CN109320630B - 一种新型仿生亲和纯化材料及其在壳聚糖酶纯化中的应用 - Google Patents

一种新型仿生亲和纯化材料及其在壳聚糖酶纯化中的应用 Download PDF

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CN109320630B
CN109320630B CN201811329059.XA CN201811329059A CN109320630B CN 109320630 B CN109320630 B CN 109320630B CN 201811329059 A CN201811329059 A CN 201811329059A CN 109320630 B CN109320630 B CN 109320630B
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李尚勇
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Qingdao University
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Abstract

本发明涉及一种新型仿生亲和纯化材料及其在壳聚糖酶纯化中的应用，属于工业生物技术领域。本发明所述仿生亲和材料的亲和配基为壳二糖，连接臂为氰尿酰氯，基础介质为活化的琼脂糖6B。所述仿生亲和材料的解离常数(Kd)及最大结合能力(Qmax)分别为24.2μg/mL和24.1mg/g。利用上述仿生亲和材料建立了一种壳聚糖酶仿生亲和纯化方法，可高效率、低成本的生产高纯度壳聚糖酶，具有良好的工业化应用潜质。

Description

一种新型仿生亲和纯化材料及其在壳聚糖酶纯化中的应用
技术领域
本发明涉及一种新型仿生亲和纯化材料及其在壳聚糖酶纯化中的应用，属于工业生物技术领域。
背景技术
壳聚糖是几丁质部分或完全脱乙酰基后的衍生物，主要由D-氨基葡萄糖通过β-1,4-糖苷键连接而成，是自然界唯一带正电荷的多糖，被誉为第六生命要素。壳寡糖是一种功能性低聚糖，具有抗菌、抗肿瘤、降血脂、降血糖、调节机体免疫、促进农作物增产等功效，是近几年功能性寡糖研究和开发的热点之一。
壳聚糖酶是一类催化氨基葡萄糖间的β-1,4糖苷键断裂，专一性降解壳聚糖的糖苷水解酶。传统的壳聚糖酶纯化需要经过多个步骤：超滤，硫酸铵沉淀，脱盐，Q柱离子交换层析，Sephacryl S-200凝胶柱层析等。整个纯化过程耗时长、成本高、回收率低。与传统的柱层析方法相比，仿生亲和纯化方法具有特异性强、操作简单、易于放大等优势。目前部分重组表达的壳聚糖酶带有组氨酸标签，可以用金属螯合层析进行纯化，可得到较高的纯化效率。但是由于金属螯合配基的特殊性，金属螯合层析过程中，洗脱过程中需要利用大量的毒性的咪唑，会影响纯化所得壳聚糖酶的生物活性，利用脱盐柱对咪唑进行处理会增加额外的步骤和成本。因此，需要开发更为特异性的仿生亲和材料去建立适用于壳聚糖酶的仿生亲和纯化方法。
发明内容
本发明针对现有技术的不足，提供了一种利用壳二糖作为仿生亲和配基，连接臂为氰尿酰氯，基础介质为活化的琼脂糖6B的新型仿生亲和材料。利用该材料建立了一种壳聚糖酶的仿生亲和纯化方法。本发明所述壳聚糖酶仿生亲和方法纯化方法简单、成本低、纯度高，适用于壳聚糖酶的规模化纯化。
