CN114958804A - 一种中性植酸酶突变体 - Google Patents

一种中性植酸酶突变体 Download PDF

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CN114958804A
CN114958804A CN202210710439.8A CN202210710439A CN114958804A CN 114958804 A CN114958804 A CN 114958804A CN 202210710439 A CN202210710439 A CN 202210710439A CN 114958804 A CN114958804 A CN 114958804A
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汪小杰
张艺达
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Shanghai Jikaixing Biotechnology Co ltd
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Abstract

本发明涉及基因工程与蛋白质改造技术领域，具体涉及一种中性植酸酶突变体。本发明以野生型中性植酸酶PhyC为基础，提供了比活力得到显著提高的突变体，突变体包含L255Q，V296T，V372T中至少一个突变位点，与野生型中性植酸酶相比，本发明提供的突变体的比活力普遍提高了26.14‑109.33%。其中，L255Q/V296T/V372T三点突变的中性植酸酶突变体的比活力最高，达45.32 U/mg，比野生型中性植酸酶PhyC提高了109.33%。本发明的中性植酸酶突变体有利于降低中性植酸酶的生产成本，促进其在饲料领域的广泛应用。

Description

一种中性植酸酶突变体
技术领域
本发明属于蛋白质改造技术领域，具体涉及一种比活得到显著提高的中性植酸酶突变体。
背景技术
植酸是豆类、谷类和油料作物等种子中磷的主要储存形式，植酸酶能够水解植酸生成无机磷和磷酸肌醇衍生物，在动物饲料中添加植酸酶可以有效地提高植酸中磷的利用效率、降低动物排泄物的环境磷污染，并能通过去除植酸的抗营养作用来提高饲料的营养价值，对具有高比活和良好热稳定性植酸酶的需求，促进了酶资源的开发利用以及植酸酶基因工程和蛋白质工程研究，也为更好应用于实践生产奠定基础。中性植酸酶也称螺旋桨植酸酶，主要来源于芽孢杆菌，是耐热性较好，最适反应呈中性（pH 7.0-7.5）,能够分解植酸释放磷元素，并且与金属离子有关的一类酶，尤其是对于植酸酶酶活性及稳定性起到至关重要的作用。这一类植酸酶可在鲤科鱼类及单胃动物等值呈中性的肠道中起作用，而酸性植酸酶在中性条件下活性基本丧失，因此，中性植酸酶弥补了酸性植酸酶性质上的不足，可提高植酸酶在动物胃肠道的效用，拓宽植酸酶的应用范围。
中性植酸酶主要指β-螺旋桨(BPP)植酸酶，即β-propeller中性植酸酶，主要来源于芽孢杆菌属。第一个报道的植酸酶是来源于Bacillus的植酸酶，该酶分子量为43 kDa，最适pH是7.0，最适温度55℃,，酶活性依赖于Ca2+的存在。Oh等推测植物来源的植酸酶可能具有与BPP相似的催化机制，Lilium longiflorum花粉和许多豆类植物来源的植酸酶活性与BPP植酸酶一样，在Ca2+存在条件下活性显著提高。此外，Zhang等和Huang等分别克隆了天牛肠道菌株Janthion-bacterium sp.TN 115的中性植酸酶基因和菌株中性植酸酶基因，并将其在大肠杆菌BL21 (DE3)中进行诱导表达。
芽孢杆菌来源的β-propeller中性植酸酶都具有相似的酶学性质，其最适范围都在6.0-8.0之间，具有相近的分子量和酶促反应最适pH（7.0左右），具有较高的热稳定性，其催化活性、热稳定性及pH稳定性均依赖于Ca2+。Ca2+具有稳定植酸酶空间结构的作用。中性植酸酶对植酸盐具存高度的特异性，其最适反应底物是植酸及其盐类复合物，将植酸酶加入到其它含有磷酸基团底物中，检测不到酶活性，且具有较小Km的值，表明对底物的亲和性较好。芽孢杆菌中性植酸酶对胰蛋白酶、木瓜蛋白酶、胰酶的抵抗力都很强，对胃蛋白酶却相当敏感，添加少量的胃蛋白酶在短时间内便可将植酸酶降解。
