CN116806830B - 一种溶杆菌来源嗜铁素的应用 - Google Patents

一种溶杆菌来源嗜铁素的应用

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CN116806830B
CN116806830B CN202310788997.0A CN202310788997A CN116806830B CN 116806830 B CN116806830 B CN 116806830B CN 202310788997 A CN202310788997 A CN 202310788997A CN 116806830 B CN116806830 B CN 116806830B
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siderophore
ferrite
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bacteria
lysobacter
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汤宝
刘凤权
赵延存
张敏
佩德罗·拉沃尔达
王波
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Jiangsu Yanjiang Agricultural Science Research Institute
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Abstract

本发明公开了一种溶杆菌来源嗜铁素的应用。经发酵培养、XAD16树脂吸附解吸、HPLC制备等从产酶溶杆菌OH11发酵液中获得了高纯度的嗜铁素，经过质谱、核磁确定了该嗜铁素为儿茶酚型，由2个儿茶酚基团和1个亚精胺组成，分子式为C21O6N3H27，分子量为417Da，命名为Sperbactin。平板拮抗试验表明，Sperbactin对水稻白叶枯病菌和水稻细菌性条斑病菌具有较强的拮抗活性，EC50分别为15.44mg/L、16.76mg/L，可开发成高效杀细菌剂。

