CN111411068B - 一种耐盐纤维素分解菌的快速分离方法 - Google Patents
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Abstract
一种耐盐纤维素分解菌的快速分离方法，样品中的耐盐菌株的分离与纯化；用荧光法快速检测筛选得到菌株的β‑葡萄糖苷酶活性；将不同β‑葡萄糖苷酶活性的菌株接入羧甲基纤维素钠（CMC‑Na）液体培养基，观察菌株生长情况，并测其粘度。本发明高效批量、操作简单和方便快捷等优点，大大提高了工作效率，结果准确可靠。
Description
技术领域
本发明涉及生物技术领域，具体涉及一种耐盐纤维素分解菌的快速分离方法。
背景技术
我国盐碱土壤面积大，分布广泛，主要分布于东北、华北、西北及沿海地区，是重要的后备耕地资源。盐碱土壤理化性质差，土壤肥力及微生物活性较正常土壤低，不利于植物生长。盐碱土壤重要的营养来源是植物凋落物，其最主要成分是纤维素。因而分离能够降解纤维素的耐盐微生物对于盐碱土壤的营养循环具有重要意义。
纤维素是构成植物细胞壁的主要物质，广泛分布于自然界中，是世界上最丰富的天然有机物，也是重要的可再生资源。纤维素是重要的造纸原料，以纤维素为原料的产品也广泛应用于塑料、化工等领域。纤维素中存在许多高能的氢键，使其水解、利用均很困难，且不完全利用会对环境造成一定的污染。目前，降解纤维素主要有两种方法，一是物理化学法，但存在设备复杂且成本高的缺点；二是微生物法，该方法因高效且易于操作的优点而受到重视。
纤维素酶是指能水解纤维素的β-1,4葡萄糖苷键，使纤维素变成纤维二糖和葡萄糖的一组酶的总称。它不是单一酶，目前一般认为：纤维素酶包括3类，一是内切1,4-β-葡聚糖苷酶，它随机切割纤维素多糖链内部的无定型区；二是纤维二糖酶，它从纤维素多糖链的末端切割释放聚糖,是一种外切葡聚糖酶；三是β-葡萄糖苷酶,它水解纤维二糖和其他水溶性的纤维糊精产生形成葡萄糖，是目前研究最广泛、使用最多的一种纤维素酶。微生物不能直接利用外界环境中的纤维素，只有通过分泌胞外酶将外界环境中的纤维素分解成可以直接吸收利用的小分子。在自然环境中，有大量微生物能够分泌可用于降解天然纤维素的纤维素酶，在真菌、放线菌和细菌中均发现有纤维素酶的存在。
关于纤维素降解菌株分离的研究很多，但研究具有耐盐特性的纤维素降解菌较少，在盐环境下高浓度盐对常规纤维素降解菌活性有强烈抑制作用，不能正常发挥其作用，因此，筛选适应盐碱地区的高效耐盐纤维素降解菌株十分必要。
发明内容
解决的技术问题：本发明公开了一种耐盐纤维素分解菌的快速分离方法，该方法用高盐培养基筛选出盐碱土壤中的耐盐菌株，用荧光法快速检测菌株β-葡萄糖苷酶的活性，筛选出β-葡萄糖苷酶活性较高的耐盐菌株，用高盐度的羧甲基纤维素钠液体培养基的粘度来表征其纤维素降解能力，从而获得具有降解纤维素能力的耐盐菌株及特性。
技术方案：一种耐盐纤维素分解菌的快速分离方法，包括以下步骤：(1)样品中的耐盐菌株的分离与纯化：称取1g盐碱土，加入9mL无菌水，30℃恒温振荡30min，取样品悬液，用无菌水稀释成10－1、10－2、10－3的溶液，分别取200μL稀释液在盐浓度为2.5％的LB固体培养基上均匀涂布，置于30℃恒温培养箱培养2～3天，直至长出单菌落，将单菌落划线纯化培养，得到纯菌株，挑取纯菌株接种于液体培养基进行富集培养；(2)用荧光法快速检测筛选得到菌株的β-葡萄糖苷酶活性：取1mL菌液，5000rpm离心5min使菌体沉淀，弃上清液，加1mL无菌水，涡旋1min，用移液枪取菌悬液100μL加到96孔板中，然后加MES缓冲液，最后加β-葡萄糖苷酶底物溶液，用移液枪混合均匀，25℃-30℃室温培养30min，4000rmp离心4min，进行荧光检测，同时做平行、标曲和底物对照实验；(3)将不同β-葡萄糖苷酶活性的菌株接入羧甲基纤维素钠(CMC-Na)液体培养基，观察菌株生长情况，并测其粘度：选取β-葡萄糖苷酶活性不同的菌株，接入羧甲基纤维素钠(CMC-Na)液体培养基，置于30℃恒温培养箱培养3-5天，用乌氏黏度计测量培养基粘度，用培养基粘度表征菌株在CMC-Na培养基中降解纤维素的能力。
