CN117210439B - 一种基于魔芋白绢病bj-y1菌株获得复合型糖苷水解酶的方法 - Google Patents
一种基于魔芋白绢病bj-y1菌株获得复合型糖苷水解酶的方法 Download PDFInfo
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- CN117210439B CN117210439B CN202311469124.XA CN202311469124A CN117210439B CN 117210439 B CN117210439 B CN 117210439B CN 202311469124 A CN202311469124 A CN 202311469124A CN 117210439 B CN117210439 B CN 117210439B
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Abstract
本发明提供了一种基于魔芋白绢病BJ‑Y1菌株获得复合型糖苷水解酶的方法，属于生物酶催化领域，将魔芋白绢病中BJ‑Y1菌株培养至Biolog FF板中并利用碳源布局图对结果进行比对与分析。利用分析结果对菌株BJ‑Y1采用单一诱导物进行水解酶的诱导发酵。发现BJ‑Y1菌株能分泌产生具有甘露聚糖酶、淀粉酶、果胶酶、纤维素酶和木聚糖酶特性的糖苷水解酶，相同催化反应pH在4‑6范围，反应温度在50℃时水解催化效果最高。范围内包括：1）制备与纯化；2）菌株碳源代谢谱分析；3）诱导物产酶分析。本发明利用BJ‑Y1菌株发酵能够产生复合型糖苷水解酶，此复合型糖苷水解酶具有甘露聚糖酶、木聚糖酶、纤维素酶和果胶酶的功能特性。
Description
技术领域
本发明属于生物酶催化领域，具体涉及一种基于魔芋白绢病BJ-Y1菌株获得复合型糖苷水解酶的方法。
背景技术
魔芋（Amorphophallus konjac）是天南星科魔芋属多年生草本植物，生长在少数亚洲国家，如中国，日本和韩国。魔芋是葡甘露聚糖来源的重要农作物之一，其球茎富含葡甘露聚糖，淀粉，维生素和矿物质。魔芋葡甘露聚糖的应用十分广泛，目前已被应用在医学、食品、化工等领域。然而随着土地种植年限以及土地使用情况和天气等因素作用下，魔芋在种植过程中会发生多种病害，导致产量降低，品质变坏，而白绢病则是危害魔芋健康的重要病害之一。
魔芋白绢病（Sclerotium rolfsii Sacc）无孢目小核菌属齐整小核菌，属于半知菌亚门丝孢纲引起的一种土传病害，通常发生在植物上，能够危害草药、蔬菜和农作物等。魔芋白绢病主要危害茎，叶柄基部和球茎，导致茎秆倒伏，叶柄基部或茎基染病后形成不规则斑，后软化致叶柄呈湿腐状。在土壤表层病面或茎基附近蔓延出大量白色绢丝状菌丝体和褐色菜籽状菌核，菌核初白色，后期变黄褐色或棕色。
1996年G.M Gübitz等人发酵培养植物担子菌，存纯化分离酸性α甘露聚糖酶研究其性质。在1998年第七届纸浆造纸工业生物技术国际会议中提及植物病原真菌为内切-β-1，4-D-甘露聚糖酶活性的优秀生产者，能够降解甘露聚糖。2007年Moussa等人对甜菜病原体（Sclerotium rolfsii Sacc）产生纤维素降解酶进行了研究，发现纤维素和木聚糖诱导了纤维素酶的合成，并且葡萄糖抑制了罗氏链球菌中的纤维素酶合成。2016年张桂宏等人对从君子兰中筛选出的罗耳阿太菌（Athelia rolfsii）进行发酵，并分离纯化出β-1,3葡聚糖酶。当前魔芋白绢病菌株的研究主要集中在生物学特性和分子鉴定、及其发生危害和防治等方面的工作。而针对于魔芋白绢病中的Athelia rolfsii菌株现阶段占无酶学性质的相关研究。
