CN111848734B - 一种具有荧光特性的铁载体pvd、制备方法及其用途 - Google Patents
一种具有荧光特性的铁载体pvd、制备方法及其用途 Download PDFInfo
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Abstract
本发明公开了一种具有荧光特性的铁载体PVD、制备方法及其用途。铁载体PVD通过假单胞菌属细菌Pseudomonas sp.SXM‑1制备得到,该铁载体PVD可耐受变性剂和蛋白酶K;耐受强还原剂DTT,强氧化剂;PVD对Ca2+、Mg2+和Zn2+稳定,对Mn2+,Co2+,Ni2+,Cu2+,Fe2+均引起荧光淬灭。
Description
技术领域
本发明涉及化合物领域,尤其涉及一种具有荧光特性的铁载体PVD、制备方法及其用途。
背景技术
荧光性假单胞菌是一种广泛存在于自然界中的革兰氏阴性细菌,在缺乏游离三价铁离子的条件下,能分泌一种水溶性,黄绿色,发荧光,分子量约为1000到2000的小肽,能够与三价铁形成紧密的复合体并将三价铁转运至细菌细胞内。该类小肽被称为铁载体(pyoverdine)。随着人们对铁载体结构越来越感兴趣,研究表明,假单胞菌属不同菌种不同菌株产生不同的铁载体,同一菌株也可能产生一种或几种不同的铁载体。铁载体所呈现的结构多样性使其成为假单胞菌属系统发育与分类的重要工具,目前,已经有60多种铁载体结构被确定。喹啉生色团是目前发现的所有假单胞菌属来源的共同组成部分,它使得脓菌素具有黄绿色和荧光的特性,研究发现,与荧光基团氨基端相连的侧链是琥珀酸或其酰胺形式、苹果酸或其酰胺形式、α-酮戊二酸或谷氨酸。
发明内容
本发明的目的在于提供一种新的具有荧光特性的铁载体PVD。
为实现上述目的,本发明提供一种具有荧光特性的铁载体PVD,其特征在于,其结构式如式(A)所示,
本发明还保护所述具有荧光特性的铁载体PVD具有耐受变性剂,蛋白酶K,还原剂,氧化剂,Ca2+、Mg2+和Zn2+的用途。
本发明还保护所述具有荧光特性的铁载体PVD具有使Mn2+,Co2+,Ni2+,Cu2+,Fe2+荧光淬灭的用途。
本发明还提供一种所述具有荧光特性的铁载体PVD的制备方法,其特征在于,包括以下步骤:
发酵种子液制备:在海水LB培养基中接种假单孢属细菌Pseudomonas sp.SXM-1进行恒温培养,所得培养物作为种子液;优选的,所述假单孢属细菌Pseudomonas sp.SXM-1为将假单孢属细菌Pseudomonas sp.SXM-1菌种接种于海水LB培养基中恒温培养后的假单孢属细菌Pseudomonas sp.SXM-1培养液;所述海水LB培养基为胰蛋白胨10g,酵母提取物 5g,氯化钠10g,1L海水;
接种培养:采用液体发酵方式,在琥珀酸钠发酵培养基中接种上述种子液,于恒温培养箱中培养得到发酵液;所述琥珀酸钠发酵培养基为每1L的海水中含磷酸氢二钾6g,磷酸二氢钾3g,硫酸铵1g,硝酸铵1g;
铁载体的富集和分离:将上述发酵液离心取上清液,将上清液与大孔树脂HP20振荡混合后,将混合液填入层析柱,以乙醇-水为洗脱剂进行梯度洗脱,收集洗脱组分,取紫外光激发下有荧光的组分合并,旋转蒸发浓缩至干,在甲醇-水溶液中,结晶,待大量棕黄色晶体析出后重结晶,过滤,冻干,即得到荧光铁载体PVD。
进一步,所述发酵种子液制备步骤中,所述恒温培养的时间为20-30小时,温度为28-32℃;优选的,培养的时间为24小时,温度为30℃。
进一步,所述发酵种子液制备步骤中,所述海水为厦门市白城海滩的海水。
进一步,所述接种培养步骤为,培养的条件为150-200rpm,28-32℃,70-75小时;优选的,培养的条件为180rpm,30℃,72小时。
进一步,所述铁载体的富集和分离步骤中,所述离心的条件为8000rpm,20分钟;
任选的,所述上清液和大孔树脂的加量比例为1L上清液:40g大孔树脂HP20;优选的,大孔树脂HP20为日本三菱大孔树脂DIAION的产品;
任选的,所述振荡混合条件为30℃下振荡4小时,振荡转速为160rpm。
进一步,所述铁载体的富集和分离步骤中,所述梯度洗脱为用蒸馏水洗脱3倍柱床体积后,以乙醇-水为洗脱剂,从体积比 10:90,20:80,30:70,40:60,50:50,60:40,70:30,80:20,90:10进行梯度洗脱,每个比例洗脱 3倍柱床体积,每0.5个柱床体积收集1个组分,共18个组分;
