CN116240187B - 脯氨酰羟化酶α1亚基突变体、编码基因及其在催化脯氨酸羟基化中的应用 - Google Patents
脯氨酰羟化酶α1亚基突变体、编码基因及其在催化脯氨酸羟基化中的应用 Download PDFInfo
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Abstract
本发明公开了一种脯氨酰羟化酶α1亚基突变体，在野生型的P4Hα1的基础上进行突变而成，突变位点为H80S、H313S、Y181A的单突变或者其组合突变，Y181A的单突变除外。还公开了其编码基因、表达载体、表达宿主菌以及在催化脯氨酸羟基化中的应用。本发明对P4Hα1亚基进行点突变，突变位点为H80S、H313S、Y181A或者其组合，从而获得了具有更高酶活和亲和力的P4Hα1亚基，可用于工业生产和实验室研究。
Description
技术领域
本发明涉及脯氨酰羟化酶α1亚基突变体、编码基因及其在催化脯氨酸羟基化中的应用，属于酶催化技术领域。
背景技术
脯氨酰羟化酶(Prolyl 4-hydroxylase，P4H)是胶原合成的关键酶，具体地说，通过将-Xaa-Pro-Bly-序列中的脯氨酸残基羟化，催化胶原和相关蛋白质中4-羟脯氨酸的形成，从而使新合成的前胶原链形成正确折叠，保持三螺旋结构。脯氨酰羟化酶是α2β2异源四聚体，由两个α亚基(P4Hα1和P4Hα2)和两个β亚基(PDI)组成。其中α1亚基是提供酶活性催化位点的主要部分，对催化至关重要，在响应胶原合成速率的变化时，该亚基似乎也更有效地受到调控。中国专利文献CN1221456A公开了脯氨酰－4－羟化酶的α2亚单位，编码此亚单位的核酸序列及其生产方法，但并未涉及到催化活性更高的α1亚基的序列。α亚基上存在大多数与酶有关的催化位点，对其进行突变以筛选酶活性更高的突变菌株，使羟化率提高或与底物亲和力增加，从而使整个反应更大限度的往羟脯氨酸合成的方向进行。。
发明内容
本发明的目的是提高一种脯氨酰羟化酶α1亚基突变体。
本发明采用的技术方案：
一种脯氨酰羟化酶α1亚基突变体，其特征在于在野生型的P4Hα1的基础上进行突变而成，突变位点为H80S、H313S、Y181A的单突变或者其组合突变，Y181A的单突变除外。
优选的，突变位点包括H80S、H313S和Y181A。
本发明还公开了上述的脯氨酰羟化酶α1亚基突变体的编码基因。
上述的脯氨酰羟化酶α1亚基突变体在催化脯氨酸羟基化中的应用。
上述的脯氨酰羟化酶α1亚基突变体的表达载体。
优选的，所述载体质粒为pSEVA321。
上述的脯氨酰羟化酶α1亚基突变体的表达宿主菌。
优选的，所述宿主菌为大肠杆菌。
本发明还公开了一种脯氨酰羟化酶，其特征在于包括上述的脯氨酰羟化酶α1亚基突变体。本发明的脯氨酰羟化酶α1亚基突变体可以采用寡核苷酸介导的定点突变或QuickChange定点突变方法获得。
其中寡核苷酸介导的定点突变，包括以下步骤：
S1.获得单链目的基因；
S2.人工合成带突变序列的引物；
S3.制备异源双链DNA；
S4.转化宿主细胞；
S5.筛选重组体；
S6.突变基因的鉴定和回收。
其中QuickChange定点突变，包括以下步骤：
S1.根据现有基因设计突变引物；
S2.合成引物并准备好模板质粒DNA；
S3.设置反应条件进行PCR；
S4.DpnI消化模板DNA；
S5.将消化产物转化宿主细胞；
S6.筛选阳性克隆并送测；
S7.突变产物的回收与保存。
本发明的有益效果：