另一方面，本发明提供了一种新型壳聚糖酶仿生亲和材料的合成方法。包括以下4个步骤：1)、表氯醇活化琼脂糖凝胶；2)、表氯醇活化琼脂糖凝胶的氨基化；3)、连接氰尿酰氯连接臂；4)、螯合壳二糖仿生亲和配基。
另一方面，本发明还提供一种所述的仿生亲和材料在壳聚糖酶的仿生亲和纯化中的应用。
另一方面，本发明还提供一种仿生亲和材料针对壳聚糖酶的解离常数和最大结合能力的测定方法。
另一方面，本发明还提供了一种壳聚糖酶的仿生亲和纯化方法，所选用的仿生亲和材料为本发明所述材料。
优选：所述纯化条件中上样缓冲液为甘氨酸-NaOH，缓冲液pH为8.0～10.0，最适上样pH为8.6；
优选：所述纯化条件中洗脱缓冲液为醋酸-醋酸钠，缓冲液pH为4.0～6.0，最适洗脱pH为4.0；
优选：所述纯化条件中上样浓度为25～150mg/mL，最适上样浓度为50mg/mL；
优选：所述纯化条件中洗脱缓冲液中NaCl浓度为0.2～1M，最适NaCl浓度为0.8M。
另一方面，本发明还提供了一种亲和柱，填料为本发明所述仿生亲和材料。
有益效果：
1.本发明的壳聚糖酶仿生亲和材料为新型仿生亲和材料，所用仿生亲和配基为壳二糖，具有以下优势：一方面，壳二糖制备方法简单，且为壳聚糖酶的最小降解产物，可与壳聚糖酶专一性结合，不能被进一步降解，为天然的优良配基；另一方面，壳二糖为碱性寡糖，天然含有两个氨基基团，可与连接臂进行牢固结合，为仿生亲和材料的大规模制备提供了便利条件。
2.本发明所述壳聚糖酶仿生亲和材料采用环状连接臂，具有很强的机械强度，配基不易脱落；而且该材料对壳聚糖酶的亲和力强，每克仿生亲和材料对壳聚糖酶CsnM的最大结合能力(Qmax)为24.1mg/g。易于洗脱，解离常数(Kd)为24.2μg/mL。
3.利用本发明所述仿生亲和材料建立的壳聚糖酶仿生亲和纯化方法简单、成本低廉、易于放大。壳聚糖酶CsnM蛋白质纯度达到95％以上。适用于壳聚糖酶的规模化纯化。
附图说明
图1为本发明仿生亲和材料的合成过程；
图2为本发明仿生亲和配基的质谱结果；
图3为本发明仿生亲和材料的解离常数和最大结合能力；
图4为本发明仿生亲和方法纯化所得壳聚糖酶的纯化测定。a为SDS-PAGE法测定壳聚糖酶CsnM的纯度；b为HPLC法检测壳聚糖酶CsnM的纯度。
具体实施方式
实施例1仿生亲和材料的合成过程
仿生亲和材料的合成过程包括有4个步骤：1)、表氯醇活化琼脂糖凝胶，使得琼脂糖凝胶处于活化状态从而连接其他基团；2)、琼脂糖凝胶的氨基化，活化后的琼脂糖凝胶添加氨基；3)、连接氰尿酰氯连接臂，利用连接的氨基基团交联氰尿酰氯连接臂；4)、螯合壳二糖仿生亲和配基，在连接臂上偶联壳二糖仿生亲和配基。具体步骤如下：
1.1表氯醇活化琼脂糖凝胶
用双蒸水按1:10(v/v)的比例对琼脂糖凝胶(Sepharose 6B)进行彻底洗涤，使得流出液的pH平衡到7.0，洗涤过的琼脂糖凝胶在室温下干燥，溶解在100mL活化溶液(1M氢氧化钠，2.5g二甲基亚砜以及10mL表氯醇)中，在40℃摇床中100rpm震荡2.5h进行活化，得到表氯醇活化琼脂糖凝胶6B(图1a)。
1.2琼脂糖凝胶的氨基化
将35％饱和氨添加到活化琼脂糖凝胶6B中，在30℃摇床中100rpm孵化过夜，使得活化琼脂糖凝胶6B加入氨基基团，得到氨基化活化琼脂糖凝胶6B(图1b)。
1.3连接氰尿酰氯连接臂