任选的,旋转蒸发的温度为40℃下,50%的甲醇-水溶液为50%的甲醇-水溶液。
假单胞菌属细菌Pseudomonas sp.SXM-1的保藏信息如下:
保藏菌种:Pseudomonas sp.SXM-1,
保藏单位:中国典型培养物保藏中心,
保藏地址:中国武汉,武汉大学,
保藏编号:CCTCC No.M2019918,
保藏日期:2019年11月11日。
实施例对荧光特性的铁载体PVD的结构进行了检测,并进行了稳定性试验:证明了PVD可耐受变性剂和蛋白酶K;可耐受强还原剂DTT;可耐受强氧化剂;PVD对Ca2+、Mg2+和Zn2+稳定,其他离子(Mn2+,Co2+,Ni2+,Cu2+,Fe2+)均引起荧光淬灭。
附图说明
图1的(A)为荧光铁载体PVD的荧光光谱结果图;
图1的(B)为UPLC-FLR-QTOF MS检测获得激发波长为340nm和发射波长为512nm条件下化合物Pvd I的荧光光谱结果图。
图1的(C)为UPLC-FLR-QTOF MS检测的总离子流图中保留时间7.38分钟处提取的质谱图。
图2是实施例2中二级质谱分析时MS/MS碎片对应的主要的B离子和Y离子示意图。
图3是实施例3的铁载体的稳定性试验结果图。
具体实施方式
下面详细描述本发明的实施例,所述实施例的示例在附图中示出,其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的,旨在用于解释本发明,而不能理解为对本发明的限制。实施例中未注明具体技术或条件者,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例1:铁载体PVD的制备
(1)菌种的准备:使用海水LB培养基,高温灭菌,制成平板,置于常温状态下,接种活化好的假单孢属细菌Pseudomonas sp.SXM-1,于恒温培养箱中培养24小时,培养温度30℃,作为菌种;所述海水LB培养基成分为胰蛋白胨10g,酵母提取物5g,氯化钠10g,1L 海水;
假单胞菌属细菌Pseudomonas sp.SXM-1的保藏信息如下:
保藏菌种:Pseudomonas sp.SXM-1,
保藏单位:中国典型培养物保藏中心,
保藏地址:中国武汉,武汉大学,
保藏编号:CCTCC No.M2019918,
保藏日期:2019年11月11日。
(2)发酵种子液制备:在多个锥形瓶中分别装入100ml海水LB液体培养基,高温灭菌后,接种上述菌种培养,于恒温培养箱中培养24小时,培养温度30℃,将该培养物作为种子液;
(3)接种:采用液体发酵方式,准备发酵瓶,加入1L琥珀酸钠发酵培养基,高温灭菌,接种上述种子液,于恒温培养箱中培养72小时(180rpm),培养温度30℃;所述琥珀酸钠发酵培养基为每1L的海水中含磷酸氢二钾6g,磷酸二氢钾3g,硫酸铵1g,硝酸铵1g (海水采自厦门市白城海滩);
(4)铁载体的富集和分离:待摇瓶培养72小时(180rpm),培养温度30℃,发酵液经8000rpm离心20分钟,取上清液,按每L上清液加入40g大孔树脂HP20(日本三菱大孔树脂DIAION)进行混合,将形成的混合液于30℃下振荡4小时,振荡转速为160rpm,接着将振荡结束后的混合液填入层析柱,用蒸馏水洗脱3倍柱床体积后,以乙醇-水为洗脱机,从体积比10:90,20:80,30:70,40:60,50:50,60:40,70:30,80:20,90:10进行梯度洗脱,每个比例洗脱3倍柱床体积,每0.5个柱床体积收集1个组分,共18个组分,取紫外光激发下有荧光的组分,合并,40℃下旋转蒸发浓缩至干,在50%的甲醇-水溶液中,4℃结晶,待大量棕黄色晶体析出后重结晶2次,过滤,冻干,即得到荧光铁载体PVD(纯度检测见图1的 B,归一化法计算纯度大于95%)。
实施例2:通过超高效液相色谱串联红外光谱及四极杆-飞行时间质谱(UPLC-FLR-QTOF MS)系统对化合物进行检测分析
UPLC-FLR-QTOF MS检测,其中,色谱条件:使用Acquity UPLC BEH C18色谱柱(2.1mm×50 mm,1.7μm,Waters),柱温:30℃,流速0.3mL/min,流动相为水(A)和甲醇(B),梯度:0-5分钟,5%B;5–9分钟,B由5%线性变化至95%;之后,95%B保持2min。