本发明对P4Hα1亚基进行点突变，突变位点为H80S、H313S、Y181A或者其组合，从而获得了具有更高酶活和亲和力的P4Hα1亚基，可用于工业生产和实验室研究。
附图说明
图1为pSEVA321-porin-P4H(α1/β)的质粒图谱。
图2为Hyp产量和转化率统计图。
具体实施方式
以下通过实施例进一步说明本发明，但不作为对本发明的限制。以下提供了本发明实施方案中所使用的具体材料及其来源。但是，应当理解的是，这些仅仅是示例性的，并不意图限制本发明，与如下试剂和仪器的类型、型号、品质、性质或功能相同或相似的材料均可以用于实施本发明。下述实施例中所使用的实验方法如无特别说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1、羟化酶突变体重组质粒的构建
1、单点突变
S1、将编码羟化酶α亚基的基因P4H(α1)在突变位点设计引物F和R，引物序列如下表所示；
表1引物序列表
S2、使用上述的引物，以野生型P4H(α1)为模板进行基因片段扩增，扩增出的片段为环p产物；
S3、使用T4 DNALigase将环p产物进行连接，反应总体系为10ul，在0.2ml的pcr小管中依次加入表2所示的成分：
表2连接体系
将以上成分混匀后，瞬时离心，16℃过夜连接(或25℃1小时)，获得连接产物；
S4、将S3反应后的产物转入E.coli BL21(DE3)感受态，从反应体系取出3-5ul于冰上与感受态混合均匀，并冰浴30min，其后水浴锅中42℃热激90s，然后立即置于冰上2min，再加入500ulLB培养基，37℃摇床中振荡培养1小时后涂布于相应抗性的平板上，在37℃摇床中倒置培养12-14小时；
S5、挑取S4中培养板的单克隆菌落进行PCR，选择阳性克隆进行测序，将测序结果与原始目的基因序列进行比对，确认是否成功突变获得突变体重组质粒，若成功突变，则进行质粒提取以及菌液保藏；
2、组合突变
以上述单点突变的基础上形成的质粒pSEVA321-porin-P4H(α1/β)-H80S为模板，使用引物F3/R3进行模板扩增，扩增后的产物使用T4连接酶进行连接，在16℃下连接过夜，将连接产物进行转化，在相应的抗性板上培养，最后挑取单克隆进行菌落pcr验证，选择阳性克隆进行送测，根据测序结果挑选出成功双突变的组合体pSEVA321-porin-P4H(α1/β)-H80SY181A。
按上述分别构建单突变重组质粒pSEVA321-porin-P4H(α1/β)-H80S、pSEVA321-porin-P4H(α1/β)-H313S、pSEVA321-porin-P4H(α1/β)-Y181A，双突变质粒pSEVA321-porin-P4H(α1/β)-H80SY181A、pSEVA321-porin-P4H(α1/β)-H80SH313S、pSEVA321-porin-P4H(α1/β)-H313SY181A、以及三突变重组质粒pSEVA321-porin-P4H(α1/β)-H80SH313SY181A。同时构建载体pSEVA321-porin-P4H(α1/β)作为对照。其中野生型的P4Hα1亚基其氨基酸序列如SEQ ID No.7所示，核苷酸序列如SEQ ID No.8所示。
野生型的P4H的β亚基氨基酸序列如SEQ ID No.9所示。
突变位点为H80S的P4Hα1亚基突变体其氨基酸序列如SEQ ID No.10所示。
突变位点为H313S的P4Hα1亚基突变体其氨基酸序列如SEQ ID No.11所示。
突变位点为Y181A的P4Hα1亚基突变体其氨基酸序列如SEQ ID No.12所示。