中性植酸酶在食品方面的应用主要是作为食品添加剂添加到食品中，降解植酸，消除其抗营养作用，而提高矿质元素的吸收率，改善人体对矿物质的吸收和提高食品加工技术。由于低磷酸肌醇在生物体内重要的生理作用，所以在医药工业中生产的肌醇及其磷酸盐也备受人们关注。中性植酸酶在农业上的用途主要作为饲料添加剂应用于水产养殖，饲料中添加外源植酸酶可以提高植酸磷的利用率，降低磷的排泄，在虹鳟、斑点叉尾鮰和条纹鲈等几种鱼的饲料中添加植酸酶尤其具有明显的效果，此外还可以提高矿物元素的生物利用率，提高鱼类对蛋白质及脂肪的利用，降低植酸盐的抗营养作用，同时提供营养物质，增加经济效益，所以中性植酸酶制剂的研发在食品、医药及水产养殖领域具有广阔的应用前景。
天然菌株所产的中性植酸酶产量低，不能满足工业化生产的需要，提高现有的中性植酸酶的酶活力和酶的性能对于其生产成本的控制效果的发挥有着至关重要的作用。因此筛选高活性的中性植酸酶是近年来的研究热点和难点。
发明内容
本发明的目的是提供一种中性植酸酶突变体。所述突变体的比活力比野生型得到显著提高，有利于其在饲料领域的广泛应用。
为了实现上述发明目的，本发明提供以下技术方案：
本发明涉及一种中性植酸酶突变体，其包含与SEQ ID NO:1具有至少90%同一性的氨基酸序列，且与SEQ ID NO:1相比在选自下组中的至少一个位置上包含氨基酸的取代：255，296，372。
在本发明的一些实施例中，所述突变体的氨基酸序列与SEQ ID NO:1相比具有至少91%，92%，93%，94%，95%，96%，97%，98%，或至少99%的同一性。
在一些更具体的实施例中，所述突变体的氨基酸序列与SEQ ID NO:1相比具有至少99.1%，99.2%，99.3%，99.4%，99.5%，99.6%，99.7%，99.8%，或至少99.9%的同一性。
在本发明的一些实施例中，所述突变体包含下组中至少一个氨基酸的取代：L255Q，V296T，V372T。
在本发明的一些实施例中，所述突变体包含的取代或取代的组合选自下述取代和取代的组合：L255Q+V296T ，V296T+V372T ，L255Q+ V372T，L255Q+V296T+V372T。
本发明还涉及编码上述中性植酸酶突变体的DNA分子。
本发明还涉及包含上述DNA分子的重组表达质粒。
本发明还涉及一种宿主细胞，包含上述重组表达质粒。
将上述的质粒转入宿主细胞中，重组表达的中性植酸酶突变体的比活力得到显著提升。
本发明还涉及一种宿主细胞，包含上述重组表达质粒。
将上述的质粒转入宿主细胞中，重组表达的中性植酸酶突变体的比活力得到显著提升。
在本发明的一些实施例中，宿主细胞为大肠杆菌（BL21（DE3））。
用IPTG 诱导大肠杆菌表达目的蛋白，表达产物利用Ni-NTA 琼脂糖凝胶进行亲和纯化。
本发明以野生型中性植酸酶为基础，提供了包含L255Q，V296T，V372T中至少一个突变位点的突变体。与野生型中性植酸酶相比，本发明提供的突变体的比活力普遍提高了26.14-109.33%。其中，L255Q/V296T/V372T三点突变的中性植酸酶突变体的比活力最高，达45.32 U/mg，比野生型中性植酸酶PhyC提高了109.33%，取得了意料不到的技术效果。
综上，本发明提供的中性植酸酶突变体的比活力得到显著提高，从而有利于降低中性植酸酶的生产成本。本发明涉及的中性植酸酶可以作为一种饲料添加剂，能有效提高动物对饲料中磷元素的利用率。
附表说明
表1中性植酸酶突变体比活力比较。
具体实施方式
本发明公开了一种中性植酸酶突变体、其制备方法及应用、编码该中性植酸酶突变体的DNA分子、载体、宿主细胞，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明用到了遗传工程和分子生物学领域使用的常规技术和方法，例如MOLECULAR CLONING：A LABORATORY MANUAL，3nd Ed. (Sambrook, 2001)和CURRENTPROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, 2003)中所记载的方法。这些一般性参考文献提供了本领域技术人员已知的定义和方法。但是，本领域的技术人员可以在本发明所记载的技术方案的基础上，采用本领域其它常规的方法、实验方案和试剂，而不限于本发明具体实施例的限定。例如，本发明可选用如下实验材料和试剂：
菌株与载体：大肠杆菌DH5α、大肠杆菌BL21（DE3）、载体pET28a、卡那霉素、购自Invitrogen公司。
酶与试剂盒：PCR酶购买自Takara公司，质粒提取试剂盒及胶纯化回收试剂盒购自Omega公司。
实施例1中性植酸酶及其突变体表达载体的构建
