CN114457057B - 一种壳聚糖酶突变体及其应用 - Google Patents
一种壳聚糖酶突变体及其应用 Download PDFInfo
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- CN114457057B CN114457057B CN202210242255.3A CN202210242255A CN114457057B CN 114457057 B CN114457057 B CN 114457057B CN 202210242255 A CN202210242255 A CN 202210242255A CN 114457057 B CN114457057 B CN 114457057B
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- chitosan
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- enzyme
- chitosan enzyme
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Abstract
本发明公开了一种壳聚糖酶突变体及其应用，所述壳聚糖酶突变体为糖苷水解酶壳聚糖酶氨基酸序列第154位点上的甘氨酸突变为缬氨酸，通过分析糖苷水解酶壳聚糖酶结构上的高去折叠自由能位点，以此选择突变位点进行饱和突变，筛选出温度稳定性提高的壳聚糖酶突变体，并在大肠杆菌中表达，制备的壳聚糖酶突变体温度稳定性显著提升，且在提高温度稳定性的同时对壳聚糖酶突变体的其他特性无影响，工业化生产具有良好的应用前景。
Description
技术领域
本发明涉及一种壳聚糖酶突变体及其应用，具体涉及到一种温度稳定性及催化活性提高的突变重组壳聚糖酶，属于基因工程技术领域。
背景技术
壳聚糖是以氨基葡萄糖（GlcN）为单体，通过β-1,4-糖苷键相连接而成的多糖。壳聚糖是由几丁质通过加碱经脱乙酰基获得的产物，几丁质广泛存在于真菌、藻类细胞、贝类、软体动物的外壳和软骨中，是地球上仅次于纤维素的第二大天然高分子化合物，属于可再生生物资源。
壳寡糖是壳聚糖的降解产物，具有多种生理功能，有抗菌、抗肿瘤、提高免疫力等生物活性，在食品、医药、农业和化妆品等领域具有广泛的应用前景。
壳聚糖酶是一种可以降解壳聚糖为壳寡糖的糖苷水解酶，具有反应专一性强、反应条件温和的优点。根据碳水化合物活性酶数据库（www.cazy.org）可知，壳聚糖酶主要来源于糖苷水解酶（GH）家族46，75，80和8。阿维链霉菌是一种革兰氏阳性细菌，主要产阿维菌素和伊维菌素等农用抗生素。有研究报道表明阿维链霉菌可以生产GH75家族壳聚糖酶，但是生产的壳聚糖酶，其催化活性及稳定性还有待提高。
发明内容
本发明的目的在于克服现有技术中的不足，提供一种壳聚糖酶突变体及其应用，提高酶活温度稳定性。
为达到上述目的，本发明是采用下述技术方案实现的：
第一方面，本发明提供一种壳聚糖酶突变体，所述壳聚糖酶突变体为野生型糖苷水解酶壳聚糖酶氨基酸序列第154位点上的甘氨酸突变为缬氨酸，所述壳聚糖酶突变体的氨基酸序列为SEQ ID NO:3，基因序列为SEQ NO. 4。
结合第一方面，进一步的，所述野生型糖苷水解酶壳聚糖酶为从阿维链霉菌（Streptomyces avermitilis）中克隆的46家族糖苷水解酶壳聚糖酶，所述野生型糖苷水解酶壳聚糖酶的氨基酸序列为SEQ ID NO:1，基因序列为SEQ NO. 2。
进一步的，所述壳聚糖酶突变体的酶切方式为内切型，能够水解由β-1,4-糖苷键连接的多糖。
进一步的，所述壳聚糖酶突变体的水解产物包括氨基葡萄糖GlcN和壳二糖（GlcN）2。
进一步的，所述壳聚糖酶突变体应用于降解壳聚糖为壳寡糖。
第二方面，本发明提供一种重组质粒，含有所述壳聚糖酶突变体的基因。
第三方面，本发明还提供一种宿主细胞，含有所述壳聚糖酶突变体的基因，或含有所述重组质粒。
与现有技术相比，本发明所达到的有益效果为：
本发明提供的一种壳聚糖酶突变体及其应用，制备的壳聚糖酶突变体温度稳定性显著提升，且在提高温度稳定性的同时并没有对壳聚糖酶突变体的其他特性造成影响，仍能将壳聚糖水解为GlcN和（GlcN）2。