荧光光谱条件:激发波长在350-600nm范围内扫描,发射波长固定在620nm,发现目标化合物Pvd I保留时间为7.38分钟,激发光谱显示其最佳激发波长为340nm(图1的A);进一步将激发波长固定在340nm,对发射波长在350-600nm范围内进行扫描,发射光谱显示其最佳发射波长为512nm(图1的A)。
质谱条件:一级质谱,正离子模式,扫描范围:50至1500m/z,毛细管电压:3kV,锥孔电压:40V,锥孔气流量:5L/h,源温度:100℃,脱溶剂温度:350℃,脱溶剂气流量:600L/h。二级质谱,正离子模式,目标离子:1278,碰撞能量范围:60~70eV。
UPLC-FLR-QTOF MS检测获得激发波长为340nm和发射波长为512nm条件下化合物Pvd I 的荧光光谱(图1的B),相同保留时间下化合物Pvd I的质谱图(图1的C)。
使用antiSMASH网站进行生物信息学分析,预测产物的详细氨基酸序列。根据A结构域 (DVWHVSLIDK)结合口袋中的签名残基,加之下游存在差向异构域,预测肽链的第一个氨基酸是D构型的丝氨酸。第二个氨基酸未注释,其签名残基为DGeacGgVtK。类似地,预测第三至第八个氨基酸是甘氨酸-苏氨酸-D-苏氨酸-谷氨酰胺-甘氨酸-D-丝氨酸。两个苏氨酸的特征残基是DFWNVGMVHK,而甘氨酸和谷氨酰胺的特征残基分别是DILQLGLIWK和DAWQ1GLIDK。由于下游存在差向异构域,第五和第八个氨基酸都为D-构型。第九个氨基酸亦未注释,它与第二个氨基酸有相同的签名残基,表明它们来自A结构域的相同的结合口袋。
对目标离子(m/z 1278)进行二级质谱分析(见图2和表1),碰撞能量范围为60eV~70 eV。在m/z 358处的片段表明存在二羟基喹啉核心,该核心包含发色团,该发色团带有琥珀酸作为侧链。m/z 445的B1片段(与m/z 358相比,Δ87Da)证实了第一个氨基酸是丝氨酸,与antiSMASH分析结果一致。m/z 617的B2片段(与m/z 445相比相比,Δ172Da),表明第二个氨基酸为N-乙酰基-N-羟基鸟氨酸(AcOHOrn)。在m/z 131处的片段Y1在肽链末端,被确定为环状N-羟基鸟氨酸(cOHOrn),是第九个氨基酸。由于下游存在差向异构域,两个未注释的氨基酸都为D构型。此外,一系列信号分别为m/z 775(B3),m/z 1061(B4)和m/z 218 (Y2)的片段确认了第三至第八个氨基酸的身份,均与antiSMASH的预测相符。化合物Pvd I 最终被表征为succa-chr-D-Ser-D-AcOHOrn-Gly-Thr-D-Thr-Gln-Gly-D-Ser-D-cOHOrn。
表1化合物Pvd I[M+H]+的MS/MS分析产生的B离子和Y离子信息表
注:Succ:succinic acid,琥珀酸;chr:chromophore,发色团。
实施例3:铁载体的稳定性试验
1.取提纯后的荧光铁载体PVD,用去离子水配置成10mg/ml,加入十二烷基硫酸钠(SDS) 至5mM,加入蛋白酶K至浓度100μg/ml,50℃温育1小时后,荧光强度没有变化,说明PVD 可耐受变性剂和蛋白酶K。结果见图3中的SDS PK管。
2.取提纯后的荧光铁载体PVD,用去离子水配置成10mg/ml,加二硫苏糖醇(DTT)至50mM, 50℃温育1小时后,荧光强度没有变化,说明PVD可耐受强还原剂DTT。结果见图3的DTT管。
3.取提纯后的荧光铁载体PVD,用去离子水配置成10mg/ml,取1ml含荧光物的溶液,加入50μl 30%H2O2溶液,结果荧光颜色和强度没有改变,说明PVD可耐受强氧化剂。结果见图3的氧化剂管。
4.取提纯后的荧光铁载体PVD,用去离子水配置成10mg/ml,取1ml含荧光物的溶液,分别加入10μl的1M的各种金属离子溶液,Ca2+(CaCl2),Mg2+(MgCl2),Zn2+(ZnCl2),Mn2+(MnCl2),Co2+(CoCl2),Ni2+(NiSO4),Cu2+(CuSO4),Fe2+(FeSO4)。结果见图3的Ca2+、 Mg2+,Zn2+,Mn2+,Co2+,Ni2+,Cu2+,Fe2+管。从结果可以看出:PVD对Ca2+、Mg2+和Zn2+稳定,其他离子均引起荧光淬灭。
