CN116121222A - 一种中性植酸酶突变体及其应用 - Google Patents
一种中性植酸酶突变体及其应用 Download PDFInfo
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- CN116121222A CN116121222A CN202211010188.9A CN202211010188A CN116121222A CN 116121222 A CN116121222 A CN 116121222A CN 202211010188 A CN202211010188 A CN 202211010188A CN 116121222 A CN116121222 A CN 116121222A
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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及其应用。本发明以野生型中性植酸酶AN为基础,获得包含G35E,E169R,T248R单点、两点和三点突变的中性植酸酶突变体,在80℃条件下处理5min后,酶活残留率得到显著提高,从而说明,本发明提供的突变位点G35E,E169R,T248R能显著提高中性植酸酶的耐热性,其中,AN‑7(G35E/E169R/T248R)三点突变的中性植酸酶突变体在80℃条件下处理5min后的酶活残留率最高,为53.22%。本发明提供的中性植酸酶突变体的耐热性得到显著提高,从而有利于其在饲料中的广泛应用。
Description
技术领域
本发明属于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及其应用。
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及其应用,该中性植酸酶突变体的耐热性与野生型中性植酸酶相比得到显著提高,有利于其在饲料中的广泛应用。
本发明一方面提供了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:35,169,248。
在本发明的一些实施例中,所述的中性植酸酶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,所述的中性植酸酶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,所述的中性植酸酶突变体包含下组中至少一个氨基酸的取代:G35E,E169R,T248R。
在本发明的一些实施例中,所述的中性植酸酶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:G35E+E169R,G35E+T248R,E169R+T248R, G35E+E169R+T248R。
本发明另一方面提供了编码上述的中性植酸酶突变体的DNA分子。
本发明再一方面提供了包含上述的DNA分子的重组表达质粒。
本发明再一方面提供了一种宿主细胞,包含上述的重组表达质粒。
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。
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,80℃条件下处理5min后,酶活残留率得到显著提高。
本发明以野生型中性植酸酶为基础,提供了包含G35E,E169R,T248R中至少一个突变位点的中性植酸酶突变体。与野生型中性植酸酶相比,本发明提供的中性植酸酶突变体在80℃条件下处理5min后的酶活残留率普遍提高了。其中,G35E/E169R/T248R三点突变的中性植酸酶突变体的酶活残留率最高,为53.22%,取得了意料不到的技术效果。
综上,本发明提供的植酸酶突变体的耐热性得到显著提高,从而有利于植酸酶在饲料中的广泛应用。本发明涉及的中性植酸酶突变体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。
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MOLECΜLAR CLONING:A LABORATORY MANUAL,3nd Ed. (Sambrook, 2001)和CURRENT PROTOCOLSIN MOLECΜLAR 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、毕赤酵母GS115、载体pPIC9k、氨苄青霉素、G418购自Invitrogen公司。
酶与试剂盒:PCR酶及连接酶购买自Takara公司,限制性内切酶购自Fermentas公司,质粒提取试剂盒及胶纯化回收试剂盒购自Omega公司,GeneMorph II随机诱变试剂盒购自北京博迈斯生物科技有限公司。
培养基配方:
大肠杆菌培养基(LB培养基):0.5%酵母提取物,1%蛋白胨,1%NaCl,pH7.0;
酵母培养基(YPD培养基):1%酵母提取物,2%蛋白胨,2%葡萄糖;
酵母筛选培养基(MD培养基):2%蛋白胨,2%琼脂糖;
BMGY培养基:2%蛋白胨,1%酵母提取物,100 mM磷酸钾缓冲液(pH6.0),1.34% YNB,4×10-5 %生物素,1%甘油;
BMMY培养基:2%蛋白胨,1%酵母提取物,100 mM磷酸钾缓冲液(pH6.0),1.34% YNB,4×10-5 %生物素,0.5%甲醇;
LB-AMP培养基:0.5%酵母提取物,1%蛋白胨,1%NaCl,100μg/mL氨苄青霉素,pH7.0;
LB-AMP平板:0.5%酵母提取物,1%蛋白胨,1%NaCl,1.5%琼脂,100μg/mL氨苄青霉素,pH7.0;
下面结合实施例,进一步阐述本发明:
实施例1 重组质粒的构建
将来源于烟曲霉(