中性植酸酶基因委托生工生物工程（上海）有限公司按照SEQ ID NO：1 进行合成，并直接连接到pET28a表达载体上，命名为pET28a-PhyC。使用如下引物进行突变体的构建：
F1-L255Q：GGCAGGCATCAGACTCGTGATATTGAAGGA
R1-L255Q：ATCACGAGTCTGATGCCTGCCGTCGGCACG
F2- V296T：AACAAATATACAGCGGATTTTCGCATAACA
R2-V296T：AAAATCCGCTGTATATTTGTTCTTTCCTTG
F3-V372T：AATGAACAGACAGACCCGAGAAAACTGACC
R3-V372T：TCTCGGGTCTGTCTGTTCATTTGCCAGCGG
以pET28a-PhyC为模板，以F1-L255Q和R1-L255Q作为引物进行PCR，将PCR产物进行DpnI消化，将消化后的产物转化E.coli DH5a 细胞，提取质粒并送生工生物工程( 上海)有限公司进行测序，测序正确的质粒命名为pET28a-Phy（L255Q）；以pET28a-Phy为模板，以F2-V296T和R2-V296T作为引物进行PCR，将PCR产物进行DpnI消化，将消化后的产物转化E.coli DH5a 细胞，提取质粒并送生工生物工程( 上海) 有限公司进行测序，测序正确的质粒命名为pET28a-Phy（V296T）；以pET28a-Phy为模板，以F3-V372T和R3-V372T作为引物进行PCR，将PCR产物进行DpnI消化，将消化后的产物转化E.coli DH5a 细胞，提取质粒并送生工生物工程( 上海) 有限公司进行测序，测序正确的质粒命名为pET28a-Phy（V372T）；以pET28a-Phy（L255Q）为模板，以F2- V296T和R2-V296T作为引物进行PCR，将PCR产物进行DpnI消化，将消化后的产物转化E.coli DH5a 细胞，提取质粒并送生工生物工程( 上海)有限公司进行测序，测序正确的质粒命名为pET28a-Phy（L255Q/V296T）；以pET28a-Phy（L255Q）为模板，以F3-V372T和R3-V372T作为引物进行PCR，将PCR产物进行DpnI消化，将消化后的产物转化E.coli DH5a 细胞，提取质粒并送生工生物工程( 上海) 有限公司进行测序，测序正确的质粒命名为pET28a-Phy（L255Q/V372T）；以pET28a-Phy（V296T）为模板，以F3-V372T和R3-V372T作为引物进行PCR，将PCR产物进行DpnI消化，将消化后的产物转化E.coli DH5a 细胞，提取质粒并送生工生物工程( 上海) 有限公司进行测序，测序正确的质粒命名为pET28a-Phy（V296T/V372T）；以pET28a-Phy（L255Q/V296T）为模板，以F3-V372T和R3-V372T作为引物进行PCR，将PCR产物进行DpnI消化，将消化后的产物转化E.coli DH5a细胞，提取质粒并送生工生物工程( 上海) 有限公司进行测序，测序正确的质粒命名为pET28a-Phy（L255Q/V296T/V372T）。
将含L255Q单点突变的中性植酸酶突变体，命名为Phy-1，其氨基酸序列为SEQ IDNO：3；
将含V296T单点突变的中性植酸酶突变体，命名为Phy-2，其氨基酸序列为SEQ IDNO：4；
将含V372T单点突变的中性植酸酶突变体，命名为Phy-3，其氨基酸序列为SEQ IDNO：5；
将含L255Q/V296T两点突变的中性植酸酶突变体，命名为Phy-4，其氨基酸序列为SEQ ID NO：6；
将含L255Q/V372T两点突变的中性制酸酶中性植酸酶突变体命名为Phy-5，其氨基酸序列为SEQ ID NO：7；
将含V296T/V372T两点突变的中性植酸酶突变体命名为Phy-6，其氨基酸序列为SEQ ID NO：8；
将含L255Q/V296T/V372T三点突变的中性植酸酶突变体命名为Phy-7，其氨基酸序列为SEQ ID NO：9。
参照上述氨基酸序列，分别得到所述突变体的编码核苷酸序列。
实施例2：中性植酸酶的诱导表达
将经鉴定后的各重组质粒转化到表达宿主E.coli BL21(DE3)中，同时将空载体pET28a（+） 转入相同宿主作为对照。各取100μL 转化液分别涂布于含Kan (50 μg/mL) 的LB 平板，置于37℃培养箱中培养至长成大小合适的菌落。挑取含有重组质粒和空质粒的转化子分别接种至含50 μg/mL Kan 的LB 液体培养液，置摇床37℃，180 r/min 振荡培养过夜。按1％接种量(V/V) 转接至新鲜LB 培养液中，37℃，180 r/min 振荡培养至对数生长期(OD600≈ 0.6-0.8) 分别加入诱导剂IPTG 至终浓度1.0 mmol/L，置20℃摇床，180r/min诱导表达6h。