糖苷水解酶是一类水解糖苷键 (glycosidic bonds)的酶，在生物体糖和糖缀合物的水解与合成过程中扮演着重要角色。糖苷酶几乎存在于所有的生物体中，是一类以内切或外切方式水解各种含糖化合物 (包括单糖苷、寡糖、多糖、皂甙和糖蛋白等)中的糖苷键,生成单糖、寡糖或糖复合物的酶。甘露聚糖酶是一种多功能的促生长剂，来源广泛，包括植物、真菌、放线菌甚至软体动物，其中，微生物是产生β-甘露聚糖酶的主要来源。纤维素酶（β-1,4-葡聚糖-4-葡聚糖水解酶）是一种复合酶，主要由外切β-葡聚糖酶、内切β-葡聚糖酶和β-葡萄糖苷酶等组成。常作用于纤维素以及从纤维素衍生出来的产物且微生物纤维素酶在转化不溶性纤维素成葡萄糖以及在果蔬汁中破坏细胞壁从而提高果汁得率等方面具有非常重要的意义。木聚糖酶 (xylan)是一种存在于植物细胞壁中的异质多糖，是植物半纤维素(hemicellose)的主要成分。大多数木聚糖是一种结构复杂的、具有高度分枝的异质多糖，含有许多不同的取代基。淀粉酶 (amylase，AMY,AMS)一般作用于可溶性淀粉、直链淀粉、糖元等α-1，4-葡聚糖，水解α-1，4-糖苷键的酶。果胶酶是指分解植物主要成分—果胶质的酶类，广泛分布于高等植物和微生物中。
吉林农业大学对于罗尔阿太菌的研究在于β-1,3葡聚糖酶以及罗耳阿太菌多糖的提取优化。江南大学研究齐整小核菌制备β-1,3-葡寡糖与硬葡聚糖。天津科技大学对于齐整小核菌胞外产多糖培养基的优化。西南农业大学主要研究关于齐整小核菌木质纤维素降解能力及硬葡聚糖合成途径。现有研究中均是对齐整小核菌的单一酶学性质进行研究，暂未发现能降解多种底物的复合酶。
发明内容
本发明的目的是提供一种基于魔芋白绢病BJ-Y1菌株获得复合型糖苷水解酶的方法，通过分离魔芋白绢病中BJ-Y1菌株，发酵产生甘露聚糖酶、淀粉酶、果胶酶、纤维素酶和木聚糖酶的一类复合糖苷水解酶，该复合酶能够为降解魔芋粉、淀粉、果胶、纤维素等底物，为植物基材料的降解提供一个方便、快捷、高效的方法，对应用于食品、饲料行业具有重要价值。
本发明提供了一种基于魔芋白绢病BJ-Y1菌株得到复合型糖苷水解酶，包括如下步骤：
1）制备与纯化：
采取具有典型性状为倒伏腐烂的魔芋标本，从病患处与健康交界处切取病变组织块，挑起病变组织放置于PDA培养基上于30℃培养2天，挑起新生长菌丝多次转到筛选培养基上进行分离、纯化，取生长状态好的纯化菌株放置于4℃冰箱保存备用；
2）菌株碳源代谢分析：
挑取PDA上的BJ-Y1菌丝在Ⅱ级生物安全柜中制备菌悬液，用移液器将菌悬液接种至FF板上，在28℃下条件下培养5天，培养完毕后进行结果获取，利用碳源布局图对结果进行比对与分析；
3）诱导物产酶分析：
利用步骤2）的分析结果，在含有1%蛋白胨的液体培养基中分别加入1%浓度的魔芋粉、CM纤维素钠盐、淀粉、果胶和木聚糖五种底物，灭菌冷却后接种BJ-Y1菌株，放置在30℃，转速160r/min的摇床中培养7天，取发酵液在12000r/min条件下离心5min获得离心上清液，取上清液测定酶活，得到诱导物产酶分析结果为：BJ-Y1菌株能分泌产生具有甘露聚糖酶、淀粉酶、果胶酶、纤维素酶和木聚糖酶特性的糖苷水解酶；
得到最适pH分析结果为：pH在4-5的区间范围内此酶活性最高，相对酶活均高于50%；当pH水平大于6之后则整体相对酶活表现随着pH的升高而逐渐下降且均低于50%；
得到最适温度分析结果为：所述复合型糖苷水解酶最适温度范围为40℃-60℃，其中温度在50℃时酶活表现效果最佳，低于40℃，高于60℃时该复合型糖苷水解酶酶活表现效果较差。
一种魔芋白绢病BJ-Y1菌株，保藏编号为CGMCC No.40885，微生物分类命名为罗耳阿太菌Athelia rolfsii，保藏在中国微生物菌种保藏管理委员会普通微生物中心，保藏地址是北京市朝阳区北辰西路1号院3号，保藏时间为2023年09月25日。
与现有技术相比本发明的有益效果是：
1、利用魔芋白绢病BJ-Y1菌株发酵能够产生复合型糖苷水解酶，此复合型糖苷水解酶具有甘露聚糖酶、木聚糖酶、纤维素酶和果胶酶的功能。
2、此复合酶在酶学特性上表现为温度在40-60℃的范围内，pH在4-6的范围内酶活稳定性较好。