Description

一种溶杆菌来源嗜铁素的应用
本申请为申请日为2022年9月14日，申请号为202211118311.9，发明名称为一种溶杆菌来源的嗜铁素及其制备方法与应用的中国发明专利的分案申请。
技术领域
本发明属于生物技术领域，具体涉及一种溶杆菌来源嗜铁素的应用
背景
溶杆菌属于黄单胞科(Xanthomonadaceae)，溶杆菌属(Lysobacter)，是一类重要的，尚未完全开发的重要植物病害生防细菌，广泛存在于土壤和淡水环境中。截止2019年，国际上报道的溶杆菌属细菌已达到50个种，其中产酶溶杆菌(Lysobacter enzymogenes)是溶杆菌属的一个典型种，也是目前作为生防细菌研究较多的一种溶杆菌。该菌能产生丰富的胞外水解酶和小分子抗菌活性产物，对农作物重要病原真菌、卵菌、细菌以及线虫具有显著的拮抗作用。。
嗜铁素(siderophore，也叫铁载体)主要是细菌、真菌在铁缺乏环境中自身合成并分泌到胞外的次生代谢产物，其本质为非蛋白质氨基酸，相对分子质量小于10KDa。对三价铁有较强的亲和力，通过降低环境pH值来增加铁的溶解性。迄今为止，还没有关于溶杆菌嗜铁素应用的报道。因此，从溶杆菌中分离鉴定嗜铁素，并对其应用进行研究，不仅丰富了溶杆菌活性产物种类和功能，有助于解析溶杆菌的作用机制，而且还能为开发新的生防制剂提供物质基础。
发明内容
本发明目的在于提供一种溶杆菌来源嗜铁素的应用。
为实现上述目标，本发明所采用的技术方案为：
第一方面，本发明提供了一种从产酶溶杆菌OH11发酵液中分离纯化的嗜铁素，进一步，鉴定了嗜铁素的化学结构如下：
本发明所述的嗜铁素类型为儿茶酚型，分子量为417Da，由2个儿茶酚基团和1个亚精胺组成，命名为Sperbactin。
第二方面，本发明还提供了第一方面所述的嗜铁素的制备方法，包括以下步骤：(1)制备产酶溶杆菌OH11的发酵液；(2)制备嗜铁素解吸液；(3)嗜铁素的分离纯化。
本发明所述的产酶溶杆菌(Lysobacter enzymogenes)OH11是本实验室自主分离的一个菌株，于2007年3月19日保藏于中国微生物菌种保藏管理委员会普通微生物中心，菌种保藏号为CGMCC NO.1978(见专利ZL200710190998.6)。
在一些实施例中，本发明步骤(1)是将产酶溶杆菌OH11接种于发酵培养基中进行发酵，在一些具体的实例中发酵过程为：将活化的菌株OH11接入到LB液体培养基中26～28℃、170～190rpm培养12-15h，按照1.0-2.5％接种量转入发酵培养基中，26-29℃、150-200rpm培养48 -60h。
在一些实施例中，本发明所述的发酵培养基成分为：葡萄糖5-15g/L、(NH4)2SO40.5-2.0g/L、MgCl2 5-20mg/L、K2HPO4 0.5-4.0g/L、KH2PO4 0.25-2.0g/L，CaCO3 0.25-1.5g/L。优选的所述的发酵培养基成分为：葡萄糖5-10g/L、(NH4)2SO40.8-1.2g/L、MgCl2 8-15mg/L、K2HPO4 1.0-1.5g/L、KH2PO4 0.5-0.75g/L，CaCO30.5-1.0g/L。更优选的所述的发酵培养基成分为：葡萄糖8g/L、(NH4)2SO4 1.0g/L、MgCl2 10mg/L、K2HPO4 1.0g/L、KH2PO4 0.5g/L，CaCO3 1.0g/L。所述的发酵培养基均用纯水配制。在本发明所述的发酵培养基下，能够有效提高嗜铁素的产量，嗜铁素产量可以达到100-220mg/L。
在一些实施例中，本发明步骤(2)具体为将步骤(1)得到的发酵液与XAD16树脂进行直接混合，置于摇床上进行吸附12～24h，过滤分离树脂和滤液，对树脂利用无水乙醇进行解吸，得到解吸液。本发明所述的XAD16树脂可以通过市面进行常规购买获得。
在一些实施例中，本发明所述的吸附条件为，树脂添加量为20-40g/L，摇床120-200rpm、26-30℃；
在一些实施例中，本发明所述的解吸条件为，无水乙醇和大孔树脂的体积质量比为20-50mL：1g(湿重)，优选25mL：1g。解吸条件为摇床120-200rpm、27-37℃、0.5-3h，在一种具体的实施例中，为摇床180rpm、28℃、2h。
在一些实施例中，本发明所述的步骤(3)具体为将步骤(2)得到的解吸液旋转蒸发，复溶于甲醇溶液中，利用制备型HPLC进行分离纯化，收集嗜铁素馏分，旋转蒸发，冷冻干燥，即得到嗜铁素纯品。
在一些实施例中，本发明HPLC制备条件为：InterSustainSwift C18 5μm,250×20mm；流动相：溶液A(0.025％ TFA水溶液)和溶液B(0.025％ TFA乙腈溶液)，流速：1～2mL/min；进样量为3mL，紫外吸收值：254nm；进样程序：0-10min，将溶液B从5％升至25％；25min，增长到80％ B；26min，增长到100％；27-30min返回到5％，整个过程溶液A和B总比例为100％；馏分收集时间为11.7-12.2min。
第三方面，本发明还提供了第一方面所述的嗜铁素在制备生防制剂中的应用。
本发明还提供了第一方面所述的嗜铁素在作为铁载体中的应用。
