CN116574157A - 一种α-淀粉酶的嵌合信号肽及其应用 - Google Patents
一种α-淀粉酶的嵌合信号肽及其应用 Download PDFInfo
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Abstract
本发明涉及一种α‑淀粉酶的嵌合信号肽，其包括如SEQ ID No.1所示的氨基酸序列，其可有效提高α‑淀粉酶的分泌效率。同时，本发明还公开了上述嵌合信号肽的应用。
Description
技术领域
本发明涉及一种α-淀粉酶的嵌合信号肽及其应用。
背景技术
重组蛋白对工业、医疗保健和可持续的生物经济发展具有重要意义，因此，建立高质、高量蛋白质高效生产平台很有必要。
枯草芽孢杆菌(Bacillus subtilis)被认为是一种生物安全菌株，并且具有遗传稳定性高和蛋白分泌能力强等优点，常被用做细胞工厂生产工业、农业、医药等领域重组蛋白，如蛋白酶、淀粉酶和木聚糖酶等。
枯草芽孢杆菌胞外蛋白分泌系统主要有一般分泌途径(Sec)、双精氨酸分泌途径(Tat)、ABC转运系统和非经典途径等，其中大部分蛋白质以Sec途径进行分泌，该途径指导分泌的蛋白质在其N端具有信号肽序列。
信号肽包含三部分，带正电荷的N端区域，含有赖氨酸或精氨酸残基；中心H区，由一连串疏水残基组成，可能采用α-螺旋构象，在这个疏水核心区的中间，经常会出现螺旋断裂的甘氨酸或脯氨酸残基，从而形成一个可以插入膜的发夹状结构；亲水性C区，具有Ⅰ型信号肽酶识别位点，包括Ala-x-Ala共识基序。
信号肽除了被相应的蛋白转位酶靶向和膜易位所需要外，还对相应的靶蛋白的生物合成、折叠动力学和稳定性有额外的影响，信号肽的净电荷、疏水性或长度的改变都会改变靶蛋白的分泌效率，且同一信号肽指导不同蛋白质的分泌效率存在很大差异，目前尚未建立预测靶蛋白最适信号肽的相关软件，因此，通过信号肽文库的构建和筛选仍是提高目的蛋白分泌量的一种有效措施。
发明内容
本发明的目的是提供一种可有效提高α-淀粉酶分泌效率的嵌合信号肽及其应用。
本发明采用如下技术方案：
一种α-淀粉酶的嵌合信号肽，其包括如SEQ ID No.1所示的氨基酸序列。
一种如权利要求1所述的α-淀粉酶的嵌合信号肽的编码基因。
优选的，所述编码基因包括如SEQ ID No.2所示的核苷酸序列。
一种包含上述编码基因的载体。
一种重组菌，其通过上述嵌合信号肽引导α-淀粉酶的表达。
进一步的，所述重组菌为枯草芽孢杆菌。
一种上述α-淀粉酶的嵌合信号肽在提高α-淀粉酶分泌效率中的应用。
一种上述α-淀粉酶的嵌合信号肽或重组菌在制备α-淀粉酶中的应用。
一种上述α-淀粉酶的嵌合信号肽的构建方法，通过筛选出引导α-淀粉酶的胞外酶活力较高的信号肽，将其N区、H区和C区进行互换，再经筛选得到。
本发明的有益效果在于：本发明从枯草芽孢杆菌的25种天然信号肽中，筛选出指导解淀粉芽孢杆菌α-淀粉酶在枯草芽孢杆菌中分泌能力较高的6种信号肽，将其中4种信号肽yvcE、yoqM、BglS和yobB的疏水区(H区)替换SacB信号肽的H区，构建4种嵌合信号肽，获得α-淀粉酶在枯草芽孢杆菌胞外酶活力提高的嵌合信号肽RSP1，其指导α-淀粉酶的分泌能力是sacB信号肽的3.07倍。
附图说明
图1为本发明的嵌合信号肽构建及筛选流程示意图。
图2为天然信号肽PCR扩增结果。
图3为α-淀粉酶天然信号肽文库刚果红平板培养结果。
图4为不同天然信号肽指导的α-淀粉酶相对酶活力。
图4中，与sacB信号肽相比，差异性显著*(P＜0.05)，差异性极显著**(P＜0.01)。
图5为不同天然信号肽指导的α-淀粉酶工程菌株发酵液SDS-PAGE电泳结果。
图6为嵌合信号肽扩增结果
