CN116656556B - 一株可以降解聚对苯二甲酸乙二醇酯的沙福芽孢杆菌及其应用 - Google Patents
一株可以降解聚对苯二甲酸乙二醇酯的沙福芽孢杆菌及其应用 Download PDFInfo
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Abstract
本发明公开了一株可以降解聚对苯二甲酸乙二醇酯的沙福芽孢杆菌及其应用。一株沙福芽孢杆菌(B.safensis)YX8,该菌株保藏于中国典型培养物保藏中心,保藏编号为CCTCCNO:M20222009,保藏日期为2023年12月23日。所述的沙福芽孢杆菌YX8在降解聚对苯二甲酸乙二醇酯中的应用。本发明沙福芽孢杆菌YX8及其菌剂适用于处理环境中PET废弃物造成的污染,尤其对于片状、颗粒状的PET的降解有效。因此,该菌株为PET的生物降解提供了新的材料,在废弃塑料回收利用、降解酶的发掘与改造等领域具备进一步研发和工程应用的前景。
Description
技术领域
本发明涉及一株可以降解聚对苯二甲酸乙二醇酯的沙福芽孢杆菌及其应用,属于微生物技术领域。
背景技术
聚对苯二甲酸乙二醇酯(PET)是对苯二甲酸(terephthalic acid,TPA)和乙二醇(Ethylene glycol,EG)以酯键依次连接形成的聚酯,因其力学性能良好,冲击强度高,同时耐折、耐油、耐脂肪、耐稀酸稀碱、耐大多数溶剂,所以它被广泛应用于包装业、电子电器、医疗卫生、建筑、汽车等领域。但PET骨架中存在重复的芳香族单元以及聚合物链有限的流动性,导致其难以降解。目前传统的PET废弃物处理方法是填埋和焚烧,这两种方法都对环境产生严重的负面影响。因此,生物降解法成为PET降解的新道路。生物降解法相对于传统方法来说有着绿色无污染及成本低等优势。生物降解PET通过寻找可降解PET的菌株来实现的。例如,Sulaiman等(Sulaiman et al..2012.Appl Environ Microbiol.78(5):1556-1562.Doi:10.1128/AEM.06725-11)通过宏基因组技术从堆肥的树叶中克隆并表达了LC-角质酶(LCC),该酶有较高的热稳定性(70℃时半衰期为40min)及PET降解活性(在50℃、pH8.0时,LCC的PET降解活性为12mg/h/mg)。Yoshida等(Yoshida et al..2016.Science.351(6278):1196-1199.Doi:10.1126/science.aad6359)从250个PET样品中筛选出菌株Ideonella sakaiensis 201-F6,并从中分离纯化了PET降解效率明显高于TfH、LCC和FsC的酶,命名为IsPETase。Yoshida等(Yoshida et al..2016.Science.351(6278):1196-1199.Doi:10.1126/science.aad6359)还从菌株Ideonella sakaiensis 201-F6中获得了能将MHET降解为TPA和EG的酶MHETase,当PETase和MHETase共同工作时,形成了一个完全降解PET的双酶系统。目前虽有PET降解菌株被报道,但是生物降解法仍未得到广泛应用,所以需要寻找更多的PET降解菌株来满足生物降解的需求。
发明内容
本发明要解决的技术问题是提供一种可降解聚对苯二甲酸乙二醇酯(PET)的沙福芽孢杆菌(B.safensis)。
本发明的另一目的是提供该菌株的应用。
本发明的目的可通过以下技术方案实现:
一株沙福芽孢杆菌(Bacillus safensis)YX8,该菌株保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:M 20222009,保藏日期为2023年12月23日。
