CN114107258A - 嗜热甘露聚糖酶ManBK突变体及其应用 - Google Patents
嗜热甘露聚糖酶ManBK突变体及其应用 Download PDFInfo
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Abstract
本发明公开了嗜热甘露聚糖酶ManBK突变体及其应用。对嗜热甘露聚糖酶ManBK的8个关键区截短或加长，采用定点突变技术构建了长度突变体，最适温度相较野生型重组酶大部分都有所下降，通过与重组原酶半衰期的对比发现，突变酶ManBK‑Ⅵ表现出最好的热稳定性。通过对ManBK敏感的氨基酸残基进行优化，同样采用定点突变的方法构建了6个突变体，突变酶ManBK‑P63G和ManBK‑P172G的最适温度为80℃，与野生型重组酶一致。其他两个突变酶最适温度相对较低：ManBK‑P169G最适温度为50℃，ManBK‑P244G最适温度为70℃。
Description
技术领域
本发明属于分子生物学技术领域，具体涉及一种嗜热甘露聚糖酶ManBK突变体及其应用。
背景技术
随着能源的日益紧张，作为自然界中继纤维素之后含量第二丰富的可再生生物质资源，半纤维素的开发利用越来越受到人们的关注，半纤维素主要由木聚糖、甘露聚糖、半乳聚糖和阿拉伯聚糖组成，甘露聚糖水解后产生的低聚甘露糖是具有优异生理活性的功能性低聚糖。甘露聚糖酶是内切型酶从甘露聚糖分子主链的内部随机切割β-1,4-D-糖苷键产生不同聚合度的甘露寡糖，是甘露聚糖降解酶系中最关键的组分。
甘露聚糖酶广泛存在于自然界的多种生物体内，一些嗜热或极嗜热微生物如海栖热袍菌也是甘露聚糖酶的来源。随着生物信息学及各种检测手段的进步，关于酶结构与功能的研究迅猛发展，但是关于甘露聚糖酶结构与功能特别是分子改造的研究报道却偏少，主要原因甘露聚糖酶较为均匀的三维结构让研究者难以寻找突破点，而其在一级结构、性质功能等方面表现出的多样性更增添了对其进行统计对比的困难。
发明内容
本发明的目的在于提供嗜热甘露聚糖酶ManBK突变体及其应用。
一种嗜热甘露聚糖酶ManBK突变体，包括突变体ManBK-Ⅰ、突变体ManBK-Ⅱ、突变体ManBK-Ⅲ、突变体ManBK-Ⅲ’、突变体ManBK-Ⅳ、突变体ManBK-Ⅵ、突变体ManBK-Ⅶ；所述突变体ManBK-Ⅱ、突变体ManBK-Ⅳ在其原有蛋白序列的基础上加长；所述ManBK-Ⅰ、突变体ManBK-Ⅲ、突变体ManBK-Ⅲ’、突变体ManBK-Ⅵ、突变体ManBK-Ⅶ在其原有蛋白序列的基础上截短。
所述嗜热甘露聚糖酶ManBK的氨基酸序列如序列表SEQ ID NO：1所示。
所述突变体ManBK-Ⅰ的氨基酸序列如序列表SEQ ID NO：2所示；所述突变体ManBK-Ⅱ的氨基酸序列如序列表SEQ ID NO：3所示；所述突变体ManBK-Ⅲ的氨基酸序列如序列表SEQ ID NO：4所示；所述突变体ManBK-Ⅲ’的氨基酸序列如序列表SEQ ID NO：5所示；所述突变体ManBK-Ⅳ的氨基酸序列如序列表SEQ ID NO：6所示；所述突变体ManBK-Ⅵ的氨基酸序列如序列表SEQ ID NO：7所示；所述突变体ManBK-Ⅶ的氨基酸序列如序列表SEQ ID NO：8所示。
一种嗜热甘露聚糖酶ManBK突变体，包括突变体包括突变体ManBK-P63G、突变体ManBK-P169G、突变体ManBK-P172G、突变体ManBK-P244G。
所述突变体ManBK-P63G由原序列中第63位氨基酸残基P突变为G；所述突变体ManBK-P169G由原序列中第169位氨基酸残基P突变为G；所述突变体ManBK-P172G由原序列中第172位氨基酸残基P突变为G；所述突变体ManBK-P244G由原序列中第244位氨基酸残基P突变为G。
所述突变体ManBK-P169G的最适pH6.0。