实施例3 ：中性植酸酶的纯化
取适量按实施例2方法进行诱导表达的重组菌培养液，8000 r/min 离心15 min收集菌体，用无菌水洗涤两次后，菌体重悬于0.5mL（pH7.5，50 mmol/L）Tris-HCl 缓冲液中，冰浴中超声破碎细胞。将超声破碎后的样品12000 r/min，4℃离心10 min 取上清即为粗酶液。由于设计的所有表达质粒的表达产物中性植酸酶上带有6 个连续的组氨酸，可以通过金属离子层析柱(Ni-NTA 琼脂糖凝胶) 亲和纯化。超声波破碎诱导后的菌体于12000r/min 离心，去除细胞碎片。上清液采用康为世纪Ni-Agarose 6x His 标签蛋白纯化试剂盒在4℃下进行纯化。将粗酶液负载上柱，流速为1 mL/min ：采用15 倍柱体积平衡液(pH7.0，20 mmol/L Tris-HCl，10mmol/L 咪唑，0.5 mol/L NaCl) 进行洗脱除去杂蛋白，流速为1 mL/min ；然后用8 倍柱体积洗脱液（pH7.0，20 mmol/L Tris-HCl，500 mmol/L 咪唑，0.5 mol/NaCl）收集目的蛋白，流速为1 mL/min ；最后将目的蛋白置透析袋中除盐，浓缩保存。
实施例5 ：中性植酸酶酶学性质测定
中性植酸酶活性测定方法参考GB/T18634-2009。取适量酶液，底物植酸钠浓度为5.0 mmol/L，反应体系中CaCl2浓度为1 mmol/L，缓冲体系为0.25 mol/L Tris-HCl（pH7.0），反应总体积为6 mL。将上述混合物置37℃反应30 min，加入4 mL显色及终止液（体积比2 ：1 ：1 的43％硝酸溶液，100 g/L 钼酸铵，2.35 g/L偏钒酸铵溶液），置分光光度计测定无机磷含量（波长415 nm）。酶活性单位（U）定义为：在一定条件下，每分钟释放出1 μmol 无机磷所需的酶量为一个酶活性单位。
表1 中性植酸酶突变体比活力比较
Figure DEST_PATH_IMAGE001
从表1的结果可以看出，与野生型中性植酸酶PhyC相比，本发明提供的中性植酸酶突变体的比活力普遍提高了26.14-109.33%。其中，L255Q/V296T/V372T三点突变的中性植酸酶突变体的比活力最高，达45.32U/mg，比野生型中性植酸酶PhyC提高了109.33%，取得了意料不到的技术效果。
本发明提供的高比活中性植酸酶突变体可广泛应用于饲料领域。
序列表
<110> 上海佶凯星生物科技有限公司
<120> 一种中性植酸酶突变体
<141> 2022-06-22
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Claims (10)

1.一种中性植酸酶突变体，其特征在于，所述突变体包含与SEQ ID NO:1具有至少90%同一性的氨基酸序列，且与SEQ ID NO:1相比在选自下组中的至少一个位置上包含氨基酸的取代：255，296，372。
2.如权利要求1所述的突变体，其特征在于，所述突变体的氨基酸序列与SEQ ID NO:1相比具有至少91%，92%，93%，94%，95%，96%，97%，98%，或至少99%的同一性。
3.如权利要求1所述的突变体，其特征在于，所述突变体的氨基酸序列与SEQ ID NO:1相比具有至少99.1%，99.2%，99.3%，99.4%，99.5%，99.6%，99.7%，99.8%，或至少99.9%的同一性。
4.如权利要求1-3任一所述的突变体，其特征在于，所述突变体包含下组中至少一个氨基酸的取代：L255Q，V296T，V372T。
5.如权利要求4所述的突变体，其特征在于，所述突变体包含的取代或取代的组合选自下述取代和取代的组合：L255Q，V296T，V372T，L255Q/V296T，L255Q/V372T, V296T/V372T,L255Q/V296T/V372T。
6.编码权利要求1-5任一所述的突变体的DNA分子。
7.包含权利要求6所述DNA分子的重组表达质粒。
8.一种宿主细胞，其特征在于，所述的宿主细胞包含权利要求7所述的重组表达质粒。
9.如权利要求8所述的宿主细胞，其特征在于，所述的宿主细胞为大肠杆菌BL21（DE3）。
10.权利要求1-5任一所述中性植酸酶突变体在饲料领域中的应用。
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