本发明提供的可降解PET的沙福芽孢杆菌(Bacillus safensis)YX8,有鞭毛,在LB平板上菌落呈米白色,不透明,形态呈圆形,边缘整齐,菌落表面光滑湿润,稍有隆起,革兰氏染色阳性(图1)。
所述PET为PET纳米级颗粒或PET膜,PET纳米级颗粒制备是参考Rodríguez-Hernández等(Rodríguez-Hernández et al..2019.Environmental Science:Nano.6:2031–2036.Doi:10.1039/c9en00365g)的方法,将PET饮料瓶底部较厚的部分剪成细小碎片,进一步筛选获得小于0.2mm的PET碎片;随后称取1g PET碎片溶解在10mL浓度为90%(v/v)的三氟乙酸中50℃搅拌至完全溶解(大约需要2h),将溶解后的溶液静置过夜;第二天加入10mL浓度为20%(v/v)的三氟乙酸剧烈搅拌2h后静置过夜,取出后12000rpm,离心1h后弃上清加入100mL的0.5%十二烷基硫酸钠(SDS)重悬剧烈搅拌后进行超声处理,静置沉淀1h后取上层50mL悬浮液,即获得PET纳米级颗粒。
本发明提供一种菌剂,该菌剂含有上述的沙福芽孢杆菌。
优选地,所述菌剂含有所述沙福芽孢杆菌的活菌体、死菌体和发酵产物中的至少一种,优选为活菌体。
优选地,所述菌剂为液态菌剂和/或固态菌剂,优选为液态菌剂。
本发明提供一种降解聚对苯二甲酸乙二醇酯(PET)的方法,将上述沙福芽孢杆菌和/或上述的菌剂与PET接触。
优选地,所述塑料为薄膜或者纳米级颗粒。
优选地,所述接触的过程包括:将所述沙福芽孢杆菌接种至含有所述塑料的发酵培养基中进行发酵培养。
本发明的有益效果为:
本发明提供了可降解聚对苯二甲酸乙二醇酯(PET)的沙福芽孢杆菌菌株或者含有该菌株的菌剂。适用于处理环境中PET废弃物造成的污染,尤其对于片状、颗粒状的PET的降解有效。因此,该菌株为PET的生物降解提供了新的材料,在废弃塑料回收利用、降解酶的发掘与改造等领域具备进一步研发和工程应用的前景。
生物材料保藏
一株沙福芽孢杆菌YX8,分类学命名为沙福芽孢杆菌YX8 Bacillus safensisYX8,已于2022年12月23日保藏于中国典型培养物保藏中心,保藏编号为CCTCCNO:M20222009,保藏湖北省武汉市武昌区武汉大学校内(武汉大学第一附属小学对面),中国典型培养物保藏中心211室,邮政编码:430072。
附图说明
图1是实施例1中菌株YX8的菌株形态特征;
图2是实施例1中菌株YX8的系统发育树;
图3是实施例2中菌株YX8在不同温度条件下的生长曲线;
图4是实施例2中菌株YX8在不同pH条件下的生长曲线;
图5是实施例3中菌株YX8降解纳米级PET颗粒的产物HPLC图;
图6是实施例3中经过菌株YX8降解的PET膜光学照片(A)和电子显微镜照片(B)。
具体实施方式
下面结合具体实施例对本发明进行进一步的阐述。
下述实施例中所述实验方法,如无特殊说明,均为常规方法;所述试剂和材料,如无特殊说明,均可从商业途径获得。
实施例1:沙福芽孢杆菌(Bacillus safensis)的筛选及鉴定
具体步骤如下:
1、筛选
从垃圾处理厂中采集PET废弃物用于分离PET降解菌株。取适量PET废弃物用超纯水冲洗表面土样,随后按5%(v/v)的接种量接种至LB培养基(蛋白胨10.0g/L,酵母粉5.0g/L,NaCl 5.0g/L,调节pH至7.0)中,置于50℃、180rpm摇床振荡培养3d后按5%(v/v)的接种量接种至新鲜的100mL LB培养基中继续富集3d,按照该方法进行3次转接富集培养。取1mL富集液稀释至10-4-10-7倍梯度后涂布于PCL筛选平板(每100mL的LB固体培养基中添加2mL的PCL母液摇匀;PCL母液:称取0.22g PCL加入10mL DMSO置于70℃水浴锅直至完全溶解,用无菌的0.22μm有机相滤头过滤。)上,置于50℃培养箱培养约3d后取出;挑取有水解圈产生的菌株进行分离纯化,并将其接种至新鲜的PCL筛选平板上进行复筛,50℃培养3d后再次观察出圈情况,将复筛仍能产生水解圈的菌株用无菌牙签点至PCL平板上并测量水解圈直径和菌落直径,最终获得解圈直径和菌落直径最大的菌株YX8。