附图说明
图1是本发明实施例提供的野生型糖苷水解酶壳聚糖酶及其突变体的不同温度稳定性柱状图。
具体实施方式
下面结合附图对本发明作进一步描述。以下实施例仅用于更加清楚地说明本发明的技术方案，而不能以此来限制本发明的保护范围。
本发明关于阿维链霉菌GH46家族壳聚糖酶基因克隆和分子改造，本发明实施例前期利用PCR技术克隆了一种新的壳聚糖酶SaCsn46A，该酶具有广泛的底物谱，但是温度稳定性有待提高，本申请利用PoPMuSiC软件筛选出可能对SaCsn46A温度稳定性有影响的位点，选取其中1个位点进行突变，并在大肠杆菌Rosetta（DE3）中进行了功能表达。对其生化性质的研究表明，相比原始酶，突变酶的温度稳定性有提高。
本发明提供的一种壳聚糖酶突变体，是将野生型糖苷水解酶壳聚糖酶氨基酸序列第154位点上的甘氨酸突变为缬氨酸，该酶由271个氨基酸组成，其中包含34个氨基酸信号肽。
壳聚糖酶突变体的氨基酸序列为SEQ ID NO:3，基因序列为SEQ NO. 4。
其中野生型糖苷水解酶壳聚糖酶为从阿维链霉菌中克隆的46家族糖苷水解酶壳聚糖酶，野生型糖苷水解酶壳聚糖酶的氨基酸序列为SEQ ID NO:1，基因序列为SEQ NO. 2。
本发明提供的壳聚糖酶突变体的酶切方式为内切型，能够水解由β-1,4-糖苷键连接的多糖，不能水解由α-1,4-糖苷键连接的多糖，应用于降解壳聚糖为壳寡糖，水解产物包括氨基葡萄糖GlcN和壳二糖（GlcN）2。
本发明提供一种重组质粒，包含壳聚糖酶突变体的基因的质粒；还提供一种宿主细胞，包含壳聚糖酶突变体的基因，或含有重组质粒。
本发明关于壳聚糖酶突变体的制备方法，包括如下步骤：
将能够甲基化的大肠杆菌宿主携带的壳聚糖酶基因的重组质粒作为模板，以带有突变位点的寡聚核苷酸序列为引物，进行反向 PCR，扩增突变质粒；
使用DpnI限制性内切酶消化作为模板的质粒；
将消化后的 PCR 产物转化至 E. coli DH5α 感受态细胞，涂布于含有卡那霉素抗性的固体 LB 平板，过夜培养；
挑取单菌落接种至含有卡那霉素抗性的 LB 液体培养基，过夜培养后提取质粒进行测序验证；
将测序结果正确的质粒转化至大肠杆菌 E.coli BL21（DE3）感受态细胞中，诱导表达，获得壳聚糖酶突变体。
具体步骤如下：
设计两条引物，扩增壳聚糖酶基因，去除信号肽，将目的基因克隆到表达载体pET-28a中，构建重组质粒pET-SaCsn46a；
利用 Swiss-Model 在线软件对野生型糖苷水解酶壳聚糖酶进行模拟，获得其空间结构；
利用PoPMuSiC预测软件计算SaCsn46A每个突变氨基酸的去折叠自由能变化，确定与SaCsn46A稳定性相关的关键氨基酸位点；
设计定点突变引物，通过反向 PCR 技术得到突变壳聚糖酶基因，将目的基因克隆到表达载体，获得含有突变壳聚糖酶基因序列的重组载体；
将重组载体转入大肠杆菌进行诱导培养，离心后收集菌体，经超声破碎细胞后，使用Ni-NTA 亲和层析柱进行蛋白纯化获得突变壳聚糖酶。
本发明实施例操作如下：
1.壳聚糖酶突变位点的确认
利用Swiss-Model以链霉菌N174（Streptomycessp. N174）壳聚糖酶（1CHK_A）为模板对SaCsn46A进行蛋白三维结构模拟，获得壳聚糖酶的空间结构，利用PoPMuSiC预测软件计算SaCsn46A每个突变氨基酸的去折叠自由能变化（ΔΔG），来辅助设计提高其稳定性，确定与SaCsn46A稳定性相关的关键氨基酸位点，由于壳聚糖酶第154位氨基酸的去折叠自由能变化相对较大，因此设计引物进行反向PCR获得相应的突变质粒。
反向PCR体系：
| 试剂名称 | 体积（μL） |
| 模板 | 2 |
| PCR Buffer | 5 |
| dNTP | 4 |
| 上、下游引物 | 各1 |
| PfuDNA聚合酶 | 1.2 |
| ddH2O | 36 |
| 总体积 | 50 |
使用到的上游引物序列：CGGGGACGGCGTCGACAGCACCAGC；下游引物序列：GCTGGTGCTGTCGACGCCGTCCCCG。
PCR扩增条件：95 ℃预变性3 min； 95 ℃ 变性 30 s，64 ℃退火1 min，68 ℃延伸10 min，15个循环，4 ℃保温。
