CN111893059B - 一种泰乐菌素降解菌及其筛选方法与应用 - Google Patents
一种泰乐菌素降解菌及其筛选方法与应用 Download PDFInfo
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Abstract
一种泰乐菌素降解菌及其筛选方法与应用，属于生物技术领域。本发明提供了一种在好氧和兼性厌氧条件下均可高效、安全地降解泰乐菌素的微生物菌株，并将其应用于泰乐菌素的降解。所述微生物菌株为解糖假苍白杆菌(Pseudochrobactrum saccharolyticum)TYL‑B35该菌株从使用泰乐菌素的养鸡场附近的土壤中筛选获得，菌株原始编号TYL‑B35，保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO：M20191028，保藏日期为2019年12月9日，保藏地址为中国，武汉，武汉大学。由该菌株制成的菌剂可应用于含泰乐菌素菌渣中泰乐菌素的降解及受泰乐菌素污染土壤或水体的修复。
Description
技术领域
本发明属于生物技术领域，具体涉及一种泰乐菌素降解菌及其筛选方法与应用。
背景技术
泰乐菌素(Tylosin)，又称泰农、泰乐霉素，是一种从弗氏链霉菌(Streptomycesfradiae)发酵液中提取的大环内酯类抗生素，其核心结构为十六元不饱和内酯，另外带有3个六碳糖衍生物结构单元(图1)。泰乐菌素可与核糖体50s亚基结合阻断转肽作用和mRNA位移，抑制细菌或支原体的蛋白质合成，从而起到杀菌作用。本品是一种广谱兽用抗生素，对支原体有特效，为畜禽支原体感染性疾病的首选药物；本品对多种革兰氏阳性菌具有很强的抗菌作用，还对部分革兰氏阴性菌及球虫具有抑制作用。此外，泰乐菌素还可用作一种饲料添加剂，连续低剂量饲用，对生长期的畜禽具有明显的促生长作用。得益于优良的药理和生理作用，泰乐菌素在畜禽养殖中得到了广泛应用，在畜禽养殖业的发展中发挥了重要作用。另一方面，泰乐菌素的大规模使用也带来了严重的抗生素残留问题。大量泰乐菌素未经处理排放到环境中，会加快诱导微生物产生抗药性，同时给人类健康带来潜在的威胁。目前，泰乐菌素残留已成为人们关注的焦点环保问题之一。如何高效、安全、低成本地降解泰乐菌素也成为科研人员研究的热点。
微生物降解抗生素，处理速度快，无二次污染，整体成本低。因此，找到高效、安全的泰乐菌素降解菌株是解决问题的关键。目前，国内只有少量针对泰乐菌素微生降解的报道。孙瑞珠等[微生物学通报,14,41(4):681-690]筛选到一株越南伯克霍尔德氏菌，72h对初始浓度为300mg/L的泰乐菌素的降解率可达99％以上。李兆君等(专利申请号：CN201710816643.7、CN201910153969.5)筛选到2株微生物，对低浓度泰乐菌素(25mg/L)的降解率达到70％以上。以上对泰乐菌素降解的报道都是在好氧条件下进行的，而在实际降解泰乐菌素的实践中，往往需要在兼性厌氧条件下进行，如泰乐菌素菌渣的堆肥。因此，找到可在兼性厌氧条件下高效、安全地降解泰乐菌素的菌株势在必行。
发明内容
本发明的目的在于提供了一种在好氧和兼性厌氧条件下均可高效、安全地降解泰乐菌素的微生物菌株，并将其应用于泰乐菌素的降解。为实现该目标，本发明从使用泰乐菌素养鸡场的浅表层土壤种筛选得到了一株泰乐菌素降解菌——解糖假苍白杆菌(Pseudochrobactrum saccharolyticum)TYL-B35，该菌株可在好氧或者兼性厌氧条件高效地降解泰乐菌素。该菌株保藏于中国典型培养物保藏中心，保藏日期为2019年12月9日，保藏地址为中国，武汉，武汉大学，保藏编号为CCTCC NO：M20191028。
本发明还提供了上述泰乐菌素降解菌的筛选方法：
1)采集使用泰乐菌素作为抗呼吸道支原体感染药物的养鸡场浅表层土壤，加入到无菌缓冲液中，摇匀后稀释，将稀释液涂布于含有泰乐菌素的LB固体培养基平板上，在培养箱中静置培养；
2)待长出菌落后，挑取单菌落，接种于含有步骤1)中2倍浓度的泰乐菌素的LB平板上，重复以上步骤直至培养基中含有步骤1)中10倍浓度的泰乐菌素，测定菌株对泰乐菌素降解情况，筛选获得泰乐菌素降解菌。
进一步地限定，步骤1)所述无菌缓冲液为50mM无菌磷酸缓冲液，pH为7.0。
进一步地限定，步骤1)所述静置培养条件为30℃恒温培养2d。
进一步地限定，步骤1)所述含有泰乐菌素的LB固体培养基平板中泰乐菌素的终浓度为50mg/L。
进一步地限定，步骤2)所述菌株对泰乐菌素降解情况测定采用高压液相色谱法。
本发明还提供了上述泰乐菌素降解菌在泰乐菌素降解上的应用。
本发明还提供了一种含有上述泰乐菌素降解菌的菌剂。
本发明还提供了上述菌剂在泰乐菌素降解上的应用。
与现有技术相比，本发明的有益效果如下：
本发明提供的菌株，在有氧或者兼性厌氧条件下均可高效地降解泰乐菌素，该菌株及其制剂可应用于泰乐菌素菌渣的处理或者用于受泰乐菌素污染环境的修复。
附图说明
图1泰乐菌素(Tylosin)化学结构；
图2P.saccharolyticum TYL-B35对泰乐菌素的降解情况：a，空白对照；b，好氧条件下泰乐菌素的降解；c，兼性厌氧条件下泰乐菌素的降解。横坐标为时间，纵坐标为响应值。