图6中，M为DNA分子量标准，泳道1-4分别为嵌合信号肽RSP1～RSP4的PCR结果。
图7为不同嵌合信号肽指导的α-淀粉酶分泌效果
图7中，与sacB信号肽相比，差异性显著*(P＜0.05)，差异性极显著**(P＜0.01)。
具体实施方式
以下结合实施例对本发明的技术方案进行详细地阐述。以下实施例仅用于说明和解释本发明，而不构成对本发明技术方案的限制。
如图1所示，嵌合信号肽的构建方法包括如下步骤：从枯草芽孢杆菌(B.subtilis)25种天然信号肽(yvcE、yoqM、yuaB、pelA、pelB、yoaW、yqxI、lipA、estB、yoqH、ybfO、ybxI、sacB、bglS、yddT、yobB、aprE、nprB、bpr、mpr、epr、nprE、vpr、wprA、amyE)中，筛选出指导解淀粉芽孢杆菌(B.amyloliquefaciens)α-淀粉酶在枯草芽孢杆菌(B.subtilis)中分泌能力较高的信号肽，将其中一个信号肽的疏水区(H区)替换另一个信号肽的H区，构建出嵌合信号肽。
其中，信号肽N区、H区和C区通过SigP 6.0(SignalP 6.0-DTU Health Tech-Bioinformatic Services)进行分析。
本发明涉及的质粒如表1所示，菌株如表2所示，引物如表3所示。
表1本发明涉及的质粒
表2本发明涉及的菌株
注：①Kawamura,F.；Doi,R.H.Construction of a Bacillus Subtilis DoubleMutant Deficient in Extracellular Alkaline and Neutral Proteases.J Bacteriol1984,160(1),442–444。
表3本发明涉及的引物
一、α-淀粉酶天然信号肽库的筛选
1.1α-淀粉酶基因amyL扩增及其表达载体构建
以解淀粉芽孢杆菌(B.amyloliquefaciens)基因组为模板，BFamy-5和BFamy-2为引物，扩增amyL基因，95℃2min；95℃20s，61℃20s，72℃25s；72℃5min，32个循环。
以Vlinker-1和VBFamy-2为引物，质粒pWB-manBl(I91N/L211I)为模板扩增载体片段，95℃2min；95℃20s，62℃20s，72℃1min，32个循环；72℃5min。
再将两PCR片段采用POE-PCR方法进行扩增，95℃2min；98℃15s，60℃20s，68℃6min；68℃10min，35个循环。
将POE-PCR产物转化至枯草芽孢杆菌B.subtilis DB104感受态细胞，获得淀粉酶表达菌株B.subtilis(pWB-amyL)。
1.2天然信号肽基因克隆
以枯草芽孢杆菌DB104基因组为模板，扩增25种天然信号肽，所用引物见表3，PCR扩增程序：95℃2min；95℃20s，45℃20s，72℃15s，5个循环；95℃20s，58℃20s，72℃15s；72℃5min。
25种天然信号PCR扩增结果如图2所示，25种天然信号PCR扩增条带单一明亮，参照表3说明中每个信号肽长度，每个天然信号肽PCR产物大小与目的条带大小一致，扩增结果与预期分子量大小一致。
1.3载体扩增
以pWB-amyL质粒为模板，Vsip-1和Vsip-2为引物，扩增5.2kb载体骨架片段，95℃2min；95℃20s，55℃20s，72℃1min30s，5个循环；95℃变性20s，61℃20s，72℃1min30s；72℃5min。
1.4天然信号肽与载体片段的融合
天然信号肽基因和载体片段纯化后，天然信号肽大小分为6组，目的基因与表达载体等摩尔比按照POE-PCR方法进行搭接，95℃2min；95℃20s，56℃20s，68℃6min；35个循环，68℃10min。将POE-PCR产物转化入至B.subtilis DB104感受态细胞，获得α-淀粉酶天然信号肽文库B.subtilis(pWB-SPn-amyL)。
