CN107236673A - 一种快速准确筛选产漆酶真菌的有效方法 - Google Patents

一种快速准确筛选产漆酶真菌的有效方法 Download PDF

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CN107236673A
CN107236673A CN201710434845.5A CN201710434845A CN107236673A CN 107236673 A CN107236673 A CN 107236673A CN 201710434845 A CN201710434845 A CN 201710434845A CN 107236673 A CN107236673 A CN 107236673A
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culture medium
fungi
quick
laccase
effective ways
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祝嫦巍
黄守程
汪雁
马玉涵
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Anhui University of Science and Technology
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Abstract

本发明公开了一种快速准确筛选产漆酶真菌的有效方法，依次包括以下步骤：1）在培养皿中配置PDA培养基，其中加入适量的ABTS，使得ABTS的终浓度在0.01~0.03%之间；2）将植物材料（如枯枝、落叶、树皮、作物秸秆、植物根部等）剪成合适的小片段并放在上述培养基中，37摄氏度恒温培养1~48小时；3）植物器官小片段周围的培养基呈现绿色或者紫色反应，则表明此处有产漆酶真菌；4）从有颜色反应处的培养基上挑选真菌菌丝，继续培养即可纯化，得到产漆酶真菌。

Description

一种快速准确筛选产漆酶真菌的有效方法
技术领域
本发明涉及一种产漆酶真菌的筛选方法，具体的是一种快速从环境中筛选产漆酶真菌的有效方法。
背景技术
漆酶是一种含铜多酚氧化酶，在真菌中普遍存在。漆酶因其底物范围很宽而被广泛应用于纸浆漂白、
染料脱色、有机物合成、木质素降解等工业及生物技术领域，具有非常重要的经济价值和社会价值。由于不同应用领域要求不同酶学性质的漆酶，因此通过开发新的漆酶基因资源来获得具有多样性的漆酶意义重大。漆酶真菌具有基因多样性的特点为我们广泛筛选产漆酶真菌提供了无限的可能。传统的筛选产漆酶真菌都是采用愈创木酚法进行筛选，利用愈创木酚与漆酶发生变色反应筛选产漆酶真菌，具体做法是采集到的样品用无菌水分别稀释成不同梯度后涂布接种于愈创木酚-PDA 平板上，恒温培养，然后挑选能使愈创木酚发生褐变的菌株，进一步划线、分离、纯化。此方法虽然为通用方法，但操作起来费时费力，效率不高；此外，愈创木酚法的灵敏度不高，即如果真菌的产漆酶量很低，显色反应不明显。因此，寻找一种快速灵敏的筛选产漆酶真菌的有效方法，对提高科研人员的研究效率具有重要的意义。
发明内容
本发明专利目的是为解决科学研究中快速有效筛选产漆酶真菌提供一个新型的方法，解决目前科学研究过程中采用常规方法筛选产漆酶真菌效率和灵敏度不高的问题。
本发明专利通过以下技术方案来实现：在培养皿中配置PDA培养基，其中加入适量的2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐，即ABTS，使得ABTS的终浓度在0.01~0.03%之间。
将植物材料（如枯枝、落叶、树皮、作物秸秆、植物根部等）剪成合适的小片段，长度不超过培养皿的直径；然后将剪碎的植物器官小片段放在上述配置的培养基中，37摄氏度恒温培养1~48小时。若植物器官小片段周围的培养基呈现绿色或者紫色反应，则表明此处有产漆酶真菌。
从有颜色反应处的培养基上挑选真菌菌丝，继续培养即可纯化。
采用上述设计方案的效果：通过以上方案的实施，能够快速灵敏的筛选出产漆酶真菌，且阳性率为100%。
附图说明
图1展示的是采用本发明专利方法筛选产漆酶真菌的结果。
具体实施方式
下述实施例中所用方法如无特别说明均为常规方法。
实施例1：首先配置含0.01% ABTS的PDA培养基，然后采集的枯枝剪成长约2cm的小片段，挑选6根放置于配好的培养基上，1小时后发现其中5个枯枝小片段旁边的培养基出现紫色反应。37摄氏度恒温培养12小时后切取菌丝体纯化培养，经分子生物学方法鉴定，均属于产漆酶真菌。
实施例2：配置含0.015% ABTS的PDA培养基，然后采集的树皮剪成长宽约1.5cm的小块，挑选4片放置于配好的培养基上，37摄氏度恒温培养18小时后发现4片树皮小块旁边的培养基出现绿色反应。切取菌丝体纯化培养，经分子生物学方法鉴定，均属于产漆酶真菌。
实施例3：配置含0.028% ABTS的PDA培养基，然后采集的水稻秸秆剪成长约3cm的小片段，挑选8根放置于配好的培养基上，37摄氏度恒温培养35小时后发现其中3根水稻秸秆旁边的培养基出现紫色反应。切取菌丝体纯化培养，经分子生物学方法鉴定，均属于产漆酶真菌。
实施效果：采用上述方案筛选产漆酶真菌，无需事先对材料进行复杂的预处理，只需要将剪碎的植物材料放在含有ABTS的培养基上，一旦出现绿色或者紫色反应，即可百分之百认定该菌是产漆酶真菌。因此，筛选的灵敏度和效率都大大的提高了。

Claims (4)

1.一种快速准确筛选产漆酶真菌的有效方法，其特征在于以下步骤：1）在培养皿中配置PDA培养基，其中加入适量的ABTS，使得ABTS的终浓度在0.01~0.03%之间；2）将植物材料（如枯枝、落叶、树皮、作物秸秆、植物根部等）剪成合适的小片段并放在上述配置的培养基中，37摄氏度恒温培养1~48小时；3）植物器官小片段周围的培养基呈现绿色或者紫色反应，则表明此处有产漆酶真菌；4）从有颜色反应处的培养基上挑选真菌菌丝，继续培养即可纯化。
2.按照权利要求1所述的一种快速准确筛选产漆酶真菌的有效方法，其特征在于：植物材料无需复杂的预处理，只需要剪成小段即可。
3.按照权利要求1所述的一种快速准确筛选产漆酶真菌的有效方法，其特征在于：只要植物材料周围的培养基出现绿色或者紫色反应，即可断定有产漆酶真菌。
4.按照权利要求1所述的一种快速准确筛选产漆酶真菌的有效方法，其特征在于：所述的材料包括但不限于上述提及的植物材料。
CN201710434845.5A 2017-06-10 2017-06-10 一种快速准确筛选产漆酶真菌的有效方法 Pending CN107236673A (zh)

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Citations (4)

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Publication number Priority date Publication date Assignee Title
CN1858236A (zh) * 2006-04-07 2006-11-08 中国科学院南京土壤研究所 漆酶高产菌的快速筛选方法
CN1948453A (zh) * 2005-10-11 2007-04-18 中国科学院过程工程研究所 从盐肤木筛选漆酶内生菌及通过固态发酵制漆酶的方法
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CA2580211C (en) * 2004-09-21 2015-12-01 Ab Enzymes Oy Novel laccase enzyme and use thereof
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Application publication date: 20171010