CN114292832A - 一种嗜热耐高温多聚半乳糖醛酸酶MlPG28B、编码基因及制备方法 - Google Patents
一种嗜热耐高温多聚半乳糖醛酸酶MlPG28B、编码基因及制备方法 Download PDFInfo
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Abstract
本发明公开了一种嗜热耐高温多聚半乳糖醛酸酶MlPG28B，氨基酸序列如SEQ ID NO.1所示；其编码基因的DNA序列如SEQ ID NO.2所示；制备方法依次按照如下步骤进行：将如SEQ ID NO.3所示的基因构建到pPICZαA真核表达载体上，在大肠杆菌DH5α中扩增培养，提取质粒并用SacI进行线性化，胶回收线性化产物并电击转化至毕赤酵母X‑33感受态细胞中，之后涂布在含100μg/mL博来霉素的固体YPDSZ培养基上，30℃培养5天；挑选平板上的单菌落菌株划含300μg/mL博来霉素的YPD的固体培养基上培养，挑取单菌落菌株进行诱导表达和纯化，获得嗜热耐高温多聚半乳糖醛酸酶MlPG28B。
Description
技术领域
本发明属于基因工程和遗传工程领域，尤其涉及一种嗜热耐高温多聚半乳糖醛酸酶MlPG28B、编码基因及制备方法。
背景技术
果胶是一种结构复杂的高分子量碳水化合物聚合物，作为结构多糖广泛存在于植物细胞壁中，并与纤维素、半纤维素和木质素相互交联形成致密的网状结构，在食品、饲料、造纸、纺织和果胶废液处理等众多工业生产中需要将果胶降解去除。
果胶多糖主链主要是通过D-GalA的O-1和O-4位置通过α-1, 4-糖苷键相连，即同聚半乳糖醛酸，其约占果胶的70 %。采用糖苷水解酶28家族（GH28）的多聚半乳糖醛酸酶(Polygalacturonase, PG)通过水解方式可使果胶主链中的α-1, 4-糖苷键断裂，进而使高度聚合的果胶发生解聚。根据NCBI和CAZy数据库统计，已经注册的PG达到9000多条，其中有近200种PG从细菌和真核生物中克隆和表征，但最适温度主要集中在30~60℃。
多年以来，人们积极挖掘嗜热耐高温的PG，如Thermotoga maritima PelB（TM0437），最适温度80℃；土壤宏基因组样品（PecJKR01）最适温度70℃；Caldicellulosiruptor bescii DSM 6725（CbPelA，最适温度72℃）；Neosartorya fischeri P1（Nf PG5）最适温度70 ℃；Penicillium occitanis （PG1）最适温度70℃；Penicillium oxalicum CZ1028（EPG4）最适温度70℃；Bispora sp. MEY-1 （BiPG28A）最适温度70℃和Talaromyces leycettanus JCM12802（TlPGA、TePG28a和TePG28b）最适温度70℃。这些PG虽为嗜热酶但并不耐高温，即酶活性最高时的温度（最适温度）至少为70℃，但在大于60℃的高温环境中会在短时间内失去催化酶活性，无法满足在饲料、造纸及纺织等有高温需求的工业上应用。
发明内容
本发明是为了解决现有技术所存在的上述技术问题，提供一种嗜热耐高温多聚半乳糖醛酸酶MlPG28B、编码基因及制备方法。
本发明的技术解决方案是：一种嗜热耐高温多聚半乳糖醛酸酶MlPG28B，其特征在于：氨基酸序列如SEQ ID NO.1所示。
一种如权利要求1所述嗜热耐高温多聚半乳糖醛酸酶MlPG28B的编码基因，其特征在于：DNA序列如SEQ ID NO.2所示。
一种如权利要求1所述嗜热耐高温多聚半乳糖醛酸酶MlPG28B的制备方法，其特征在于依次按照如下步骤进行：将如SEQ ID NO.3所示的基因构建到pPICZαA真核表达载体上，在大肠杆菌DH5α中扩增培养，提取质粒并用SacI进行线性化，胶回收线性化产物并电击转化至毕赤酵母X-33感受态细胞中，之后涂布在含100 μg/mL博来霉素的固体YPDSZ培养基上，30℃培养5天；挑选平板上的单菌落菌株划含300 μg/mL博来霉素的YPD固体培养基培养，挑取单菌落菌株进行诱导表达和纯化，获得获得氨基酸序列如SEQ ID NO.4所示的重组嗜热耐高温多聚半乳糖醛酸酶MlPG28B。