所述突变体ManBK-P169G最适温度为50℃，突变体ManBK-P244G最适温度为70℃。
所述嗜热甘露聚糖酶ManBK突变体在食品发酵工业中的应用。
本发明的有益效果：本发明对嗜热甘露聚糖酶ManBK的8个关键区截短或加长，采用定点突变技术构建了长度突变体，并在毕赤酵母GS115成功的分泌表达。最适温度相较野生型重组酶大部分都有所下降，ManBK-Ⅲ、ManBK-Ⅳ和ManBK-Ⅵ的最适温度为70℃，突变体ManBK-Ⅱ的最适温度为60℃，ManBK-Ⅶ的最适温度为50℃。通过与重组原酶半衰期的对比发现，突变酶ManBK-Ⅵ表现出最好的热稳定性。通过对温度较敏感的氨基酸残基进行优化，同样采用定点突变的方法构建了6个突变体，并在毕赤酵母GS115成功的分泌表达。除突变重组酶ManBK-P169G(最适pH6.0)之外，其余3个突变体的最适pH均为5.0，与野生型重组酶一致。突变酶ManBK-P63G和ManBK-P172G的最适温度为80℃，与野生型重组酶一致。其他两个突变酶最适温度相对较低：ManBK-P169G最适温度为50℃，ManBK-P244G最适温度为70℃。
附图说明
图1野生型和长度突变体的SDS-PAGE分析；M表示标准分子量，1表示重组野生酶ManBK，2-9分别表示突变酶ManBK-Ⅰ、ManBK-Ⅱ、ManBK-Ⅲ、ManBK-Ⅲ’、ManBK-Ⅳ、ManBK-Ⅴ、ManBK-Ⅵ和ManBK-Ⅶ。
图2为ManBK长度突变体最适pH。
图3为ManBK长度突变体最适温度。
图4为ManBK长度突变体动力学曲线。
图5为野生型和ManBK长度突变体的半衰期。
图6为野生型和ManBK柔性突变体的SDS-PAGE分析；
M表示标准分子量，1表示重组野生酶ManBK，2-7分别表示突变酶ManBK-P63G、ManBK-P169G、ManBK-P172G、ManBK-P217G、ManBK-P244G和ManBK-P318G。
图7为ManBK柔性突变体最适pH。
图8为ManBK柔性突变体最适温度。
图9为ManBK柔性突变体动力学曲线。
图10野生型和ManBK柔性突变体的半衰期。
具体实施方式
为了便于理解本发明，下面将对本发明进行更全面的描述。但是，本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
实施例1嗜热甘露聚糖酶ManBK关键区域长度对其热稳定性的影响
(1)采用定点突变法获得突变体：依据嗜热甘露聚糖酶中关键结构区域的分析结果，对关键结构区域进行加长或截断，研究关键区域长度对嗜热甘露聚糖酶热稳定的影响。设计嗜热甘露聚糖酶ManBK 8个不同长度的突变体引物(表1)，采用Fast Mutagenesissystem试剂盒，进行定点突变。
扩增条件：94℃5min，94℃30s，52℃30s，72℃4min，共30个循环，72℃10min。琼脂糖凝胶电泳检测，将目的大小正确的条带进行切胶回收。
表1ManBK关键区域长度突变引物
(2)甘露聚糖酶ManBK长度突变体真核表达载体的构建
嗜热甘露聚糖酶ManBK长度突变体ManBK-Ⅰ、ManBK-Ⅱ、ManBK-Ⅲ、ManBK-Ⅲ’、ManBK-Ⅳ、ManBK-Ⅴ、ManBK-Ⅵ和ManBK-Ⅶ的成熟蛋白编码基因用EcoR I和Not I双酶切后，连接到表达载体pPIC9K并转化至Trans 5α中，使用AOX引物菌液PCR验证正确后，送测序公司进一步鉴定。得到序列正确的8个突变体的酵母表达质粒载体pPIC9K-ManBK-Ⅰ、pPIC9K-ManBK-Ⅱ、pPIC9K-ManBK-Ⅲ、pPIC9K-ManBK-Ⅲ’、pPIC9K-ManBK-Ⅳ、pPIC9K-ManBK-Ⅴ、pPIC9K-ManBK-Ⅵ和pPIC9K-ManBK-Ⅶ。
