CN107129958B - 一种β-甘露聚糖酶工程菌的筛选方法 - Google Patents

一种β-甘露聚糖酶工程菌的筛选方法 Download PDF

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CN107129958B
CN107129958B CN201710432097.7A CN201710432097A CN107129958B CN 107129958 B CN107129958 B CN 107129958B CN 201710432097 A CN201710432097 A CN 201710432097A CN 107129958 B CN107129958 B CN 107129958B
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李爽
樊吴迪
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Abstract

本发明公开了一种β‑甘露聚糖酶工程菌的筛选方法，包括如下步骤：(1)将β‑甘露聚糖酶的表达菌在含有台酚蓝的KT平板上培养，直至平板上产生水解圈；(2)测量水解圈直径与微生物菌落直径，以二者大小的比值作为β‑甘露聚糖酶的酶活指数，该指数与酶活正相关。本发明无需对工程菌裂解，即可实现胞内β‑甘露聚糖酶酶活的快速检测。所需步骤简单，耗时短，成本低廉，且利于高通量筛选。

Description

一种β-甘露聚糖酶工程菌的筛选方法
技术领域
本发明涉及一种β-甘露聚糖酶突变体的原位快速半定量筛选方法，属于生物工程领域。
背景技术
β-甘露聚糖酶(β-mannanase,EC 3.2.1.78)属于半纤维素酶，可以在β-1,4-D-甘露聚糖分子的主链内部随机切割β-1,4-D-甘露糖苷键，从而产生不同长度的低聚甘露糖。低聚甘露糖作为添加剂在动物饲料行业有广泛的应用，也是膳食纤维的重要成分，此外在造纸、纺织业、医学、药学、食品及石油开采等领域也具有重要的应用价值。
大肠杆菌表达β-甘露聚糖酶具表达量高、培养周期短、成本低等优势。但由于重组的β-甘露聚糖酶多为胞内表达，而底物甘露聚糖属于大分子化合物，无法自由进出大肠杆菌细胞壁，酶活的检测通常需要对细胞进行繁琐、耗时、耗能的破壁处理。特别的，当对β-甘露聚糖酶引入随机突变，而这种突变库通常达到103以上数量级，对每个突变体逐一进行细胞破壁、重组酶纯化、酶活检测，检测周期以及人力、物力需要都非常巨大，无法高效的实现突变体库的大规模筛选。
发明内容
本发明的目的是提供一种基于大肠杆菌重组表达β-甘露聚糖酶酶活的半定量筛选方法，该方法无需细胞破壁、蛋白纯化，且可以批量处理，大大缩短了突变体的初筛时间，提高了效率，且操作简便、快速，成本低廉，有利于β-甘露聚糖酶突变体库的高效筛选获得候选突变体。
为实现发明目的采用如下技术方案：
一种β-甘露聚糖酶工程菌的筛选方法，包括如下步骤：
(1)将β-甘露聚糖酶的表达菌在含有台酚蓝的KT平板上培养，直至平板上产生水解圈；
(2)测量水解圈直径与微生物菌落直径，以二者大小的比值作为β-甘露聚糖酶的酶活指数(EI)，该指数与酶活正相关。
Figure BDA0001317626280000021
步骤(1)所述的培养包括三个阶段：第一阶段为37±2℃保温6-12h；第二阶段为25±2℃保温1-3h；第三阶段为30-60℃保温8-20h。
所述第一阶段为37℃保温10h。
所述第二阶段为25℃保温2h。
所述第三阶段的温度为37℃～50℃。
所述第三阶段的保温时间为12h。
所述KT平板的原料组分为：1-2％蛋白胨、0.5-1％酵母提取物、1％氯化钠、1-3％琼脂和0.3-1％魔芋胶溶液。
所述KT平板的配制：将上述原料混合后115℃条件下高压灭菌20min，冷却后加入预先配制好的1％台酚蓝溶液至终浓度为0.02-0.05％。
所述β-甘露聚糖酶表达菌，其宿主菌为E.coli BL21(DE3)或E.coli BL21。
所述β-甘露聚糖酶表达菌，其表达载体为pET30(a)。
本发明的基本原理是台酚蓝可结合甘露聚糖将其染成蓝色，而β-甘露聚糖酶具有降解甘露聚糖的能力，从而在KT平板上产生透明水解圈，且透明圈直径/微生物菌落直径的比值(EI值)与酶活力正相关。
与现有技术相比，本发明具有如下有益效果：
(1)无需对工程菌裂解，即可实现胞内β-甘露聚糖酶酶活的快速检测。所需步骤简单，耗时短，成本低廉，且利于高通量筛选。
(2)通过透明圈直径/微生物菌落直径的比值(EI值)的检测，可以半定量检测工程菌内β-甘露聚糖酶酶活，有利于该酶突变体的高通量筛选的展开。
附图说明
图1为β-甘露聚糖酶胞内产生菌的筛选平板的应用图。A-C：平板在37℃培养10h，后继续在25℃培养2h拍照；D-F：将A-C分别对应转移至50、37和25℃培养12h。其中“0”表示的是E.coli BL21(DE3)，1、2、3、4、5标号表示E.coli BL21(DE3)/pET30-man25。
具体实施方式
下面结合具体实施例对本发明作进一步具体详细描述，但本发明的实施方式不限于此，对于未特别注明的工艺参数，可参照常规技术进行。
实施例1
一、重组β-甘露聚糖酶活性初步原位验证
首先配制筛选用KT平板。配制1％台酚蓝溶液，过滤除菌以备用。配制含1％蛋白胨、0.5％酵母提取物、1％氯化钠、1.5％琼脂和0.5％魔芋胶溶液，于115℃灭菌20分钟后加入一定量的1％台酚蓝溶液至终浓度为0.03％。每20mL培养基铺制一块90mm平板。台酚蓝与魔芋胶可以紧密结合，配制好的平板呈均匀蓝色。
用无菌牙签挑取本实验室保存的β-甘露聚糖酶表达菌株E.coli BL21(DE3)/pET30-man25与E.coli BL21(DE3)到新制备的KT平板上。其中，E.coli BL21(DE3)作为对照组，β-甘露聚糖酶表达菌株E.coli BL21(DE3)/pET30-man25是将β-甘露聚糖酶编码基因插入到pET30a表达载体Nde I和Xho I酶切位点中构建获得。
按照以下方案进行β-甘露聚糖酶的表达和活性半定量筛选：
1.菌体培养。37℃培养箱内培养10h。
2.β-甘露聚糖酶表达。25℃培养箱内培养2h。
3.β-甘露聚糖酶催化反应。将KT平板分别转移到25、37或50℃培养箱内，继续培养12h。
此时平板上呈现出不同大小、亮度的水解圈(图1)。结果表明，在50℃条件下进行酶解反应，水解圈直径最大、最明显。后续酶活半定量检测将采用此条件进行。
二、重组β-甘露聚糖酶紫外诱变
取对数生长中期的5mLE.coli BL21(DE3)/pET30-man25菌液，添加至无菌培养皿中，将培养皿置于磁力搅拌器上，调整距离为30cm，紫外灯照射剂量为60s。照射的同时进行磁力搅拌。随后将菌液继续在37℃条件下继续培养6-8h后，涂布在LB/Kan平板上，于37℃培养过夜，作为原始突变库。最终共分离获得167个单菌落。
三、重组β-甘露聚糖酶突变体的透明圈法在突变株筛选中的应用
分别挑取步骤二所得的单克隆菌落至KT平板上，其中约9株透明圈较大。分别用游标卡尺测定菌落直径和透明圈直径，并计算EI值。
Figure BDA0001317626280000041
测定结果如表1所示。其中UV15，UV22和UV101的EI值较大，选择这3个菌株为优良突变株进行β-甘露聚糖酶酶活的验证测定。
表1E.coli BL21(DE3)/pET30-man25突变株台酚蓝法透明水解圈测定
Figure BDA0001317626280000051
将原始菌株和UV15，UV22和UV101四个菌株，按照常规大肠杆菌重组表达外源酶方法进行蛋白的诱导表达，对其进行β-甘露聚糖酶酶活力测定结果见表2。透明圈大的突变株，其在液体培养基中β-甘露聚糖酶酶活也高，其中UV101的β-甘露聚糖酶酶活较原始菌株提高了1.9倍。
表2突变菌株β-甘露聚糖酶酶活测定
Figure BDA0001317626280000052
Figure BDA0001317626280000061
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
SEQUENCE LISTING
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Claims (7)