1.5α-淀粉酶天然信号肽文库的筛选
将天然信号肽文库B.subtilis(pWB-SPn-amyL)在刚果红筛选培养基(氯化钠10g/L，酵母粉0.5g/L，蛋白胨10g/L，玉米淀粉10g/L，琼脂15g/L，刚果红1g/L，卡那霉素10mg/L)培养，于37℃恒温培养14h进行初筛；从每组转化平板中挑取水解圈直径(D)与菌落直径(d)比值较大的10个单克隆进行复筛，结果如图3所示，每个克隆所产透明圈有明显差异，再每组挑取水解圈D/d比值较大的3个菌落，进行液体培养，测定α-淀粉酶活力，共挑取10个酶活力较高的菌株进行测序。测序结果表明，筛选得到对α-淀粉酶分泌效率较高的信号肽是yoqM、yobB、BglS、sacB、nprB和yvcE。
1.6α-淀粉酶天然信号肽工程菌株的发酵
每种天然信号肽工程菌B.subtilis(pWB-SPn-amyL，其中SPn代表yoqM、yobB、BglS、sacB、nprB和yvcE 6种天然信号肽)培养3个单克隆，首先在LB液体培养基，37℃180rpm培养12h进行种子培养，再按10％比例转接至发酵培养基(酵母粉20g/L，蛋白胨25g/L，K2HPO4 3g/L，葡萄糖30g/L)，相同培养条件下继续培养5d，每隔1d进行取样。
1.7α-淀粉酶活力测定方法
采用DNS法测定酶活力。将玉米淀粉溶于pH 7.0的磷酸缓冲液，配制20g/L的底物，在1.5mL离心管当中加入底物0.27mL，再加入0.03mL适当稀释的粗酶液，混合均匀后放入60℃恒温水浴锅，反应10min，取出后迅速加入0.6mL的DNS试剂，混合均匀后放入100℃沸水浴5min，迅速取出进行冷水浴，测定样品540nm吸光值。配制不同浓度的葡萄糖，与DNS试剂混匀，沸水浴5min，测定OD540制备标准曲线。
1.8天然信号肽工程菌胞外α-淀粉酶活力
对发酵1～5d的6种天然信号肽工程菌B.subtilis(pWB-yoqM-amyL)、B.subtilis(pWB-yobB-amyL)、B.subtilis(pWB-BglS-amyL)、B.subtilis(pWB-sacB-amyL)、B.subtilis(pWB-nprB-amyL)和B.subtilis(pWB-yvcE-amyL)发酵液样品，调至相同OD600，12000rpm离心10min，取上清作为粗酶液，进行适当稀释后测定胞外α-淀粉酶活力，图4所示为上述6种不同天然信号肽工程菌B.subtilis(pWB-SPn-amyL)发酵第4天胞外α-淀粉酶活力，yoqM信号肽指导的α-淀粉酶分泌效率最高，酶活力在第4天时达763.65U/mL，是所筛选得到的最佳天然信号肽，分泌效率为sacB信号肽的2.42倍，其它天然信号肽指导α-淀粉酶分泌效率由高到低分别是sacB、BglS、nprB、yvcE及yobB信号。同时对上述6种天然信号肽工程菌株第4d粗酶液样品进行SDS-PAGE电泳，可以看到6种工程菌在55.4kDa位置都有目的蛋白(如图5)，与α-淀粉酶预期分子量大小一致，但蛋白表达量明显有差异，与酶活力测定结果一致，即yoqM信号肽指导的α-淀粉酶分泌量最高，信号肽yobB指导的α-淀粉酶分泌量最低。
二、嵌合信号肽的构建
由于sacB是B.subtilis常用信号肽，我们以该信号肽为基础，将α-淀粉酶筛选得到的胞外酶活力较高的4种天然信号肽yvcE、yoqM、BglS和yobB信号肽的H区替换sacB信号肽的H区，构建嵌合信号肽RSP1、RSP2、RSP3和RSP4(如表4所示)，以期通过α-淀粉酶信号肽N区、H区和C区的强强组合，实现α-淀粉酶更加高效分泌。