(3)甘露聚糖酶ManBK长度突变体的表达
将突变体的重组质粒经Bgl II完全线性化后电击转化到GS115感受态细胞中进行诱导表达，经甲醇诱导48h后，以角豆胶为底物分别检测八个突变体培养液上清中的甘露聚糖酶活性，结果除pPIC9K-ManBK-Ⅴ外，均检测到甘露聚糖酶活性。
将野生型和8个突变体培养液离心取上清，将取得的上清液浓缩后，SDS-PAGE发现酵母表达的野生型和8个突变体表观分子量均为47KDa左右(图1)。
(4)甘露聚糖酶ManBK长度突变体与野生型酶学性质分析与比较
甘露聚糖酶ManBK长度突变体的最适pH：7个甘露聚糖酶ManBK长度突变体在60℃，pH(2.0～12.0)缓冲体系中测得的结果，如图2。除ManBK-Ⅳ(最适pH为5.0)之外，其余6个突变体的最适pH均为6.0，较野生型的最适pH 5.0高。且7个突变体在pH5.0～8.0范围内，均能够维持60％以上的酶活力。
甘露聚糖酶ManBK长度突变体的最适温度：7个甘露聚糖酶ManBK长度突变体在最适pH的条件下，不同温度(20℃～90℃)下的酶活力，实验结果如图3所示。突变体ManBK-Ⅰ和ManBK-Ⅲ’的最适温度为80℃；ManBK-Ⅲ、ManBK-Ⅳ和ManBK-Ⅵ的最适温度为70℃，突变体ManBK-Ⅱ的最适温度为60℃，ManBK-Ⅶ的最适温度为50℃，均低于野生型(80℃)。甘露聚糖酶ManBK长度突变体动力学常数的测定：按照双倒数法绘制以角豆胶为底物的甘露聚糖酶ManBK长度突变体的动力学曲线，由图4中不同甘露聚糖酶ManBK长度突变体所对应的动力学曲线计算出其Km和Vmax。其中4个长度突变体的Km值比野生型(Km值为1.40mg/mL)都要大，均在2.0mg/mL以上。Km值与酶的浓度无关，与底物的浓度也无关，同一种酶随着反应条件(温度、pH)的改变而变化，由于7个长度突变体与野生型之间的最适反应条件均不相同，因此Km值也各有差异。
甘露聚糖酶ManBK长度突变体半衰期的测定：为了探究甘露聚糖酶ManBK长度突变体的热稳定性，发明人分别测定了突变体70℃、85℃和95℃条件下的半衰期，如图5所示。
7个突变体在70℃和85℃条件下，半衰期均低于野生型；95℃下，ManBK-Ⅰ、ManBK-Ⅲ、ManBK-Ⅲ’和ManBK-Ⅶ却有所提升，和野生型相比分别提高了5.67％、32.27％、28.08％和45.57％。突变体ManBK-Ⅱ在70℃下的半衰期低于野生型，85℃和95℃条件下半衰期几乎与野生型相同。突变体ManBK-Ⅵ在85℃和95℃条件下半衰期比野生型分别提高了1.8％和15.27％。相较与野生型，突变体ManBK-Ⅵ表现出较好的热稳定性。
实施例2嗜热甘露聚糖酶ManBK关键区域柔性对其热稳定性的影响
(1)甘露聚糖酶ManBK关键区域柔性突变体真核表达载体的构建
嗜热甘露聚糖酶ManBK柔性突变体ManBK-P63G、ManBK-P169G、ManBK-P172G、ManBK-P217G、ManBK-P244G和ManBK-P318G的成熟蛋白编码基因用EcoR I和Not I双酶切后，连接到表达载体pPIC9K并转化Trans5α中，使用AOX引物菌落PCR验证正确后，送测序公司进一步鉴定。得到序列正确的6个突变体的酵母表达质粒载体pPIC9K-ManBK-P63G、pPIC9K-ManBK-P169G、pPIC9K-ManBK-P172G、pPIC9K-ManBK-P217G、pPIC9K-ManBK-P244G和pPIC9K-ManBK-P318G。
(2)甘露聚糖酶ManBK柔性突变体的表达
将突变体的重组质粒经Bgl II完全线性化后电击转化到GS115感受态细胞中进行诱导表达，经甲醇诱导48h后，以角豆胶为底物分别检测6个突变体培养液上清中的甘露聚糖酶活性，结果除pPIC9K-ManBK-P217G和pPIC9K-ManBK-P318G外，均检测到甘露聚糖酶活性。将野生型和6个突变体培养液离心取上清，将取得的上清液浓缩后，SDS-PAGE发现酵母表达的野生型和6个突变体表观分子量均为47KDa左右(图6)。
