板放置4℃条件下静置12h后,转移到37℃培养箱培养直到出现明显的抑菌圈;用游标卡尺测量记录抑菌圈大小。
(4)取5个梯度所有5000U/mL浓度的抑菌圈大小的总平均值作为标准值,单个梯度的5000U/mL浓度的抑菌圈大小的平均值与标准值的差为校正误差;各梯度浓度的抑菌圈大小平均值与校正误差的和即为梯度校正值。
表1标准曲线数据(单位/mm)
注:5000U/mL多粘菌素E抑菌圈大小直径总平均值17.50mm。
(5)根据多粘菌素E浓度效价的对数与抑菌圈直径的大小呈线性关系,用Original8.1绘制拟合方程如图2所示。
实施例4枯草芽孢杆菌WB800dc在污染水土环境中的应用。
环境中受抗生素污染的地方多为水土混合淤泥,因此从环境中取50mL水和50g土壤(此处的水和土壤就是广泛环境中选取的,没有受抗生素污染,主要是含有一些菌体生长的基本物质如有机质等,能够保证菌体在模拟自然环境时能正常生长)配制成水土混合淤泥,在其中加入多粘菌素E并混合均匀,配置成多粘菌素E终浓度为16000U/mL的受多粘菌素污染的模拟环境。
按照实施例3步骤1的方法制备OD600为2.0的枯草芽孢杆菌WB800dc菌悬液,按照体积浓度8%的接种量加入上述受16000U/mL多粘菌素污染的模拟环境中,并设置对照组(含有空载质粒pWBUC01的WB800),放置于30℃、200r/min培养箱中,培养0、2、4、6和8天取样测定污水中多粘菌素的残留量,具体检测方法同实施例3。
表2枯草芽孢杆菌WB800dc对污染环境中多粘菌素的降解率(单位/U·mL-1)
上述实验表明,该枯草芽孢杆菌WB800dc能够有效降解受污染淤泥中的多粘菌素残留,16000U/mL的多粘菌素在8天时间基本降解完全,而含有空载质粒pWBUC01的WB800对多粘菌素的降解率仅为约38%。
上述实施例为本发明一般的实施方式,其他任何未背离本发明实质与原理的改变均视为本发明的保护范围之内。
序列表
<110> 浙江工业大学
<120> 一种重组枯草芽孢杆菌及其应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1539
<212> DNA
<213> 枯草芽孢杆菌(Bacillus subtilis )
<400> 1
ccgctcgaga tctttcaccc gtttctgtat gcgatatatt gcatatttta atagatgatc 60
gacaaggccg caacctcctt cggcaaaaaa tgatctcata aaataaatga atagtatttt 120
cataaaatga atcagatgga gcaatctcct gtcattcgcg gccctcggga cctctttccc 180
tgccaggctg aagcggtcta ttcatacttt cgaactgaac atttttctaa aacagttatt 240
aataaccaaa aaattttaaa ttggtcctcc aaaaaaatag gcctaccata taattcattt 300
tttttctata ataaattaac agaataattg gaatagatta tattatcctt ctatttaaat 360
tattctgaat aaagaggagg agagtgagta atgatgagga aaaagagttt ttggcttggg 420
atgctgacgg ccttcatgct cgtgttcacg atggcattca gcgattccgc ttctgctgct 480
caaccggcga aaaatgttga aaaggattat attgtcggat ttaagtcagg agtgaaaacc 540
gcatctgtca aaaaggacat catcaaagag agcggcggaa aagtggacaa gcagtttaga 600
atcatcaacg cggcaaaagc gaagctagac aaagaagcgc ttaaggaagt caaaaatgat 660
ccggatgtcg cttatgtgga agaggatcat gtggcccatg ccttggcgca aaccgttcct 720
tacggcattc ctctcattaa agcggacaaa gtgcaggctc aaggctttaa gggagcgaat 780
gtaaaagtag ccgtcctgga tacaggaatc caagcttctc atccggactt gaacgtagtc 840
ggcggagcaa gctttgtggc tggcgaagct tataacaccg acggcaacgg acacggcaca 900
catgttgccg gtacagtagc tgcgcttgac aatacaacgg gtgtattagg cgttgcgcca 960
agcgtatcct tgtacgcggt taaagtactg aattcaagcg gaagcggatc atacagcggc 1020
attgtaagcg gaatcgagtg ggcgacaaca aacggcatgg atgttatcaa tatgagcctt 1080
gggggagcat caggctcgac agcgatgaaa caggcagtcg acaatgcata tgcaagaggg 1140
gttgtcgttg tagctgcagc agggaacagc ggatcttcag gaaacacgaa tacaattggc 1200
tatcctgcga aatacgattc tgtcatcgct gttggcgcgg tagactctaa cagcaacaga 1260
gcttcatttt ccagtgtggg agcagagctt gaagtcatgg ctcctggcgc aggcgtatac 1320
agcacttacc caacgaacac ttatgcaaca ttgaacggaa cgtcaatggc ttctcctcat 1380
gtagcgggag cagcagcttt gatcttgtca aaacatccga acctttcagc ttcacaagtc 1440
cgcaaccgtc tctccagcac ggcgacttat ttgggaagct ccttctacta tgggaaaggt 1500
ctgatcaatg tcgaagctgc cgctcaataa ggatccgcg 1539
<210> 2
<211> 379
<212> PRT
<213> 枯草芽孢杆菌(Bacillus subtilis )
<400> 2
Met Met Arg Lys Lys Ser Phe Trp Leu Gly Met Leu Thr Ala Phe Met
1 5 10 15
Leu Val Phe Thr Met Ala Phe Ser Asp Ser Ala Ser Ala Ala Gln Pro
20 25 30
Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys Ser Gly Val
35 40 45
Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser Gly Gly Lys
50 55 60
Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala Lys Leu Asp
65 70 75 80
Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val Ala Tyr Val
85 90 95
Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val Pro Tyr Gly
100 105 110
Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly Phe Lys Gly
115 120 125
Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln Ala Ser His
130 135 140
Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala Gly Glu Ala
145 150 155 160
Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala Gly Thr Val
165 170 175
Ala Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala Pro Ser Val
180 185 190
Ser Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser Gly Ser Tyr
195 200 205
Ser Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn Gly Met Asp
210 215 220
Val Ile Asn Met Ser Leu Gly Gly Ala Ser Gly Ser Thr Ala Met Lys
225 230 235 240
Gln Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val Val Ala Ala
245 250 255
Ala Gly Asn Ser Gly Ser Ser Gly Asn Thr Asn Thr Ile Gly Tyr Pro
260 265 270
Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp Ser Asn Ser
275 280 285
Asn Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu Val Met Ala
290 295 300
Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Asn Thr Tyr Ala Thr
305 310 315 320
Leu Asn Gly Thr Ser Met Ala Ser Pro His Val Ala Gly Ala Ala Ala
325 330 335
Leu Ile Leu Ser Lys His Pro Asn Leu Ser Ala Ser Gln Val Arg Asn
340 345 350
Arg Leu Ser Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe Tyr Tyr Gly
355 360 365
Lys Gly Leu Ile Asn Val Glu Ala Ala Ala Gln
370 375
Claims (8)
1.一种重组枯草芽孢杆菌在降解多肽类抗生素中的应用,其特征在于所述重组枯草芽孢杆菌是将SEQ ID NO.1所示基因片段与线性化pWBUC01载体连接后转入枯草芽孢杆菌获得的;所述多肽类抗生素为多粘菌素E。
2.如权利要求1所述的应用,其特征在于所述枯草芽孢杆菌为枯草芽孢杆菌(Bacillus subtilis )WB800。
3.如权利要求1-2之一所述的应用,其特征在于所述重组枯草芽孢杆菌为枯草芽孢杆菌(Bacillus subtilis)WB800/pWBUC01-apr,保藏于中国典型培养物保藏中心,保藏日期2017年11月27日,保藏编号为:CCTCC NO: M 2017732,保藏地址为中国武汉,武汉大学,邮编430072。
4.如权利要求1所述的应用,其特征在于所述应用方法为:将重组枯草芽孢杆菌经发酵培养获得的湿菌体用pH为5~10的磷酸缓冲溶液清洗后悬浮配制成OD600为0.4~5.0的菌悬液;将菌悬液以体积浓度2-16%的接种量接种至含终浓度为5000~15000U/mL多粘菌素E的LB液体培养基中,在37℃、50~250r/min条件下培养,获得多粘菌素E降解的培养液;所述LB液体培养基组成为:酵母粉 1.0~10.0 g/L、胰蛋白胨 2.0~25.0 g/L、氯化钠2.0~25.0 g/L,溶剂为去离子水,pH 5~10。
5.如权利要求4所述的应用,其特征在于所述湿菌体按如下方法制备:将重组枯草芽孢杆菌接种至含5~100μg/mL卡那霉素LB固体培养基,于37℃倒置培养12~72h后,挑选单菌落接种在LB液体培养基中,37℃、50~250r/min培养12~72h,获得种子液;随后按体积浓度2~16%的接种量接种至LB液体培养基,37℃、50~250r/min培养12~72h,取培养结束后的菌液在4000~12000r/min的转速下离心2~20min后,弃上清液,收集湿菌体;LB固体培养基终浓度组成:酵母粉 1.0~10.0 g/L、胰蛋白胨 2.0~25.0 g/L、氯化钠2.0~25.0 g/L,琼脂15.0~25.0g/L,溶剂为去离子水,pH 5~10。
6.一种重组枯草芽孢杆菌在修复多肽类抗生素污染水体中的应用,其特征在于所述重组枯草芽孢杆菌是将SEQ ID NO.1所示基因片段与线性化pWBUC01载体连接后转入枯草芽孢杆菌获得的;所述多肽类抗生素为多粘菌素E。
7.如权利要求6所述的应用,其特征在于所述的应用是将重组枯草芽孢杆菌经发酵培养获得的湿菌体用pH为5~10的磷酸缓冲溶液清洗后悬浮配制成OD600为0.4~5.0的菌悬液;将菌悬液以体积浓度2-16%的接种量接种至多肽类抗生素污染水体中,在30℃、50~250r/min条件下培养,获得多肽类抗生素降解的培养液。
8.如权利要求6所述的应用,其特征在于所述多肽类抗生素污染水体中多肽类抗生素终浓度为5000~20000U/mL。
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