本发明还提供了第一方面所述的嗜铁素在防治植物病原细菌方面的应用。
本发明所述的细菌为水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae PXO99)、水稻细菌性条斑病菌(Xanthomonas oryzae pv.oryzicola Rs105)、丁香假单胞菌(Pseudomonas syringae)、黄瓜细菌性角斑病菌(Pseudomonas syringaepv.Lachrymans)。进一步，所述的细菌为水稻白叶枯病菌(Xanthomonas oryzaepv.oryzae PXO99)或水稻细菌性条斑病菌(Xanthomonas oryzae pv.oryzicola Rs105)，EC50分别为15.44mg/L、16.76mg/L。
本发明所述的方法相对于现有技术的有益效果：
(1)目前还没有从溶杆菌中产生嗜铁素的报道。本发明所述的嗜铁素是从溶杆菌中分离鉴定出一种新的活性化合物，可开发成新型的生防制剂或抗菌药物，还能够为植物的生长提供铁营养元素，在生物防治领域具有重要的应用价值；
(2)本发明所述的嗜铁素对水稻白叶枯病菌和水稻细菌性条斑病菌具有较强的拮抗效果，可开发成专一性杀菌剂，用于水稻细菌病害防治。
附图说明
图1CAS平板试验；
图2发酵液上清的Arnow反应；
图3滤液和解吸液的Arnow反应；
图4HPLC分析解吸液成分；
图5质谱分析；
图6核磁分析；
图7嗜铁素的定量分析标曲；
图8嗜铁素对病原菌的EC50测定。
具体实施方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。
实施例1：产酶溶杆菌OH11产生嗜铁素的能力检测
铁载体检测染液：将0.079g CAS溶于50mL去离子水中，再加入10mL 1mmol/LFeCl3溶液(含12mmol/L HCl)，溶液A。将0.069g十六烷基三甲基溴化铵(HDTMA)溶于40mL的去离子水中，得溶液B。将A溶液沿烧杯壁缓缓加入B溶液中，搅拌混匀即得100mL CAS蓝色检测液；
CAS培养基：每1L含20％蔗糖溶液10mL，10％酸水解酪素30mL，1mmol/LCaCl2 1000μL，1mmol/L MgSO4 20mL，琼脂18g，在约60℃时缓慢加入磷酸盐缓冲液和CAS检测染液各50mL；
划取LB固体平板上的产酶溶杆菌OH11单菌落菌株于50mL LB培养液中，28℃下180rpm振荡培养12h，即得到菌液。取3μL菌液点在CAS固体平板四周，置于28℃培养箱中培养2天。结果如图1所示，可看到在菌落周围产生橘黄色晕圈，表明OH11菌株具有产生嗜铁素的能力。
实施例2：产酶溶杆菌OH11产生嗜铁素类型的确定
Arnow方法检测儿茶酚型嗜铁素，其配方为：0.5M HCl；10g亚硝酸钠和10g钼酸钠溶于少量水后加水至100mL；1M NaOH。
发酵培养基配方：葡萄糖5g/L、(NH4)2SO4 2g/L、K2HPO4 1.5g/L、KH2PO40.75g/L，用纯水配制1L，分装50mL于250mL三角瓶中，121℃灭菌20min。同时用添加FeCl3 8mg/L作为对照。种子液制备方法同实例1，按照2.5％的接种量接入发酵培养基中，28℃、180rpm下培养48h，得到发酵液。
发酵液10000rpm离心5min，取1mL上清与1mL0.5 M HCl、1mL钼酸-亚硝酸钠、1mLNaOH混合，如果有儿茶酚型嗜铁素存在，该混合液的颜色会变成红色。如图2所示，在不加Fe3+的发酵液中，有明显的红色反应，说明OH11产生的嗜铁素为儿茶酚类型。
实施例3：嗜铁素解吸液的制备
分别向实施例2发酵液中加入2g(湿重)预处理好的XAD16树脂，置于摇床中，28℃、180rpm、振荡过夜，使其达到充分吸附。利用3层纱布分离树脂和滤液，对滤液进行Arnow反应检测嗜铁素含量。对吸附的树脂进行解吸：加入无水乙醇50mL，置于摇床中，28℃、180rpm、解吸2h。将解吸液收集一起，进行Arnow反应，同时旋转蒸发仪50℃进行浓缩，蒸干后用甲醇溶解，0.22μm滤膜过滤后用于HPLC分析。进样量为20μL，检测条件为，紫外吸收值：254nm，反相柱：InterSustainSwift C18 5μm,250×4.6mm；流动相：溶液A(0.025％TFA水溶液)和溶液B(0.025％ TFA乙腈溶液)，流速：1mL/min；进样程序：0-10min，将溶液B从5％至25％；25min，增长到80％ B；26min，增长到100％；30min返回到5％，整个过程溶液A和B总比例为100％。
由图3可知，滤液中几乎不含有嗜铁素，而解吸液中呈现明显的红色，说明发酵液中嗜铁素能够通过树脂吸附转入解吸液中。图4可知，与对照相比，不添加Fe3+的解吸液在HPLC色谱图上有一个明显的吸收峰，保留时间为11.9min，此峰即为OH11产生的儿茶酚型嗜铁素吸收峰。
实施例4：嗜铁素的纯化及鉴定
将实施例3中嗜铁素解吸液采用HPLC进一步分离纯化，收集保留时间为11.7-12.2min的馏分，冷冻干燥后，用于下一步结构鉴定。制备条件为，反相柱：InterSustainSwift C18 5μm,250×20mm，流速：5mL/min；进样量：3mL，其它同分析程序。