上述盐浓度为2.5％的LB固体培养基组分为：胰蛋白胨10g，酵母提取物5g，氯化钠25g，琼脂粉10-20g，加去离子水至1L，调节pH至7.4。
上述羧甲基纤维素钠液体培养基的组成为：CMC-Na 20g，Na2HPO4 2.5g，KH2PO41.5g，蛋白胨2.5g，加去离子水至1L，调节pH至7.0-7.5。
上述CMC-Na液体培养基组分为：CMC-Na 20g，Na2HPO4 2.5g，KH2PO4 1.5g，蛋白胨2.5g，根据比例分别加入NaCl，去离子水至1L，得到盐浓度为0wt.％、0.5wt.％、1wt.％、1.5wt.％、2wt.％、3wt.％的CMC-Na液体培养基，调节pH至7.0-7.5。
有益效果：本发明提供了一种快速分离耐盐纤维素分解菌的方法，通过培养基初筛，再测定酶活，最后用CMC-Na液体培养基的粘度变化来验证(表1)，传统的传统纤维素分解菌的分离方法包括选择培养基初筛、刚果红染色复筛、测定发酵培养基酶活进行验证。本发明的分离方法与传统的方法相比，具有能定量菌株酶活、高效批量、操作简单和方便快捷等优点，大大提高了工作效率，结果准确可靠，且与传统方法具有可比性，两种方法具体的比较如表2所示。
表1
表2
本发明中筛选获得的菌株具有耐盐及分解纤维素的特性，在高盐环境下可以发挥降解纤维素的能力。
附图说明
图1为筛选获得耐盐菌株β-葡萄糖苷酶活性图。
图2为15株菌在CMC-Na液体培养基中菌液的特性粘度图。
图3为15株菌的β-葡萄糖苷酶活性和菌液的特性粘度相关性图。
图4为3菌株在不同盐浓度CMC-Na液体培养基的菌液的特性粘度图。
图5为3菌株在不同盐浓度CMC-Na液体培养基的菌液的特性粘度的变化图。
具体实施方式
下面结合附图和具体实施方式对技术方案作进一步详细说明。本发明所选的目标样品为盐碱土壤，对非本发明所述样品中的纤维素降解菌的分离具有通用性，可参考本实施方式进行。以下实施方式仅用于说明本发明，而不用于限制本发明所要求保护的范围。
实施例1：样品中的耐盐菌株的分离与纯化
称取1g盐碱土，加入用9mL无菌水，30℃恒温振荡30min，使样品充分散开，取样品悬液，用无菌水稀释成10－1、10－2、10－3的溶液，分别取200μL稀释液在盐浓度为2.5wt.％的LB固体培养基均匀涂布上，置于30℃恒温培养箱，其中2.5％的LB固体培养基配制方法为胰蛋白胨10g，酵母提取物5g，氯化钠25g，琼脂粉10-20g，加去离子水至1L，调节pH至7.4，121℃灭菌20min。培养2～3天，直至长出单菌落，将单菌落划线纯化培养，得到纯菌株，挑取纯菌株接种于液体培养基进行富集培养。本实施例共分离出29株耐盐菌株。
实施例2：用荧光法快速检测筛选得到菌株的β-葡萄糖苷酶活性
取1mL菌液，5000rpm离心5min使菌体沉淀，弃上清液，加1mL无菌水，涡旋1min，用移液枪取菌悬液100μL加到96孔板中，然后加缓冲液，加β-葡萄糖苷酶底物溶液，用移液枪混合均匀，25℃-30℃室温培养30min，4000rmp离心4min,进行荧光检测。结果显示有8株编号为1-1、1-2、1-9、1-11、1-15、3-6、3-8和3-13的菌没有β-葡萄糖苷酶活性，其余菌株酶活结果如图1所示。
实施例3
选取β-葡萄糖苷酶活性不同的15株菌，接入羧甲基纤维素钠(CMC-Na)液体培养基，对照不接菌株(CK)置于30℃恒温摇床培养3-5天，用培养基粘度表征菌株在CMC-Na培养基中降解纤维素的能力。β-葡萄糖苷酶活性不同的15株菌的菌液特性粘度如表3和图2所示，β-葡萄糖苷酶活性和菌液的特性粘度相关性如图3所示。
表3