附图说明
图1是本发明一实施例中碳源代谢图谱的碳源代谢测试图；
图2是本发明一实施例中碳源代谢图谱的碳源分布位置图；
图3是本发明一实施例中摇瓶发酵形成的酶液进行的底物亲和能力测试图；
图4是本发明一实施例中摇瓶发酵形成的酶液进行的酶活性的测定图；
图5是本发明一实施例中最适反应pH图；
图6是本发明一实施例中最适反应温度图。
实施方式
下面结合附图所示的各实施方式对本发明进行详细说明，但应当说明的是，这些实施方式并非对本发明的限制，本领域普通技术人员根据这些实施方式所作的功能、方法、或者结构上的等效变换或替代，均属于本发明的保护范围之内。
本实施例提供了一种基于魔芋白绢病BJ-Y1菌株获得复合型糖苷水解酶的方法，来源于魔芋白绢病菌株BJ-Y1发酵出具多种糖苷水解功能的复合型酶，具有共有的催化反应pH和反应温度，和其他的复合酶具有明显的差异性。将魔芋白绢病中BJ-Y1菌株培养至Biolog FF板中并利用碳源布局图对结果进行比对与分析。利用分析结果对BJ-Y1菌株采用单一诱导物进行水解酶的诱导发酵。发现BJ-Y1菌株能够产生复合型糖苷水解酶，具体包括如下步骤：
1）制备与纯化：采取具有典型性状为倒伏腐烂的魔芋标本，从病患处与健康交界处切取病变组织块，挑起病变组织放置于PDA培养基上于30℃培养2天，挑起新生长菌丝多次转到筛选培养基上进行分离、纯化，取生长状态好的纯化菌株放置于4℃冰箱保存备用。
2）菌株碳源代谢分析：挑取PDA上的BJ-Y1菌丝在Ⅱ级生物安全柜中制备菌悬液，用移液器将菌悬液接种至FF板上（每孔100μl）。按照Biolog FF MicroPlate使用说明书完成操作。在28℃下条件下培养5天,培养完毕后使用BIOLOG鉴定仪MicrologTM3软件进行结果获取，利用碳源布局图对结果进行比对与分析。
3）诱导物产酶分析：利用步骤2）的分析结果，在含有1%蛋白胨的液体培养基中分别加入1%浓度的魔芋粉、CM纤维素钠盐、淀粉、果胶和木聚糖五种底物，灭菌冷却后接种BJ-Y1菌株，放置在30℃，转速160r/min的摇床中培养7天，取发酵液在12000r/min条件下离心5min获得离心上清液，取上清液测定酶活，得到诱导物产酶分析结果为：BJ-Y1菌株能分泌产生具有甘露聚糖酶、淀粉酶、果胶酶、纤维素酶和木聚糖酶特性的糖苷水解酶；
最适pH，最适温度分析：①分别用柠檬酸与磷酸氢二钾配制出pH为2.0、3.0、4.0、5.0、6.0、7.0、8.0、9.0的缓冲液。②取不同pH的缓冲液分别配制浓度为1%魔芋粉、CM纤维素钠盐、淀粉、果胶和木聚糖底物并做标记。③测定步骤，每个梯度依照如下步骤进行：取3支试管（2支为样品管，1支为空白管），分别加入1.8mL底物在50℃的水浴锅中预热5min，2支样品管中加入0.2mL酶液，在50℃水浴反应30min后，三支管中加入3mL的DNS，空白管补加0.2mL酶液，三支管放置于沸水浴中显色反应5min，冷却后蒸馏水定容至15mL摇匀，在540nm下测定其吸光度，判断最佳反应pH范围；
取柠檬酸与磷酸氢二钾pH为4的缓冲液，分别配制浓度为1%魔芋粉、CM纤维素钠盐、淀粉、果胶和木聚糖底物并做标记。取对应试管加入1.8mL底物与0.2mL酶液在10℃、20℃、30℃、40℃、50℃、60℃、70℃、80℃、水浴反应30min后加3mL的DNS沸水浴5min冷却后蒸馏水定容至15mL摇匀；以空白管调零，在540nm下比色分析；
得到最适pH分析结果为：pH在4-5的区间范围内此酶活性最高，相对酶活均高于50%；当pH水平大于6之后则整体相对酶活表现随着pH的升高逐渐下降且均低于50%；
得到最适温度分析结果为：所述此复合型糖苷水解酶最适温度范围为40℃-60℃，其中温度在50℃时酶活表现效果最佳，低于40℃，高于60℃时该复合型糖苷水解酶酶活表现效果较差。