(3)甘露聚糖酶ManBK柔性突变体与野生型酶学性质分析与比较
甘露聚糖酶ManBK柔性突变体的最适pH：4个甘露聚糖酶ManBK柔性突变体在60℃，pH(2.0～12.0)缓冲体系中测得的结果，如图7。除了突变体ManBK-P169G(最适pH为6.0)之外，其余3个突变体的最适pH均为5.0，与野生型一致。且4个突变体在pH4.0～7.0范围内，均能够维持60％以上的酶活力。
甘露聚糖酶ManBK柔性突变体的最适温度：测定4个Loop长度突变体在最适pH的条件下，不同温度(20℃～90℃)下的酶活力，实验结果如图8所示。突变体ManBK-P63G和ManBK-P172G的最适温度为80℃；ManBK-P244G的最适温度为70℃，ManBK-P169G的最适温度为50℃，均低于野生型(80℃)。
甘露聚糖酶ManBK柔性突变体动力学常数的测定：按照双倒数法绘制以角豆胶为底物的甘露聚糖酶ManBK柔性突变体的动力学曲线，由图9中不同柔性突变体所对应的动力学曲线计算出其Km和Vmax。在相同的温度和pH条件下，突变体ManBK-P63G和ManBK-P244G的Km值(0.49mg/mL、0.88mg/mL)比野生型(1.40mg/mL)低；突变体ManBK-P169G和ManBK-P172G的Km值(5.73mg/mL、3.09mg/mL)比野生型高。Km值为酶促反应速度等于酶促反应最大速度的一半时所需的底物浓度。它表示酶和底物之间的亲和能力，Km值越大，说明需要更多的底物才能达到最大反应速度的一半，即酶的亲和能力越弱，反之亦然。因此突变体ManBK-P63G与底物的亲和能力最强，突变体ManBK-P169G与底物的亲和能力最弱，柔性突变在一定程度上改变了酶与底物的亲和力。
甘露聚糖酶ManBK柔性突变体半衰期的测定：为了探究甘露聚糖酶ManBK长度突变体的热稳定性，我们分别测定了突变体70℃、85℃和95℃下的半衰期，如图10所示。
4个突变酶ManBK-P63G、ManBK-P169G、ManBK-P172G和ManBK-P244G在70℃、85℃和95℃条件下，半衰期均低于野生型。相对于野生重组酶，这四个柔性突变体热稳定性均有所下降，其原因可能是将对温度敏感的脯氨酸突变为甘氨酸，使得甘露聚糖酶ManBK的蛋白质结构柔性增强、稳定性降低，因此柔性突变体的热稳定性比野生型差。
本发明通过嗜热和嗜温甘露聚糖酶氨基酸多序列比对，结合二级结构预测结果和两类甘露聚糖酶分子特征的比较，以嗜热甘露聚糖酶ManBK的为研究对象，找到了影响嗜热甘露聚糖酶稳定性的8个关键关键区域：Ⅰ:61-SQPSSG TVWYQ-70,Ⅱ:168-EPRCPSCDT-176,Ⅲ:206-FGLNIDSDGSY-216,Ⅲ’:226-FTMNLDTDT-234,Ⅳ:241-HLYPDSWGTSDD-252,Ⅴ:278-GVTSNH-283,Ⅵ:311-DLSTGKSPDDGNT-321和Ⅶ:43-LKSSG-47，为后续研究奠定了良好的基础。
对嗜热甘露聚糖酶ManBK的8个关键区截短或加长(Ⅱ、Ⅳ和Ⅴ加长，其余截短)，采用定点突变技术构建了长度突变体ManBK-Ⅰ、ManBK-Ⅱ、ManBK-Ⅲ、ManBK-Ⅲ’、ManBK-Ⅳ、ManBK-Ⅴ、ManBK-Ⅵ和ManBK-Ⅶ，并在毕赤酵母GS115成功的分泌表达。重组酶ManBK-Ⅴ经测定没有甘露聚糖酶活性，但经SDS-PAGE电泳检测确已表达。其他重组突变酶的pH特性相比于野生型重组酶变化不大，除突变重组酶ManBK-Ⅳ(最适pH5.0)之外，其余6个突变体的最适pH均为6.0，偏酸性。最适温度相较野生型重组酶大部分都有所下降，ManBK-Ⅲ、ManBK-Ⅳ和ManBK-Ⅵ的最适温度为70℃，突变体ManBK-Ⅱ的最适温度为60℃，ManBK-Ⅶ的最适温度为50℃。通过与重组原酶半衰期的对比发现，突变酶ManBK-Ⅵ表现出最好的热稳定性。