2.DpnI 消化模板质粒
取 20 μLPCR 产物，在 PCR 产物种直接添加 1 μLDpnI 限制性内切酶。
3.转入大肠杆菌
取 10 μL 酶切产物直接转化 E. coli DH5α 感受态细胞。将携带突变质粒的重组细胞送至上海生物工程有限公司测序，将测序正确的突变质粒转化至 E. coli BL21。
4.蛋白纯化
将超声破碎细胞获得的粗酶液上样至 Ni-NTA 亲和层析柱，用0.02M咪唑洗脱液（0.02M Tris-HCl，pH 8.0，0.5M NaCl，0.02M咪唑和10%甘油）洗脱后，再使用0.08M咪唑洗脱液（0.02M Tris-HCl，pH 8.0，0.5M NaCl，0.08M咪唑和10%甘油）进行洗脱，收集含有壳聚糖酶活性的洗脱液，通过透析除去咪唑，酶保存在-20 ℃。
5.酶学性质检测
使用DNS法测定酶活，在2 mL的反应体系中加入1475 μL pH缓冲液、500 μL胶体壳聚糖溶液，加入25 μL充分混匀酶液，立即在40 ℃水浴锅中反应10 min，取出，加入1.5 mLDNS溶液终止反应，再用沸水煮5 min，最后使用蒸馏水定容至25 mL，冷却至室温后，混匀，转入50 mL离心管中，设定8000 rpm离心5 min，取上清测定其在520 nm下的OD值，将加入25μLddH2O的溶液设为空白对照组调零。
酶活定义：在40 ℃条件下，1分钟内催化生产相当于1 µmol氨基葡萄糖的还原糖所需要的酶量为1个酶活单位（U）。
（1）最适pH
在 40 ℃的温度条件下，在不同的 pH缓冲液（pH 3.0-8.0）中，测定壳聚糖酶的酶活。以酶活最高点作为100%。
（2）pH稳定性
在 0 ℃的条件下，将壳聚糖酶置于pH 6.2的磷酸盐缓冲液中保存 2 h，最开始测定的壳聚糖酶酶活作为100%。
（3）最适温度
在最适 pH 条件下，将反应体系分别置于 25-80 ℃下进行反应，每5 ℃为一个温度梯度，测定壳聚糖酶的酶活。以酶活最高点作为100%。
（4）温度稳定性
将壳聚糖酶置于60 ℃、pH 6.2条件下保存2 h，最开始时测定的壳聚糖酶酶活作为100%。
6. 野生型糖苷水解酶壳聚糖酶及其突变体的酶学性质
表 1 野生型糖苷水解酶壳聚糖酶及其突变体的酶学性质
| 酶 | 比酶活（U/mg） | 最适pH | 最适温度（℃） |
| SaCsn46A | 435.36 | 6.2 | 45 |
| G154V | 734.40 | 6.4 | 50 |
野生型糖苷水解酶壳聚糖酶及其突变体的酶学性质如图1和表1所示，从图1及表1可以看出：本发明通过分析该酶蛋白结构上的高去折叠自由能位点，选择突变位点进行饱和突变，本发明将原始酶SaCsn46A第154位甘氨酸突变为缬氨酸（G154V），突变体在大肠杆菌Rosetta中进行表达。通过与野生型糖苷水解酶壳聚糖酶比较，发现突变酶的温度稳定性相比于野生菌型的温度稳定性提高了1.8倍。薄层色谱分析结果表明，该酶水解产物主要为氨基葡萄糖GlcN和（GlcN）2，说明突变体壳聚糖酶在温和条件下工业化生产GlcN和（GlcN）2方面具有良好的应用前景。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和变形，这些改进和变形也应视为本发明的保护范围。
序列表
<110> 常州大学
<120> 一种壳聚糖酶突变体及其应用
<141> 2022-03-10
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Claims (4)
1. 一种壳聚糖酶突变体，其特征在于，所述壳聚糖酶突变体为野生型糖苷水解酶壳聚糖酶氨基酸序列第154位点上的甘氨酸突变为缬氨酸，所述壳聚糖酶突变体的氨基酸序列为SEQ ID NO:3。
2.根据权利要求1所述的一种壳聚糖酶突变体在降解壳聚糖为壳寡糖中的应用。
3.一种重组质粒，其特征在于，含有权利要求1所述壳聚糖酶突变体的基因。
4.一种宿主细胞，其特征在于，含有权利要求1所述壳聚糖酶突变体的基因，或含有权利要求3所述的重组质粒，所述宿主细胞非动物或植物品种。
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