将氨基化活化琼脂糖凝胶6B按等体积加入50％(v/v)丙酮，在冰浴搅拌的条件下以0.5mL/min的流速缓慢加入氰尿酰氯溶液(溶入70mL丙酮)，用1M的氢氧化钠溶液调节该溶液的pH值至7.0，再用50％(v/v)丙酮洗涤去除未结合的氨基基团，氰尿酰氯(连接臂)连接至活化琼脂糖凝胶6B上，得到连接氰尿酰氯连接臂的仿生亲和配基(图1c)。
1.4螯合壳二糖仿生亲和配基
将上述步骤1.3的过饱和的连接氰尿酰氯连接臂的仿生亲和材料溶解在2M的碳酸钠溶液中，然后在碳酸钠溶液中缓慢添加两倍质量的壳二糖。室温搅拌24h，用双蒸水洗涤凝胶，保存在0.02％(W/V)的叠氮钠，得到仿生亲和材料(图1d)。
实施例2仿生亲合材料的性能表征
实施例1合成的仿生亲和材料为大分子的仿生亲和凝胶，为了确定合成的仿生亲和材料的配基密度，将实施例1中1.2步骤中合成的仿生亲和材料，利用茚三酮法检测仿生亲和材料中的氨基密度，从而表征合成的仿生亲和材料的配基密度。通过茚三酮法检测发现，合成的仿生亲和配基的密度达到20.9μmol/ml。
为了确定合成的仿生亲和材料中仿生亲和配基结构，将合成的仿生亲和凝胶等比例(w/v)加入6M浓盐酸，作用6h后，4000rpm离心10min，取上清液，用双蒸水稀释100倍，然后加入等体积(v/v)的乙腈，进行质谱分析。理论上，利用6M的HCl切割下的仿生亲和配基为C18H31ClN6O12，它的相对分子量为558.9。但是，由于氯离子在酸性环境中不稳定，在6M的HCl中会被羟基取代，因此，其分子式应变构为C18H32N6O12，它的相对分子量为524.5。质谱结果(图2)显示，质谱所得仿生亲和配基的相对分子量为525.01，与其正离子一级质谱的理论分子量保持一致。
实施例3仿生亲和材料的解离常数和最大结合能力
利用斯卡查德(Scatchard)方程法对实施例1中所述仿生亲和材料进行解离常数(Kd)及最大结合能力(Qmax)评价，确定合成的仿生亲和材料对壳聚糖酶CsnM的吸附及解离能力。所用壳聚糖酶CsnM购自青岛艾菲特生物科技有限公司。
具体测定方法为：取1mL不同浓度(0.1-0.9mg/mL，20mM Gly-NaOH，pH 8.6)的壳聚糖酶CsnM与0.5g的实施例1中所述仿生亲和材料混合，在4℃条件下100rpm震荡培养2h，达到吸附平衡。混合液1500g离心5min，检测上清中剩余的蛋白酶活性及蛋白含量。所检测数据按照Scatchard方程法计算：
其中，Q代表吸附在仿生亲和材料中的蛋白量(mg/g)，Qmax代表理论上与亲和材料吸附壳聚糖酶的最大蛋白量(mg/g)，[C*]溶液中剩余的蛋白量(mg/mL)，Kd代表吸附常数。如图3所示，通过Scatchard方程法做图，计算得到实施例1中所述仿生亲和材料对壳聚糖酶CsnM的解离常数Kd值为24.2μg/ml；最大吸附量Qmax为24.1μg/g。
实施例4壳聚糖酶的仿生亲和纯化方法的条件筛选
取30mg的壳聚糖酶CsnM酶粉，稀释到1mL的样品缓冲液(0.1M Tris-HCl,pH 8.0)，经过0.22μm的滤膜过滤至平衡后的装载有实施例1中所述仿生亲和材料的亲和柱(柱长25mm×内径7mm)。用吸附平衡缓冲液直至体系平衡，直至无蛋白峰出现。然后利用洗脱缓冲液洗脱柱子，收集活性组分。本发明针对纯化过程中各种洗脱和上样条件进行了优化。
4.1仿生亲和柱上样条件筛选
上样前，用5-10个柱体积的用0.22μm过滤过的双蒸水洗涤实施例1中所述仿生亲和材料，然后用5-10个柱体积的上样缓冲液平衡柱子，流速为1mL/min，柱子压力设置保护(低于0.3mPa)。根据纯化结果筛选最适的上样缓冲液种类，缓冲液的pH，以及上样量。