2.鉴定
提取菌株YX8的基因组总DNA进行16S rDNA的扩增和测序(由生工生物工程(上海)股份有限公司完成),采用细菌通用引物27F:5’-AGAGTTTGATCCTGGCTCAG-3’和1492R:5’-TACGGCTACCTTGTTACGACTT-3’作为扩增引物进行PCR扩增,将测序结果在EzBioCloud(https://www.ezbiocloud.net/)中进行核酸序列比对,结果显示与沙福芽孢杆菌模式菌FO-36b核酸序列相似度高达100%。将菌株YX8的16S rDNA序列(序列如SEQ ID NO.1所示)与其相似度高的菌株构建系统进化树(图2),结果显示菌株YX8与同B.safensis FO-36b属于一个分支。此外,还测定了菌株YX8的基因组序列,分别使用EzBioCloud(https://www.ezbiocloud.net/)和GGDC(http://ggdc.dsmz.de/distcalc2.php)计算了菌株YX8基因组与沙福芽孢杆菌模式菌FO-36b基因组间的平均核酸统一性(ANI)(Yoon etal..2017.Antonie Van Leeuwenhoek.110(10):1281-1286.Doi:10.1007/s10482-017-0844-4)和DNA-DNA杂交值(dDDH)(Meier-Kolthoff et al..2013.BMCBioinformatics.14:60.Published 2013Feb 21.Doi:10.1186/1471-2105-14-60),分别为97.64%和78.8%,通常将ANI值>95%、dDDH值≥70%的菌株的菌株定义为同一个种。
因此,鉴定菌株YX8属于沙福芽孢杆菌(Bacillus safensis),命名为沙福芽孢杆菌YX8。
实施例2:评估沙福芽孢杆菌(Bacillus safensis)YX8的生长特性
为了探究温度对菌株YX8生长的影响,将菌株YX8接种至液体LB培养基中,37℃、200rpm培养至对数期。以1%(v/v)转接量转接至30mL新鲜的液体LB培养基中,分别置于20℃、30℃、37℃、45℃及55℃摇床,200rpm培养,每隔2h取样测量菌液的OD600,每组设置3个重复。由图3可知,30℃、37℃、45℃条件下培养时菌株YX8生长状况较好,无明显生长延滞期,且30℃、37℃条件下培养至第4天时仍处于稳定期;20℃条件下培养时菌株YX8的生长出现了明显的延滞期,培养6h后才进入指数增长期;而55℃条件下培养时对菌株YX8生长影响较大,OD600几乎无增长。
为了探究初始pH对菌株YX8生长的影响,将菌株YX8接种至液体LB培养基中,37℃、200rpm培养至对数期。以1%((v/v)转接量转接至30mL初始pH值为3.0、4.0、5.0、6.0、7.0、8.0、9.0、10.0、11.0、12.0的液体LB培养基中,45℃、200rpm培养,每隔2h取样测量菌液的OD600,每组设置3个重复。由图4可知,pH 5.0-10.0范围内菌株YX8生长状况良好,培养24h后进入衰退期;pH 3.0-4.0范围内菌株YX8几乎未生长;pH 11.0-12.0时菌株YX8仍能生长但增长速度更慢,24h才进入稳定期。
根据以上结果,菌株YX8最适生长条件为30℃,pH 6.0。