一种魔芋白绢病BJ-Y1菌株，保藏编号为CGMCC No.40885，微生物分类命名为罗耳阿太菌Athelia rolfsii，保藏在中国微生物菌种保藏管理委员会普通微生物中心，保藏地址是北京市朝阳区北辰西路1号院3号，保藏时间为2023年09月25日。
魔芋白绢病BJ-Y1菌株，其中BJ-Y1为自编号。
由图1、图2可知 BJ-Y1菌株能利用D-半乳糖醛酸、D-核糖、D-木糖、乳果糖和麦芽三糖、D-阿洛酮糖、麦芽糖、肝糖、熊果苷、D-山梨醇、水苏糖。我们把这些利用碳源分类为五碳糖和六碳糖及其衍生聚糖类，其中D-核糖和D-木糖属于五碳糖；D-半乳糖醛酸、D-阿洛酮糖、熊果苷、D-山梨醇为六碳糖；乳果糖、麦芽二糖、麦芽三糖、水苏糖和肝糖是由六碳糖通过糖苷键醛缩反应形成的聚糖产物。而其他85种碳源却不能利用或利用率极低。
图3、图4分别为摇瓶发酵形成的酶液进行的底物亲和能力测试图和酶活性的测定图。由图3可知，BJ-Y1在魔芋粉培养基发酵分泌产生的水解酶能水解魔芋粉、果胶、CMC纤维素钠、木聚糖和淀粉底物，但对海藻酸钠多糖没有效果。根据酶的专一性，表明BJ-Y1菌株产生了一系列糖苷水解酶，才能降解淀粉、纤维素、葡甘露聚糖、木聚糖。为进一步探索不同多糖底物对BJ-Y1进行诱导产酶的特性，我们把BJ-Y1菌株分别接种到魔芋精粉、玉米淀粉、果胶、CMC纤维素和木聚糖的培养基中进行摇瓶发酵并测定酶活。结果表明BJ-Y1菌株分泌产生了具有甘露聚糖酶、淀粉酶、果胶酶、纤维素酶和木聚糖酶特性的糖苷水解酶。图4为在复合型糖苷水解酶中以甘露聚糖酶和淀粉酶活力最高。
图5为该复合酶的pH的分布，该复合酶在pH为4时表现的酶活性最为良好，得到最适pH分析结果为：pH在4-5的区间范围内此酶活性最高，相对酶活均高于50%。当pH水平大于6之后则整体相对酶活表现随着pH的升高而逐渐下降且均低于50%；
参图6所示，根据BJ-Y1菌株产酶性能表征验证出BJ-Y1菌株分泌的糖苷水解酶，与此同时对此复合糖苷水解酶的最适pH与最适温度进行分析。该复合酶稳定温度范围为40℃-60℃，其中温度在50℃时酶活表现效果最佳，低于40℃，高于60℃时该复合型糖苷水解酶酶活表现效果较差。
该基于魔芋白绢病BJ-Y1菌株得到复合型糖苷水解酶的方法具有如下技术效果：
1、利用魔芋白绢病BJ-Y1菌株发酵能够产生复合型糖苷水解酶，此复合型糖苷水解酶具有甘露聚糖酶、木聚糖酶、纤维素酶和果胶酶的功能特性。
2、此复合酶在酶学特性上表现为温度在40-60℃的范围内，pH在4-6的范围内酶活稳定性较好。
对于本领域技术人员而言，显然本发明不限于上述示范性实施例的细节，而且在不背离本发明的精神或基本特征的情况下，能够以其他的具体形式实现本发明。因此，无论从哪一点来看，均应将实施例看作是示范性的，而且是非限制性的，本发明的范围由所附权利要求而不是上述说明限定，因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。
Claims (2)
1.一种魔芋白绢病BJ-Y1菌株，其特征在于，保藏编号为CGMCC No.40885，微生物分类命名为罗耳阿太菌（Athelia rolfsii），保藏在中国微生物菌种保藏管理委员会普通微生物中心，保藏地址是北京市朝阳区北辰西路1号院3号，保藏时间为2023年09月25日。
2.一种基于权利要求1所述魔芋白绢病BJ-Y1菌株获得复合型糖苷水解酶的方法，其包括如下步骤：
在含有1%蛋白胨的液体培养基中分别加入1%浓度的魔芋粉、CM纤维素钠盐、淀粉、果胶和木聚糖五种底物，灭菌冷却后接种权利要求1所述的BJ-Y1菌株，放置在30℃，转速160r/min的摇床中培养7天，取发酵液在12000r/min条件下离心5min获得离心上清液，取上清液测定酶活，得到诱导物产酶分析结果为：BJ-Y1菌株能分泌产生甘露聚糖酶、淀粉酶、果胶酶、纤维素酶和木聚糖酶。
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