通过对关键区域温度较敏感的氨基酸残基进行优化(脯氨酸替换)，同样采用定点突变的方法构建了6个突变体ManBK-P63G、ManBK-P169G、ManBK-P172G、ManBK-P217G、ManBK-P244G和ManBK-P318G，并在毕赤酵母GS115成功的分泌表达。重组酶ManBK-P217G和ManBK-P318G经测定没有甘露聚糖酶活性，但经SDS-PAGE电泳检测确已表达。其他重组突变酶的pH特性相比于野生型重组酶变化不大，除突变重组酶ManBK-P169G(最适pH6.0)之外，其余3个突变体的最适pH均为5.0，与野生型重组酶一致。突变酶ManBK-P63G和ManBK-P172G的最适温度为80℃，与野生型重组酶一致。其他两个突变酶最适温度相对较低：ManBK-P169G最适温度为50℃，ManBK-P244G最适温度为70℃。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
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Claims (7)
1.一种嗜热甘露聚糖酶ManBK突变体，其特征在于，包括突变体ManBK-Ⅰ、突变体ManBK-Ⅱ、突变体ManBK-Ⅲ、突变体ManBK-Ⅲ’、突变体ManBK-Ⅳ、突变体ManBK-Ⅵ、突变体ManBK-Ⅶ；所述突变体ManBK-Ⅱ、突变体ManBK-Ⅳ在其原有蛋白序列的基础上加长；所述ManBK-Ⅰ、突变体ManBK-Ⅲ、突变体ManBK-Ⅲ’、突变体ManBK-Ⅵ、突变体ManBK-Ⅶ在其原有蛋白序列的基础上截短。
2.根据权利要求1所述嗜热甘露聚糖酶ManBK突变体，其特征在于，所述突变体ManBK-Ⅰ的氨基酸序列如序列表SEQ ID NO：2所示；所述突变体ManBK-Ⅱ的氨基酸序列如序列表SEQID NO：3所示；所述突变体ManBK-Ⅲ的氨基酸序列如序列表SEQ ID NO：4所示；所述突变体ManBK-Ⅲ’的氨基酸序列如序列表SEQ ID NO：5所示；所述突变体ManBK-Ⅳ的氨基酸序列如序列表SEQ ID NO：6所示；所述突变体ManBK-Ⅵ的氨基酸序列如序列表SEQ ID NO：7所示；所述突变体ManBK-Ⅶ的氨基酸序列如序列表SEQ ID NO：8所示。
3.一种嗜热甘露聚糖酶ManBK突变体，其特征在于，包括突变体包括突变体ManBK-P63G、突变体ManBK-P169G、突变体ManBK-P172G、突变体ManBK-P244G。
4.根据权利要求3所述嗜热甘露聚糖酶ManBK突变体，其特征在于，所述突变体ManBK-P63G由原序列中第63位氨基酸残基P突变为G；所述突变体ManBK-P169G由原序列中第169位氨基酸残基P突变为G；所述突变体ManBK-P172G由原序列中第172位氨基酸残基P突变为G；所述突变体ManBK-P244G由原序列中第244位氨基酸残基P突变为G。
5.根据权利要求3所述嗜热甘露聚糖酶ManBK突变体，其特征在于，所述突变体ManBK-P169G的最适pH6.0。
6.根据权利要求3所述嗜热甘露聚糖酶ManBK突变体，其特征在于，所述突变体ManBK-P169G最适温度为50℃，突变体ManBK-P244G最适温度为70℃。
7.权利要求1-6所述嗜热甘露聚糖酶ManBK突变体在食品发酵工业中的应用。
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