实施例3:沙福芽孢杆菌(Bacillus safensis)YX8对PET塑料的降解能力
为探究菌株YX8对PET塑料的降解能力,将复筛得到的菌株接种至液体LB培养基中,培养至OD600=1.0时5000rpm、4℃离心5min收集菌体。用新鲜的LB培养基洗涤两次后将菌体用LB培养基重悬至OD600=3.0。将菌悬液接种至含15mg PET纳米级颗粒的30mL液体LB培养基中至OD600=1.0,45℃、200rpm条件下培养15d后取5mL菌液,12000rpm离心10min取上清转移至另一离心管中。用盐酸调节至pH=2.0后加入两倍体积的乙酸乙酯进行萃取,取上层溶液置于通风橱中风干,最后加入500μL甲醇重悬样品经0.22μm有机相滤膜过滤后利用高效液相色谱(high performance liquid chromatography,HPLC)进行检测。
利用HPLC检测降解产物的产生,由图5可知,经菌株YX8处理后的样品中检测到了新的产物峰。其中代谢产物Ⅰ保留时间(9.300min)与标准样品TPA保留时间(9.300min)一致;代谢产物Ⅱ保留时间(10.240min)与标准样品MHET保留时间(10.233min)基本一致。代谢产物Ⅲ保留时间(10.763min)与标准样品BHET保留时间(10.690min)基本一致。这些结果表明菌株YX8具有PET纳米级颗粒降解能力。
为探究菌株YX8的半结晶PET降解能力,将PET膜(购自德国Goodfellow GmbH)(Weiet al..2014.AMB Express.2014.4:44.Published 2014Jun 3.Doi:10.1186/s13568-014-0044-9)与菌株YX8进行孵育,由图6A可以看出,经过2个月的处理后,与对照组相比经菌株YX8处理后PET膜表面一部分由透明变成了白色且产生了肉眼可见的类似划痕的侵蚀痕迹,由图6B知,在电子显微镜下观察菌株YX8处理后的PET膜表面,发现PET膜表面产生了多个侵蚀斑,且在侵蚀斑表面有多个块状结构,推测该块状结构是PET解聚产生的;放大5000倍后观察到侵蚀斑与周围形成了明显的分界,进一步放大观察发现侵蚀斑处PET膜产生明显裂痕。这表明菌株YX8具有PET膜降解能力。
以上详细描述了本发明的优选实施方式,但是,本发明并不限于此。在本发明的技术构思范围内,可以对本发明的技术方案进行多种简单变型,包括各个技术特征以任何其它的合适方式进行组合,这些简单变型和组合同样应当视为本发明所公开的内容,均属于本发明的保护范围。
Claims (8)
1.一株沙福芽孢杆菌(Bacillus safensis)YX8,其特征在于,所述沙福芽孢杆菌保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:M 20222009,保藏日期为2022年12月23日。
2.权利要求1所述的沙福芽孢杆菌YX8在降解聚对苯二甲酸乙二醇酯中的应用。
3.根据权利要求2所述的应用,其特征在于,权利要求1所述的沙福芽孢杆菌YX8在液体或固体培养基中降解聚对苯二甲酸乙二醇酯。
4.根据权利要求3所述的应用,其特征在于,降解产物为对苯二甲酸和乙二醇。
5.一种菌剂,其特征在于,该菌剂含有权利要求1所述沙福芽孢杆菌YX8的活菌体。
6.根据权利要求5所述的菌剂,其特征在于,所述菌剂为液态菌剂和/或固态菌剂。
7.一种降解塑料的方法,其特征在于,该方法包括:将权利要求1所述的沙福芽孢杆菌YX8和/或权利要求5所述的菌剂与聚对苯二甲酸乙二醇酯塑料接触。
8.根据权利要求7所述的方法,其特征在于,所述塑料为聚对苯二甲酸乙二醇酯薄膜或者聚对苯二甲酸乙二醇酯纳米级颗粒。
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