将HPLC纯化后的嗜铁素组分进行质谱分析，结果如图5所示(HRMS(ESI):calculated for C21H28N3O6[M+H]+418.1978,found 418.2000；calculated for C21H26N3O6[M-H]-416.1822,found 416.2000)，该组分的分子量为417Da。核磁结果如图6所示(1H NMR(400MHz,CD3OD):δ7.21(dd,1H,J＝8.0,1.6Hz),7.21(dd,1H,J＝8.4,1.6Hz),6.95(dd,1H,J＝6.0,1.6Hz),6.93(dd,1H,J＝6.0,1.6Hz),6.93(t,1H,J＝8.0Hz),6.72(t,1H,J＝7.6Hz),3.52(t,2H,J＝6.4Hz),3.46(t,2H,J＝6.0Hz),3.11-3.04(m,4H),1.99(quint,2H,J＝7.2Hz),1.82-1.70(m,4H).
13C NMR(100MHz,CD3OD):δ170.78,170.30,148.81,148.64,145.97,145.95,118.47,118.43,118.29,118.27,117.52,117.29,115.36,115.25,47.30,45.16,38.08,35.71,26.30,26.17,23.27)，综合以上分析，推断的嗜铁素结构式为C21O6N3H27，化学结构如下所示，含有2个儿茶酚活性基团，1个亚精胺结构，我们将其命名为Sperbactin。
实施例5：产酶溶杆菌OH11发酵液中嗜铁素的定量检测
建立嗜铁素浓度与吸收峰面积间的标准曲线，具体过程如下：：准备称取10mg实施例4制得的嗜铁素纯品加入10mL甲醇溶液中，配置成1000mg/L母液，并依次稀释不同梯度浓度，最后通过HPLC分析柱检测，条件同实施例3。记录峰面积，以配置溶液中嗜铁素的浓度为纵坐标(Y)，吸收峰面积为横坐标(X)绘制标准曲线，如图7所示，可得嗜铁素浓度与峰面积间的线性方程为Y＝0.0483*X-7.0726，R2＝0.9999。
实施例6：产酶溶杆菌OH11高产嗜铁素的发酵培养基
高产嗜铁素的发酵培养基配方：葡萄糖8g/L、(NH4)2SO4 1g/L、MgCl2 10mg/L、K2HPO4 1g/L、KH2PO4 0.5g/L，CaCO31 g/L，用纯水配制1L，分装50mL于250mL三角瓶中，121℃灭菌20min。种子液制备方法同实例实施例2，按照2.5％的接种量接入发酵培养基中，28℃、180rpm下培养48h，得到发酵液。结果显示，OH11菌株在此培养基中嗜铁素产量达到213.99mg/L，而实施例2的嗜铁素产量为108.24mg/L，可见实施例6的培养基有效提高了嗜铁素的产量，为其进一步开发奠定基础。
实施例7：嗜铁素的应用
准确称取10mg实施例4中得到的嗜铁素纯品加入1mL DMSO溶液中，配置成10mg/mL母液。
所测试的病原细菌包括黄瓜细菌性角斑病菌Pseudomonas syringaepv.Lachrymans,水稻白叶枯病菌Xanthomonas oryzae pv.oryzae PXO99,水稻细菌性条斑病菌Xanthomonas oryzae pv.oryzicola Rs105,和丁香假单胞菌Pseudomonas syringae。上述病原菌现保存于本实验室中，发明人和申请人承诺永久向公众提供。
病原细菌EC50的测定：将病原细菌在NB培养液中培养到OD600＝0.5，按照体积比例1:1000混合，吸取1mL混合液到24孔板中，并添加嗜铁素母液，使其终浓度分别为0μg/mL、5μg/mL、8μg/mL、10μg/mL、15μg/mL、20μg/mL、35μg/mL、50μg/mL、80μg/mL、100μg/mL。放置摇床中28℃、180rpm培养12-24h，测量OD600，并计算EC50。
由图8可知，OH11嗜铁素对水稻白叶枯病菌Xanthomonas oryzae pv.oryzaePXO99、水稻细菌性条斑病菌Xanthomonas oryzae pv.oryzicola Rs105、丁香假单胞菌Pseudomonas syringae、黄瓜细菌性角斑病菌Pseudomonas syringae pv.Lachrymans均具有良好的抑制效果，EC50分别为15.44mg/L、16.76mg/L、62.74mg/L、73.78mg/L，特别是对水水稻白叶枯病菌和水稻细菌性条斑病菌，可开发成高效杀菌剂，用于水稻细菌病害防治。

Claims (2)

1.一种嗜铁素在防治植物病原细菌方面的应用，其特征在于，所述的细菌为水稻白叶枯病菌、水稻细菌性条斑病菌、丁香假单胞菌、黄瓜细菌性角斑病菌中的一种或多种，所述嗜铁素的化学结构如下：
。
2.根据权利要求1所述的应用，其特征在于，所述的细菌为水稻白叶枯病菌或水稻细菌性条斑病菌。
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