4.1.1最适上样缓冲液
选取最高能达到pH 9.0的五种常见碱性缓冲液，巴比妥钠-盐酸、甘氨酸-氢氧化钠、硼酸-硼砂、磷酸氢二钠-磷酸氢二钾、碳酸钠-碳酸氢钠，利用FPLC对壳聚糖酶进行纯化。收集洗脱峰样品并测定纯度，对比五种洗脱缓冲液的纯化效果。通过分析纯化结果可知，甘氨酸-氢氧化钠缓冲液纯化所得蛋白量最大，且该缓冲液对亲和柱损伤较小。因此，选用甘氨酸-氢氧化钠为上样缓冲液。
4.1.2最适上样缓冲液pH
配制不同pH的甘氨酸-氢氧化钠缓冲液，利用FPLC对壳聚糖酶进行纯化。测洗脱峰样品的酶比活力，分析不同pH的上样缓冲液对纯化效果的影响。通过纯化效率分析，发现甘氨酸-氢氧化钠缓冲液pH为8.6时，洗脱效果最好，因此选用缓冲液的pH为8.6。
4.1.3最适上样量
配制25mg/mL、50mg/mL、75mg/mL、100mg/mL、125mg/mL、150mg/mL六种不同浓度的酶溶液，考察不同的上样浓度对纯化效果的影响。结果发现，最适上样浓度为50mg/mL，上样体积为1mL。
4.2洗涤去除杂蛋白
按照4.1.3中所述壳聚糖酶的样本样，上样至按4.1中平衡过的仿生亲和柱，上样时流速为1mL/min，用洗涤缓冲液A冲洗5-10个柱体积，去除杂蛋白，洗涤流速为1.5mL/min，柱子压力不超过0.3mPa，所述洗涤缓冲液A为含有100mM NaCl的100mM甘氨酸-氢氧化钠缓冲液(pH 8.6)。
4.3洗脱获得壳聚糖酶纯品
按照4.2所述方法去除杂蛋白之后，用洗涤缓冲液B洗脱，收取活性峰，即为壳聚糖酶的纯酶，洗脱流速为1.5ml/min，柱子压力不超过0.3mPa，选取最低能达到pH 4.0的五种常见酸性缓冲液醋酸-醋酸钠、柠檬酸-柠檬酸钠、磷酸氢二钠-柠檬酸、柠檬酸-氢氧化钠-盐酸、醋酸-醋酸钾，利用实施例1中所述仿生亲和材料对壳聚糖酶CsnM进行分离纯化，收集洗脱峰并测其酶比活力。综合洗脱结果及成本等因素，洗脱缓冲液确定为醋酸-醋酸钠缓冲液。
配制不同pH的洗脱缓冲液，在其他条件不变的前提下利用蛋白纯化色谱仪(FPLC)对壳聚糖酶进行纯化。测洗脱峰样品的酶比活力，分析不同pH的洗脱缓冲液对纯化效果的影响，确定最适的洗脱缓冲液pH。利用醋酸-醋酸钠缓冲液配制三种不同的pH条件(3.0，4.0，5.0，6.0)，进行色谱纯化，结果显示pH越低纯化所得蛋白量越高，但是考虑到低的pH会对酶的活力产生破坏，选择洗脱pH为4.0。
在洗脱缓冲液中添加NaCl能显著提高洗脱壳聚糖酶的效率，通过优化NaCl浓度，确定最适的NaCl浓度为0.8M。确定洗涤缓冲液为含有0.8M的NaCl的100mM醋酸-醋酸钠缓冲液(pH 4.0)，所述洗涤缓冲液B为含有0.8MNaCl的100mMTris-HCl缓冲液(pH 8.0)。
4.4洗涤并保存柱子
用洗涤缓冲液C洗涤柱子，去除牢固结合在柱子上的蛋白，再用0.22μm过滤过的双蒸水冲洗5-10个柱体积，去除洗涤缓冲液中的各种盐离子，再用20％乙醇保存柱子。所述洗涤缓冲液C为含有2M NaCl的100mMTris-HCl缓冲液(pH 8.0)。
实施例5壳聚糖酶CsnM的仿生亲和纯化方法