具体实施方式
本发明所述的泰乐菌素降解菌的筛选方法如下：采集山东某散养型养鸡场浅表层土壤。该养鸡场使用泰乐菌素作为抗呼吸道支原体感染的药物。将采集到的土样带回实验室，加入到无菌缓冲液中，在摇床中振荡摇匀，稀释后涂布于含有泰乐菌素的LB固体培养基平板上，在培养箱中静置培养。待长出菌落后，挑取单菌落，接种于含有更高浓度泰乐菌素的LB平板上，重复以上步骤直至培养基中含有高浓度泰乐菌素。在含有高浓度泰乐菌素的LB液体培养基中好氧或者兼性厌氧培养筛选得到的菌株，高压液相色谱法(HPLC)测定菌株对泰乐菌素降解情况。从中筛选得到一株对泰乐菌素有较好降解效果的菌株TYL-B35，经鉴定所得菌株为解糖假苍白杆菌(P.saccharolyticum)。将该菌株应用于泰乐菌素的降解，在LB液体培养基中振荡培养5d后，泰乐菌素的降解率可以达到96％以上。
下面具体描述本发明。
实施例1.泰乐菌素抗逆菌株的分离与筛选。
1)采集山东某散养型养鸡场浅表层(表面以下10cm)湿润土壤，密封于自封袋中，置于低温保温箱(箱中温度约4℃)中带回实验室。该养鸡场建成约5年，使用泰乐菌素作为禽类抗呼吸道感染的药物。所得土样带回实验室后，称取5.0g加入含有100mL 50mM无菌磷酸缓冲液(pH7.0)的500mL挡板摇瓶中，在30℃恒温摇床中振荡20min，转速180rpm。取悬浊液，分别稀释10倍、102倍、103倍、104倍，取100μL均匀涂布于含有50mg/L磷酸泰乐菌素的LB固体培养基(胰蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L，琼脂粉20g/L，121℃高温高压灭菌20min)平板上，30℃恒温静置培养2d。根据菌落形态、大小、颜色的不同，挑取菌落在含有50mg/L磷酸泰乐菌素的LB平板上划线，直至菌落形态单一。
2)挑取单菌落在含有更高浓度磷酸泰乐菌素(100mg/L)的LB固体培养基上划线，30℃恒温静置培养2d。依次将培养基中磷酸泰乐菌素升高至200mg/L、500mg/L，重复上述操作。通过以上操作得到的菌株初步认定为可耐受高浓度泰乐菌素。
将上述得到菌株，分别接种于含有500mg/L磷酸泰乐菌素的LB液体培养基中，在30℃恒温摇床中振荡(转速180rpm)培养5d。取2mL菌液，12000×g离心1min，收集上清，过0.22μm滤膜，HPLC法测定菌液中残留的泰乐菌素并计算降解率。以含有相同浓度的泰乐菌素但不接种菌落的LB液体培养基为对照。
HPLC条件如下：Waters 1525高效液相色谱仪配备W2489UVD双通道紫外可见检测器；色谱柱为Agilent HC-C18柱(5μm，250mm×4.6mm)；流动相为乙腈:KH2PO4溶液(0.02M，pH2.5)＝30:70(体积比)等度洗脱，流速1mL/min；柱温25℃；进样量20μL；检测波长285nm。
经过上述筛选步骤，最终得到1株可以高效降解泰乐菌素的菌株TYL-B35。提取TYL-B35菌株基因组，PCR扩增菌株的16S rDNA基因序列，PCR反应体系如下：
表1 PCR反应体系
27F(5'-AGAGTTTGATCCTGGCTCAG-3')和1492R(5'-TACCTTGTTACGACTT-3')是克隆细菌16S rDNA基因序列的通用引物。PCR条件如下：98℃预变性30s，98℃变性10s，52℃复性10s，72℃延伸10s，32个循环，72℃保真延伸3min。
PCR产物经琼脂糖凝胶电泳纯化后，送青岛擎科梓煕生物技术有限公司测序，序列如SEQID No.1所示。将序列在GenBank中比对，结果显示，TYL-B35序列与数据库中Pseudochrobactrum saccharolyticum 16S rDNA基因序列相似性高达99.8％，因此，TYL-B35鉴定为解糖假苍白杆菌(Pseudochrobactrum saccharolyticum)。该菌株于2019年12月9日，送交中国典型培养物保藏中心保藏，保藏编号为CCTCC NO:M20191028，保藏地址为中国，武汉，武汉大学。
实施例2.P.saccharolyticum TYL-B35对泰乐菌素的降解。
挑取实施例1筛选获得的P.saccharolyticum TYL-B35单菌落转接于5mL含有500mg/L磷酸泰乐菌素的LB液体培养基中，30℃和180rpm条件下振荡培养18h。分别吸取50μL菌液转接于5mL含有500mg/L磷酸泰乐菌素的LB液体培养基中。培养基分别装于两种小瓶中，两种小瓶均带有硅胶塞，一种容积为25mL(好氧培养)，另一种为5mL(兼性厌氧)，分别用于验证好氧和兼性厌氧条件下泰乐菌素的降解。接种好的小瓶分别置于30℃恒温摇床中以180rpm振荡培养5d，HPLC法测定菌液中残留的泰乐菌素并计算降解率，结果显示泰乐菌素的降解率达到96％以上。
以上所述实施例仅表达了本申请的具体实施方式，其描述较为具体和详细，但并不能因此而理解为对本申请保护范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本申请技术方案构思的前提下，还可以做出若干变形和改进，这些都属于本申请的保护范围。