其中，CMC-Na液体培养基：CMC-Na 20g，Na2HPO4 2.5g，KH2PO4 1.5g，蛋白胨2.5g，加去离子水至1L，调节pH至7.0-7.5，121℃灭菌20min。
实施例4
选取β-葡萄糖苷酶活性较高的菌株1-16、1-6和1-13，分别接入盐浓度为0wt.％、0.5wt.％、1wt.％、1.5wt.％、2wt.％、3wt.％的CMC-Na液体培养基，对照不接菌株(CK)，置于30℃恒温摇床，待菌株生长3-5天后，用乌氏黏度计测量培养基粘度。1-16、1-6和1-13菌株在不同盐浓度CMC-Na液体培养基的菌液的特性粘度如图4和图5所示。3株菌均在含盐量为1wt.％的CMC-Na培养基中生长的菌液特性粘度最低，说明该条件下这三株菌生长最好，具有较好的降解纤维素的能力，其中菌株1-16在各盐浓度的CMC-Na培养基中生长的菌液特性粘度较低，表明其在各盐浓度的CMC-Na培养基中生长较好，在高盐环境下降解纤维素的能力也较好。
其中，不同盐浓度CMC-Na液体培养基：CMC-Na 20g，Na2HPO4 2.5g，KH2PO4 1.5g，蛋白胨2.5g，分别加入0wt.％、0.5wt.％、1wt.％、1.5wt.％、2wt.％、3wt.％的NaCl，去离子水至1L，配制成盐浓度为0wt.％、0.5wt.％、1wt.％、1.5wt.％、2wt.％、3wt.％的CMC-Na液体培养基，调节pH至7.0-7.5，121℃灭菌20min。
Claims (4)
1.一种耐盐纤维素分解菌的快速分离方法，其特征在于包括以下步骤：
(1)样品中的耐盐菌株的分离与纯化：称取1g盐碱土，加入9mL无菌水，30℃恒温振荡30min，取样品悬液，用无菌水稀释成10-1、10-2、10-3的溶液，分别取200μL稀释液在盐浓度为2.5％的LB固体培养基上均匀涂布，置于30℃恒温培养箱培养2～3天，直至长出单菌落，将单菌落划线纯化培养，得到纯菌株，挑取纯菌株接种于液体培养基进行富集培养；
(2)用荧光法快速检测筛选得到菌株的β-葡萄糖苷酶活性：取1mL菌液，5000rpm离心5min使菌体沉淀，弃上清液，加1mL无菌水，涡旋1min，用移液枪取菌悬液100μL加到96孔板中，然后加MES缓冲液，最后加β-葡萄糖苷酶底物溶液，用移液枪混合均匀，25℃-30℃室温培养30min，4000rmp离心4min，进行荧光检测，同时做平行、标曲和底物对照实验，用菌株的β-葡萄糖苷酶活性表征菌株的纤维素能力；
(3)将不同β-葡萄糖苷酶活性的菌株接入羧甲基纤维素钠(CMC-Na)液体培养基，观察菌株生长情况，并测其粘度：选取β-葡萄糖苷酶活性不同的菌株，接入羧甲基纤维素钠(CMC-Na)液体培养基，置于30℃恒温培养箱培养3-5天，用乌氏黏度计测量培养基粘度，用培养基粘度表征菌株在CMC-Na培养基中降解纤维素的能力。
2.根据权利要求1所述耐盐纤维素分解菌的快速分离方法，其特征在于所述盐浓度为2.5％的LB固体培养基组分为：胰蛋白胨10g，酵母提取物5g，氯化钠25g，琼脂粉10-20g，加去离子水至1L，调节pH至7.4。
3.根据权利要求1所述耐盐纤维素分解菌的快速分离方法，其特征在于所述羧甲基纤维素钠液体培养基的组成为：CMC-Na 20g，Na2HPO4 2.5g，KH2PO4 1.5g，蛋白胨2.5g，加去离子水至1L，调节pH至7.0-7.5。
4.根据权利要求1所述耐盐纤维素分解菌的快速分离方法，其特征在于所述羧甲基纤维素钠液体培养基的组成为：CMC-Na 20g，Na2HPO4 2.5g，KH2PO4 1.5g，蛋白胨2.5g，根据比例分别加入NaCl，去离子水至1L，得到盐浓度为0wt.％、0.5wt.％、1wt.％、1.5wt.％、2wt.％、3wt.％的羧甲基纤维素钠液体培养基，调节pH至7.0-7.5。
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