本发明的多聚半乳糖醛酸酶MlPG28B不仅嗜热耐高温，而且还具有宽泛的的pH耐受性，其酶活性最高时的温度（最适温度）为60℃；在60℃热处理2 h后，仍可以保留30 %以上的相对酶活力，在100℃热处理60 min仍有酶活性；在pH 2.0~11.0时相对酶活性保持在90%以上。可满足中低温甚至大于60℃的高温环境下酶解果胶之需要。
附图说明
图1是本发明实施例嗜热耐高温多聚半乳糖醛酸酶MlPG28B诱导表达96 h纯化后的SDS-PAGE电泳图。
图2是本发明实施例的嗜热耐高温多聚半乳糖醛酸酶MlPG28B的温度影响活性曲线示意图。
图3是本发明实施例的嗜热耐高温多聚半乳糖醛酸酶MlPG28B的pH影响活性曲线示意图。
图4是本发明实施例的嗜热耐高温多聚半乳糖醛酸酶MlPG28B的温度稳定性示意图。
图5是本发明实施例的嗜热耐高温多聚半乳糖醛酸酶MlPG28B的pH稳定性示意图。
图6是本发明实施例的嗜热耐高温多聚半乳糖醛酸酶MlPG28B的酶动力学拟合曲线图。
图7是本发明实施例的嗜热耐高温多聚半乳糖醛酸酶MlPG28B对聚半乳糖醛酸降解产物的ESI-MS分析图。
具体实施方式
本发明是将来源于拉斯坦毛霉（Mucor lusitanicus）的基因序列采用GenBank数据库中的Basic Local Alignment Search Tool (BLAST )分析，DNAMAN软件进行多序列比对，Vector NTI分析序列信息。加His标签和密码子优化，人工合成获得如SEQ ID NO.3所示的基因，属于糖苷水解酶第28 家族（GH28）。该基因编码区长1098bp，单链，序列从第1-1077位为编码嗜热耐高温多聚半乳糖醛酸酶MlPG28B的DNA序列（如SEQ ID NO.2所示），1078-1098位为His-Tag标签和终止密码子的DNA序列。将该基因序列构建到pPICZαA真核表达载体上，在大肠杆菌DH5α中扩增培养，提取质粒并用SacI进行线性化，胶回收线性化产物并电击转化至毕赤酵母X-33感受态细胞中，之后涂布在含100 μg/mL博来霉素的固体YPDSZ培养基上，30℃培养5天；挑取平板上的单菌落菌株划含300 μg/mL博来霉素的YPD固体培养基培养，进行高浓度抗生素筛选，挑取单菌落生长较大的菌株进行按毕赤酵母表达手册方法进行诱导表达。
诱导表达96h后，用聚丙烯酰胺凝胶电泳检测，电泳图结果如图1所示，嗜热耐高温多聚半乳糖醛酸酶MlPG28B在电泳胶上呈现单一条带，分子量约为38 kDa。最后将菌体破碎、裂解、纯化，收集纯化液，即获得重组表达的嗜热耐高温多聚半乳糖醛酸酶MlPG28B。氨基酸序列如SEQ ID NO.4所示，从氨基端开始的第1-365位氨基酸残基序列，单链，其中1-359位为具有嗜热耐高温多聚半乳糖醛酸酶MlPG28B活性的氨基酸序列（如SEQ ID NO.1所示），360-365位为His-Tag的氨基酸标签序列。
实验：
1. 温度对本发明实施例嗜热耐高温多聚半乳糖醛酸酶MlPG28B（重组酶MlPG28B，下同）活性影响实验
在pH 5.0（HAc-NaAc）的条件下，以200 μL 0.5% （w/v）的聚半乳糖醛酸（PGA）为底物，加入10 μL重组酶MlPG28B，反应30 min，采用3, 5-二硝基水杨酸（DNS）法测定其活性，分别在20-100 ℃，每隔10℃设置一个温度来测定聚半乳糖醛酸酶MlPG28B的活力。以灭活的酶为对照，以反应最高酶活力为100％计算相对酶活，根据酶在不同温度下的相对活性绘制曲线。结果如图2所示，重组酶MlPG28B的最适反应温度为60 ℃，说明其为嗜热酶。
2. pH对重组酶MlPG28B活性影响实验
在60 ℃的条件下，以200 μL pH分别为2.0-11.0的0.5 % (w/v)的PGA为底物，加入10 μL重组酶MlPG28B，反应30 min，采用3, 5-二硝基水杨酸（DNS）法测定其活性。以灭活的酶为对照，以反应最高酶活力为100％计算相对酶活，根据酶在不同温度下的相对活性绘制曲线。结果如图3所示，重组酶MlPG28B的最适反应pH为5.0。
3.重组酶MlPG28B的温度稳定性实验