依据实施例4中摸索的各影响因素的最适条件，将壳聚糖酶酶粉经过0.22μm的滤膜过滤至平衡后的装载有实施例1中所述仿生亲和材料的亲和柱。用吸附平衡缓冲液(甘氨酸-氢氧化钠pH 8.6)洗涤5-10个柱体积直至体系平衡，无蛋白峰出现，流速为1mL/min，柱子压力设置保护(低于0.3mPa)。上样浓度为50mg/mL，上样体积为1mL。上样后，先用洗涤缓冲液A洗涤柱子，去除杂蛋白，再用洗涤缓冲液B冲洗柱子，获得目的蛋白。再用洗涤缓冲液C洗涤柱子，去除牢固结合的杂蛋白。柱子保存于20％乙醇。
实施例6壳聚糖酶的纯度测定
蛋白质变性电泳(SDS-PAGE)法检测实施例5中纯化所得壳聚糖酶CsnM进行SDS-PAGE纯度分析：分离胶的浓度为10％，浓缩胶的浓度为5％。将纯化所得的样品中等体积的加入2×Loading Buffer，在100℃沸水中煮沸5min，在BioRad垂直电泳槽中中进行SDS-PAGE电泳分析，浓缩胶电压为80V，在分离胶中的电压为120V，以溴酚蓝的颜色为指示控制电泳时间。电泳完成后利用考马斯亮蓝R-350染色，并用脱色液脱色后在全自动凝胶成像系统拍照并分析纯度。如图4a所示，利用SDS-PAGE分析所得壳聚糖酶CsnM的蛋白纯度为96.7％。
利用高效液相色谱(HPLC)法对按照实施例5中纯化方法所得的样品进行纯度分析。利用TSK3000SW凝胶过滤柱在波长280nm处检测。流动相为100mM PBS,100mM Na2SO4,0.05％NaN3，流速为0.6mL/min。如图4b所示，通过计算HPLC峰面积，纯化所得壳聚糖酶CsnM的蛋白纯度为95.9％。

Claims (6)

1.一种新型仿生亲和材料，其特征是，仿生亲和配基为壳二糖，连接臂为氰尿酰氯，基础介质为活化的琼脂糖6B，其连接结构式为。
2.如权利要求1所述的仿生亲和材料的制备方法，其特征是步骤如下：
1）、表氯醇活化琼脂糖凝胶：
用双蒸水按1:10的比例对琼脂糖凝胶 Sepharose 6B进行彻底洗涤，使得流出液的pH平衡到7.0，洗涤过的琼脂糖凝胶在室温下干燥，溶解在100 mL活化溶液中，在40℃摇床中100 rpm震荡2.5 h进行活化，得到表氯醇活化琼脂糖凝胶6B，所述活化溶液为1 M氢氧化钠，2.5 g二甲基亚砜以及10 mL表氯醇；
2)、琼脂糖凝胶的氨基化：
将35%饱和氨添加到活化琼脂糖凝胶6B中，在30℃摇床中100 rpm孵化过夜，使得活化琼脂糖凝胶6B加入氨基基团，得到氨基化活化琼脂糖凝胶6B；
3）、连接氰尿酰氯连接臂：
将氨基化活化琼脂糖凝胶6B按等体积加入50%丙酮，在冰浴搅拌的条件下以0.5 mL/min的流速缓慢加入氰尿酰氯溶液，用1 M的氢氧化钠溶液调节该溶液的pH值至7.0，再用50%丙酮洗涤去除未结合的氨基基团，连接臂氰尿酰氯连接至活化琼脂糖凝胶6B上，得到连接氰尿酰氯连接臂的仿生亲和配基；
4）、螯合壳二糖仿生亲和配基：
将上述步骤3）的过饱和的连接氰尿酰氯连接臂的仿生亲和材料溶解在2M的碳酸钠溶液中，然后在碳酸钠溶液中缓慢添加两倍质量的壳二糖，室温搅拌24h，用双蒸水洗涤凝胶，保存在0.02%的叠氮钠，得到仿生亲和材料。
3.如权利要求1所述的仿生亲和材料在壳聚糖酶的仿生亲和纯化中的应用。
4.一种壳聚糖酶的仿生亲和纯化方法，所选用的仿生亲和材料为权利要求1所述仿生亲和材料。
5.如权利要求4所述纯化方法，其特征是，纯化条件中上样缓冲液为甘氨酸-NaOH，缓冲液pH为8.0~10.0。
6.如权利要求4所述纯化方法，其特征是，纯化条件中洗脱缓冲液为醋酸-醋酸钠，缓冲液pH为4.0~6.0。
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