表4本发明构建的四种嵌合信号肽
| 嵌合信号肽名称 | 结构 | 氨基酸序列 | 核苷酸序列 |
| RSP1 | N(SacB)-H(yvcE)-C(SacB) | SEQ ID No.1 | SEQ ID No.2 |
| RSP2 | N(SacB)-H(yoqM)-C(SacB) | SEQ ID No.3 | SEQ ID No.4 |
| RSP3 | N(SacB)-H(BglS)-C(SacB) | SEQ ID No.5 | SEQ ID No.6 |
| RSP4 | N(SacB)-H(yobB)-C(SacB) | SEQ ID No.7 | SEQ ID No.8 |
三、嵌合信号肽的筛选
3.1淀粉酶基因amyL扩增及其表达载体构建：同步骤1.1。
3.2嵌合信号肽基因克隆：根据信号肽N、H、C三个区域的预测结果，依据表4的结构，委托北京六合华大基因科技有限公司合成模板DNA序列，嵌合信号肽RSP1～RSP4克隆所用引物为P25和P26，PCR扩增程序同步骤1.2的天然信号肽的PCR扩增程序。嵌合信号肽RSP1～RSP4扩增结果如图6所示，与期望结果一致。
3.3载体扩增：同步骤1.3。
3.4嵌合信号肽与载体片段融合：同步骤1.4，将POE-PCR产物转化入至B.subtilisDB104感受态细胞，获得α-淀粉酶嵌合信号肽文库B.subtilis(pWB-RSPn-amyL)。
3.5嵌合信号肽指导α-淀粉酶工程菌株发酵：将4种嵌合信号肽工程菌，B.subtilis(pWB-RSP1-amyL)、B.subtilis(pWB-RSP2-amyL)、B.subtilis(pWB-RSP3-amyL)和B.subtilis(pWB-RSP4-amyL)按步骤1.6方法进行发酵。
3.6α-淀粉酶活力测定：同步骤1.7。
3.7天然信号肽工程菌胞外α-淀粉酶活力：同步骤1.8。以sacB信号肽所测酶活力为对照，计算各嵌合信号肽指导的α-淀粉酶的相对酶活力，筛选得到的适合α-淀粉酶的嵌合信号肽为RSP1，其指导的胞外α-淀粉酶活力达968.52U/mL，是sacB信号肽的3.07倍，与sacB信号肽指导的胞外α-淀粉酶差异显著，而其它嵌合信号肽RSP2、RSP3和RSP4指导的α-淀粉酶胞外酶活力约为sacB信号肽的10％左右，也与sacB信号肽指导的胞外α-淀粉酶差异显著(图7)。
根据上述的实施例对本发明作了详细描述。需说明的是，以上的实施例仅仅为了举例说明发明而已。在不偏离本发明的精神和实质的前提下，本领域技术人员可以设计出本发明的多种替换方案和改进方案，其均应被理解为在本发明的保护范围之内。
Claims (9)
1.一种α-淀粉酶的嵌合信号肽，其特征在于，其包括如SEQ ID No.1所示的氨基酸序列。
2.一种如权利要求1所述的α-淀粉酶的嵌合信号肽的编码基因。
3.根据权利要求2所述的编码基因，其特征在于，其包括如SEQ ID No.2所示的核苷酸序列。
4.一种包含如权利要求2所述的编码基因的载体。
5.一种重组菌，其特征在于，其通过如权利要求1所述的嵌合信号肽引导α-淀粉酶的表达。
6.根据权利要求5所述的重组菌，其特征在于，其为枯草芽孢杆菌。
7.一种权利要求1所述的α-淀粉酶的嵌合信号肽在提高α-淀粉酶分泌效率中的应用。
8.一种权利要求1所述的α-淀粉酶的嵌合信号肽或权利要求5所述的重组菌在制备α-淀粉酶中的应用。
9.一种如权利要求1所述的α-淀粉酶的嵌合信号肽的构建方法，其特征在于，通过筛选出引导α-淀粉酶的分泌效率较高的信号肽，将其N区、H区和C区进行互换，再经筛选得到。
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