尽管上面已经示出和描述了本发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在不脱离本发明的原理和宗旨的情况下在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
Claims (16)
2.权利要求1所述具有荧光特性的铁载体PVD具有耐受变性剂SDS、蛋白酶K、还原剂DTT、氧化剂H2O2、以及Ca2+、Mg2+和Zn2+的用途。
3.权利要求1所述具有荧光特性的铁载体PVD具有加入Mn2+、Co2+、Ni2+、Cu2+、Fe2+后引起铁载体PVD荧光淬灭的用途。
4.一种权利要求1所述具有荧光特性的铁载体PVD的制备方法,其特征在于,包括以下步骤:
发酵种子液制备:在海水LB培养基中接种假单孢属细菌Pseudomonas sp.SXM-1进行恒温培养,所得培养物作为种子液;所述假单孢属细菌Pseudomonas sp.SXM-1的保藏编号为CCTCC No.M2019918;
接种培养:采用液体发酵方式,在琥珀酸钠发酵培养基中接种上述种子液,于恒温培养箱中培养得到发酵液;所述琥珀酸钠发酵培养基为每1L的海水中含磷酸氢二钾6g,磷酸二氢钾3g,硫酸铵1g,硝酸铵1g;
铁载体的富集和分离:将上述发酵液离心取上清液,将上清液与大孔树脂HP20振荡混合后,将混合液填入层析柱,以乙醇-水为洗脱剂进行梯度洗脱,收集洗脱组分,取紫外光激发下有荧光的组分合并,旋转蒸发浓缩至干,在甲醇-水溶液中,结晶,待大量棕黄色晶体析出后重结晶,过滤,冻干,即得到荧光铁载体PVD。
5.如权利要求4所述,其特征在于,所述假单孢属细菌Pseudomonas sp.SXM-1为将假单孢属细菌Pseudomonas sp.SXM-1菌种接种于海水LB培养基中恒温培养后的假单孢属细菌Pseudomonas sp.SXM-1培养液;所述海水LB培养基为胰蛋白胨10g,酵母提取物5g,氯化钠10g,1L海水。
6.如权利要求4所述具有荧光特性的铁载体PVD的制备方法,其特征在于,所述发酵种子液制备步骤中,所述恒温培养的时间为20-30小时,温度为28-32℃。
7.如权利要求6所述具有荧光特性的铁载体PVD的制备方法,其特征在于,所述培养的时间为24小时,温度为30℃。
8.如权利要求4所述具有荧光特性的铁载体PVD的制备方法,其特征在于,所述发酵种子液制备步骤中,所述海水为厦门市白城海滩的海水。
9.如权利要求4所述具有荧光特性的铁载体PVD的制备方法,其特征在于,所述接种培养步骤为,培养的条件为150-200rpm,28-32℃,70-75小时。
10.如权利要求9所述具有荧光特性的铁载体PVD的制备方法,其特征在于,所述培养的条件为180rpm,30℃,72小时。
11.如权利要求4所述具有荧光特性的铁载体PVD的制备方法,其特征在于,所述铁载体的富集和分离步骤中,所述离心的条件为8000rpm,20分钟。
12.如权利要求4所述具有荧光特性的铁载体PVD的制备方法,其特征在于,所述上清液和大孔树脂的加量比例为1L上清液:40g大孔树脂HP20。
13.如权利要求12所述具有荧光特性的铁载体PVD的制备方法,其特征在于,所述大孔树脂HP20为日本三菱大孔树脂DIAION的产品。
14.如权利要求4所述具有荧光特性的铁载体PVD的制备方法,其特征在于,所述振荡混合条件为30℃下振荡4小时,振荡转速为160rpm。
15.如权利要求4所述具有荧光特性的铁载体PVD的制备方法,其特征在于,所述铁载体的富集和分离步骤中,所述梯度洗脱为用蒸馏水洗脱3倍柱床体积后,以乙醇-水为洗脱剂,从体积比10:90,20:80,30:70,40:60,50:50,60:40,70:30,80:20,90:10进行梯度洗脱,每个比例洗脱3倍柱床体积,每0.5个柱床体积收集1个组分,共18个组分。
16.如权利要求4所述具有荧光特性的铁载体PVD的制备方法,其特征在于,所述旋转蒸发的温度为40℃下,甲醇-水溶液为50%的甲醇-水溶液。
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