1.一种β-甘露聚糖酶工程菌的筛选方法，其特征在于，包括如下步骤：
（1）将β-甘露聚糖酶的表达菌在含有台酚蓝的KT平板上培养，直至平板上产生水解圈；所述的培养包括三个阶段：第一阶段为37±2℃ 保温6-12 h；第二阶段为25±2℃ 保温1-3h；第三阶段为37oC~50℃ 保温8-20 h；
（2）测量水解圈直径与微生物菌落直径，以二者大小的比值作为β-甘露聚糖酶的酶活指数，该指数与酶活正相关；
所述β-甘露聚糖酶表达菌，其宿主菌为E. coli BL21(DE3)或E. coli BL21。
2.根据权利要求1所述的筛选方法，其特征在于，所述第一阶段为37 ℃ 保温10 h。
3.根据权利要求1所述的筛选方法，其特征在于，所述第二阶段为25℃ 保温2 h。
4.根据权利要求3所述的筛选方法，其特征在于，所述第三阶段的保温时间为12 h。
5.根据权利要求1~4任意一项所述的筛选方法，其特征在于，所述KT平板的原料组分为：1-2%蛋白胨、0.5-1%酵母提取物、1%氯化钠、1-3%琼脂和0.3-1%魔芋胶溶液。
6.根据权利要求5所述的筛选方法，其特征在于，所述KT平板的配制：将上述原料混合后115℃ 条件下高压灭菌20 min，冷却后加入预先配制好的1%台酚蓝溶液至终浓度为0.02-0.05%。
7.根据权利要求5所述的筛选方法，其特征在于，所述β-甘露聚糖酶表达菌，其表达载体为pET30(a)。
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