突变位点为H80S、H313S的P4Hα1亚基突变体其氨基酸序列如SEQ IDNo.13所示。
突变位点为H313S、Y181A的P4Hα1亚基突变体其氨基酸序列如SEQ IDNo.14所示。
突变位点为H80S、H313S、Y181A的P4Hα1亚基突变体其氨基酸序列如SEQ ID No.15所示。
突变位点为H80S、Y181A的P4Hα1亚基突变体其氨基酸序列如SEQ IDNo.16所示。
实施例2、生物转化法生产羟脯氨酸
S1、将实施例1中构建完成的所有突变体重组质粒与对照组质粒一起进行摇瓶培养，即挑取突变重组质粒的单克隆加入到5ml带有相应抗性的LB培养基中，37℃培养12小时，为一级种子液；
S2、取1％一级种子液接种到20ml带有相应抗性的LB培养基中，37℃培养12小时，为二级种子液；
S3、取5％二级种子液到50ml带有相应抗性的摇瓶发酵培养基中，37℃培养48小时，分别在12、24、36、48小时时取1ml样保存，最后发酵结束收集菌体细胞。按照羟脯氨酸含量检测试剂盒要求进行羟脯氨酸产量测定，而α-酮戊二酸与L-脯氨酸的含量由HPLC测定，测定结束后进行转化率的计算，对不同突变位点及不同突变组合的重组质粒进行评估。
转化率计算公式：
(M1：转化前的脯氨酸的含量；M2：转化后剩余的脯氨酸含量；M3：羟脯氨酸浓度。)
结果如图2所示，除了Y181A单突变体与野生型相比几乎一样之外，其余突变体其产物含量与转化率都有所提升，其中提升最明显的是三突体重组质粒pSEVA321-porin-P4H(α1)-H80SH313SY181A。
实施例3、羟化酶酶活性能表征
当实施例2中的所有重组质粒发酵结束后，将反应混合物(含有80mM MES缓冲液(pH 6.5)、4mM L-脯氨酸、8mMα-KG、2mM FeSO4、4mM l-抗坏血酸和细胞或纯化的P4H)在35℃下摇晃孵育10分钟，然后在100℃下热处理5分钟使细胞活性完全灭活，离心后取上清测定其在560nm处的吸光度。一个单位P4H活性定义为：在一分钟内形成1nmol Hyp的酶的量，对突变后羟化酶的酶活与酶对底物(Pro-Pro-Gly)10亲和力变化进行检测。
结果如表3所示，除了Y181A突变体的亲和力与野生型的几乎一致外，其余所有的突变体其对底物(Pro-Pro-Gly)10的亲和力都增强了，其中三突变体对底物的亲和力是增强最多的。此外，对于酶活力，将野生型设为100作为对照，其余突变体相比野生型，三突变重组质粒的酶活力是最高的。
表3羟脯氨酸酶活及动力学参数
Claims (8)
1.一种脯氨酰羟化酶，由两个α1亚基和两个β亚基组成，其中β亚基的氨基酸序列如SEQID No.9所示，其特征在于，α1亚基系在野生型的P4Hα1亚基的基础上进行突变而成的P4Hα1亚基突变体，突变位点为H313S，其中野生型的P4Hα1亚基的氨基酸序列如SEQ ID No.7所示。
2.根据权利要求1所述的脯氨酰羟化酶，其特征在于，所述P4Hα1亚基突变体还包括突变位点H80S，或者还包括突变位点H80S和Y181A。
3.权利要求1或2所述的脯氨酰羟化酶的编码基因。
4.权利要求1或2所述的脯氨酰羟化酶在催化脯氨酸羟基化中的应用。
5.权利要求1或2所述的脯氨酰羟化酶的表达载体。
6.根据权利要求5所述的表达载体，其特征在于，所述载体质粒为pSEVA321。
7.权利要求1所述的脯氨酰羟化酶的表达宿主菌。
8.根据权利要求7所述的表达宿主菌，其特征在于，所述宿主菌为大肠杆菌。
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