核苷酸序列表
<110> 中国科学院青岛生物能源与过程研究所
<120> 一种泰乐菌素降解菌及其筛选方法与应用
<130>
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 1361
<212> DNA
<213> 16S rDNA基因序列
<400> 1
ttaactgcag tcgaacggtc tcttcggagg cagtggcaga cgggtgagta atgcatggga 60
atctaccgtt ctctacggaa taactcaggg aaacttgtgc taataccgta tacgcccttt 120
tggggaaaga tttatcggag aatgatgagc ccatgttgga ttagctagtt ggtagggtaa 180
aggcctacca aggcgacgat ccatagctgg tctgagagga tgatcagcca cactgggact 240
gagacacggc ccagactcct acgggaggca gcagtgggga atattggaca atgggcgcaa 300
gcctgatcca gccatgccgc gtgagtgatg aaggccctag ggttgtaaag ctctttcacc 360
ggtgaagata atgacggtaa ccggagaaga agccccggct aacttcgtgc cagcagccgc 420
ggtaatacga agggggctag cgttgttcgg atttactggg cgtaaagcgc acgtaggcgg 480
acttttaagt caggggtgaa atcccggggc tcaaccccgg aactgccttt gatactggaa 540
gtcttgagta tggaagaggt aagtggaatt gcgagtgtag aggtgaaatt cgtagatatt 600
cgcaggaaca ccagtggcga aggcggctta ctggtccatt actgacgctg aggtgcgaaa 660
gcgtggggag caaacaggat tagataccct ggtagtccac gccgtaaacg atgaatgtta 720
gccgtcgggg tgtttacact tcggtggcgc agctaacgca ttaaacattc cgcctgggga 780
gtacggtcgc aagattaaaa ctcaaaggaa ttgacggggg cccgcacaag cggtggagca 840
tgtggtttaa ttcgaagcaa cgcgcagaac cttaccagct cttgacatgc ctatgaatgt 900
tagtggagac actttcagcc tttcggggcg taggacacag gtgctgcatg gctgtcgtca 960
gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaaccctcg cctttagttg 1020
ccatcattta gttgggcact ctaaagggac tgccagtgat aagctggagg aaggtgggga 1080
tgacgtcaag tcctcatggc ccttacgggc tgggctacac acgtgctaca atggtggtga 1140
cagtgggcag cgagaccgcg aggtcgagct aatctccaaa agccatctca gttcggattg 1200
cactctgcaa ctcgagtgca tgaagttgga atcgctagta atcgcggatc agaatgccgc 1260
ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac accatgggag ttggttttac 1320
ccgaaggcgc tgtgctaacc gcaaggaggc agcgaccacc g 1361
<210> 2
<211> 20
<212> DNA
<213> 27F
<400> 2
agagtttgat cctggctcag 20
<210> 3
<211> 16
<212> DNA
<213> 1492R
<400> 3
taccttgtta cgactt 16
Claims (4)
1.一株泰乐菌素降解菌，其特征在于，所述泰乐菌素降解菌为解糖假苍白杆菌(Pseudochrobactrum saccharolyticum)TYL-B35，保藏编号为CCTCC NO:M20191028。
2.权利要求1所述泰乐菌素降解菌在泰乐菌素降解上的应用。
3.一种含有权利要求1所述泰乐菌素降解菌的菌剂。
4.权利要求3所述菌剂在泰乐菌素降解上的应用。
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