将重组酶MlPG28B分别在不同温度（60 ℃、70 ℃、80 ℃、90 ℃、100 ℃）条件下孵育不同的时间，在上述不同温度处理的不同时间节点分别取出一定量热处理的酶液置于4℃下待用，随后在60 ℃的条件下，以200 μL 0.5 % (w/v)的PGA为底物，分别加入上述热处理的10 μL重组酶MlPG28B，反应30 min，随后通过DNS法测定酶活性，检测残余酶活，并和未处理的酶活进行比较，计算相对酶活性。结果如图4所示，重组酶MlPG28B在60 ℃热处理2 h后，仍可以保留30 %以上的相对酶活力；在100 ℃热处理60 min仍有酶活性，说明其为嗜热耐高温酶。
4.重组酶MlPG28B的pH稳定性实验
将重组酶MlPG28B分别置于不同（pH 2.0~11.0）的pH缓冲液中稀释5倍，于4 ℃条件下放置24 h；随后将10 μL酶溶液与200 μL 5 mg/mL的PGA底物在最适反应条件（60 ℃，pH 5.0）下孵育30 min，随后通过DNS法测定酶活性，并和未处理的酶活进行比较，计算相对酶活性。结果如图5所示，重组酶MlPG28B在pH 2.0~11.0时相对酶活性保持在90 %以上，说明重组酶 MlPG28B具有宽泛的的pH耐受性。
5.重组酶MlPG28B的底物特异性实验
选取PGA、低酯果胶、果胶、高酯果胶、海藻酸钠和亚麻耔胶6种底物考察重组酶MlPG28B的底物特异性。在最适温度60 ℃和pH 5.0条件下，反应30 min，按标准方法测得重组酶MlPG28B的相对酶活力。结果如表1所示，重组酶MlPG28B对PGA和低酯果胶均具有很高的降解酶活性。
表1
6.重组酶MlPG28B的酶动力学参数的测定
将ApPel1（0.0159 mg/ml）酶液10 μl与200 μl不同浓度（0.5 mg/mL、1.5 mg/mL、2.5 mg/mL、3.5 mg/mL、4.5 mg/mL和6.5 mg/mL）的PGA底物（pH 5.0）混合，在60 ℃条件下反应30 min，采用DNS法测定其活性。所得数据在GraphPad Prism 9.0.0软件用米氏方程进行拟合，得到反应速率与底物浓浓度的拟合曲线，就可以得到相关的酶动力学参数。结果如图6所示，通过计算得出Vmax为1.47±0.24 mg/L/s，Km为3055±1104 mg/L，Kcat为11.667±1.905 s-1，Kcat/Km为4.655±2.305 mL/mg/s。
7.重组酶MlPG28B对聚半乳糖醛酸降解产物的ESI-MS分析
在35℃的条件下，以500 μL，pH=5.0的0.5 %（w/v）的PGA为底物，加入10 μL重组酶MlPG28B混匀，700 rpm震荡反应21h，对其降解产物进行ESI-MS分析。结果如图7所示：在进行ESI-MS的阴离子模式分析其降解产物时发现其降解产物为寡聚半乳糖醛酸，主要由DP1、DP2、DP3、DP4、DP5、DP6和DP7组成。说明重组酶MlPG28B可以有效的对富含果胶多糖的原料进行高效降解来制备寡聚半乳糖醛酸。
序列表
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<120> 一种嗜热耐高温多聚半乳糖醛酸酶MlPG28B、编码基因及制备方法
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Claims (3)
1. 一种嗜热耐高温多聚半乳糖醛酸酶MlPG28B，其特征在于：氨基酸序列如SEQ IDNO.1所示。
2. 一种如权利要求1所述嗜热耐高温多聚半乳糖醛酸酶MlPG28B的编码基因，其特征在于：DNA序列如SEQ ID NO.2所示。
3. 一种如权利要求1所述嗜热耐高温多聚半乳糖醛酸酶MlPG28B的制备方法，其特征在于依次按照如下步骤进行：将如SEQ ID NO.3所示的基因构建到pPICZαA真核表达载体上，在大肠杆菌DH5α中扩增培养，提取质粒并用SacI进行线性化，胶回收线性化产物并电击转化至毕赤酵母X-33感受态细胞中，之后涂布在含100 μg/mL博来霉素的固体YPDSZ培养基上，30℃培养5天；挑选平板上的单菌落菌株划含300 μg/mL博来霉素的YPD固体培养基上培养，挑取单菌落菌株进行诱导表达和纯化，获得氨基酸序列如SEQ ID NO.4所示的重组嗜热耐高温多聚半乳糖醛酸酶MlPG28B。
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