Aspergillus fumigatus)的植酸酶基因(GeneBankKAF4251155)根据毕赤酵母密码子偏好性进行优化,并在其起始密码子ATG前增加6个碱基GAATTC(EcoR I酶切位点),在其终止密码子TAA后增加GCGGCCGC(Not I酶切位点)。优化后的核苷酸序列由生工生物工程(上海)股份有限公司合成。将该植酸酶命名AN,其氨基酸序列为SEQ ID NO:1,编码核苷酸序列为SEQ ID NO:2。
用限制性内切酶EcoR I和Not I(Fermentas)对植酸酶AN基因进行酶切;同时,用限制性内切酶EcoR I和Not I对质粒pPIC9K进行酶切。使用凝胶纯化试剂盒将酶切产物纯化,并用T4 DNA连接酶(Fermentas)将上述两个酶切产物连接。将连接产物转化进DH5α大肠杆菌(Invitrogen),用氨苄青霉素进行选择。为确保准确,对若干克隆进行测序。
使用质粒小量制备试剂盒(Omega)从测序结果正确的大肠杆菌克隆中纯化质粒,获得1个重组质粒,将其命名为pPIC9K-AN。
实施例2 耐温中性植酸酶突变体的筛选
为了进一步提高植酸酶AN的耐热性,申请人通过定向进化技术对该酶进行了大量突变的筛选。
1.1设计PCR引物AN-F1、AN-R1:
AN-F1:GGCGAATTCGCTCCATCTTCTGCTGGTTCTAAGTC(下划线为限制性内切酶EcoRI识别位点);
AN-R1:ATAGCGGCCGCTTAGGAGAAACATTCACCCCAG(下划线为限制性内切酶NotI识别位点)。
以AN基因(SEQ ID NO:1)为模板,利用上述引物用GeneMorph II随机突变PCR试剂盒(博迈斯)进行PCR扩增,胶回收PCR产物,EcoRI、NotI进行酶切处理后与经同样酶切后的pET21a载体连接,转化至大肠杆菌BL21(DE3)中,涂布于LB+Amp平板,37℃倒置培养,待转化子出现后,用牙签逐个挑至96孔板,每个孔中加入150 μL含有0.1 mM IPTG的LB+Amp培养基,37℃、220 rpm培养6 h左右,离心弃上清,菌体用缓冲液重悬,反复冻融破壁,获得含有植酸酶的大肠杆菌细胞裂解液。
分别取出40 ul裂解液至两块新的96孔板,将其中一块96孔板于75℃处理5 min;然后向两块96孔板中各加入80 ul底物,于37℃反应30 min后加入80 ul终止液(钒酸铵:钼酸铵:硝酸=1:1:2),测定生成的无机磷含量。不同的突变子高温处理后保持的活性不同。
实验结果表明,有些突变对植酸酶APPA-N0的耐热性没有影响,有些突变甚至使其耐热性或酶活变得更差了,另外还有些突变虽然能提高APPA-N0对温度的耐受性,但突变后其酶学性质发生了显著的变化,这些均不符合要求。最终,申请人得到既能显著提高AN耐热性,又不会影响其酶活及原有酶学性质的突变位点:G35E,E169R,T248R。
实施例3 植酸酶在毕赤酵母中的表达
3.1表达载体的构建
依照毕赤酵母的密码偏爱性分别对植酸酶AN及其突变体的基因序列进行优化,由上海捷瑞生物工程有限公司合成,并且在合成序列5’和3’两端分别加上EcoRI和NotI两个酶切位点。
按照实施例1中所述方法,将合成的植酸酶AN及其突变体的基因序列分别进行EcoRI和NotI双酶切,然后与经同样酶切后的pPIC-9K载体16℃过夜连接,并转化大肠杆菌DH5a,涂布于LB+Amp平板,37℃倒置培养,待转化子出现后,菌落PCR(反应体系:模板挑取的单克隆,rTaqDNA聚合酶 0.5 μL,10×Buffer 2.0 μL,dNTPs(2.5 mM) 2.0 μL,5’AOX引物(10 mM):0.5 μL,3’AOX引物:0.5 μL,ddH2O 14.5 μL,反应程序:95℃预变性5 min,30个循环: 94℃ 30 sec,55℃ 30 sec,72℃ 2 min,72℃ 10 min)。验证阳性克隆子,经测序验证后获得了正确的重组表达质粒。
3.2毕赤酵母工程菌株的构建
3.2.1酵母感受态制备
将毕赤酵母GS115菌株进行YPD平板活化,30℃培养48 h后接种活化的GS115单克隆于6 mL YPD液体培养基中,30℃、220 rpm,培养约12 h后转接菌液于装有30 mL YPD液体培养基的三角瓶中,30℃、220 rpm培养约5h,经紫外分光光度计检测其菌体密度,待其OD600值在1.1–1.3范围后,4℃ 9000 rpm离心2 min分别收集4 mL菌体至灭菌EP管中,轻轻弃上清,用灭菌的滤纸吸干残留的上清后用预冷的1 mL灭菌水重悬菌体,4℃、9000 rpm离心2 min,轻轻弃上清,重复用1 mL灭菌水洗一遍后,4℃、9000 rpm离心2 min,轻轻弃上清,预冷的1 mL山梨醇(1 mol/L)重悬菌体;4℃、9000 rpm离心2 min,轻轻弃上清,预冷的100-150 μL山梨醇(1 mol/L)轻柔重悬菌体。
3.2.2转化和筛选
分别将3.1构建得到的重组表达质粒用Sac I进行线性化,线性化片段纯化回收后通过电穿孔法分别转化毕赤酵母GS115,在MD平板上筛选得到毕赤酵母重组菌株,然后在含不同浓度G418的YPD平板(0.5 mg/mL-8 mg/mL)上筛选多拷贝的转化子。
将获得的转化子分别转接于BMGY培养基中,30℃、250rpm振荡培养1天;再转入BMMY培养基中,30℃、250 rpm振荡培养;每天添加0.5%的甲醇,诱导表达4 天;9000 rpm离心10 min去除菌体,即得到分别含植酸酶AN和植酸酶突变体的发酵上清液。
(1)植酸酶酶活单位的定义
在温度为37℃、pH为5.0的条件下,每分钟从浓度为5.0 mmol/L植酸钠中释放1 μmol无机磷,即为一个植酸酶活性单位,以U表示。
(2)植酸酶酶活测定方法
取甲、乙两支25 mL比色管,各加入1.8 mL乙酸缓冲液(pH 5.0)、0.2 mL样品反应液,混匀,37℃预热5 min。在甲管中加入4mL底物溶液,乙管中加入4 mL终止液,混匀,37℃反应30 min,反应结束后甲管中加入4 mL终止液,乙管中加入4 mL底物溶液,混匀。静置10min,分别在415 nm波长处测定吸光值。每种样品作3个平行,取吸光值的平均值,通过标准曲线用回归直线方程计算植酸酶活性。
酶活X=F×C/(m×30)
其中:X——酶活力单位,U/g(mL);
F——试样溶液反应前的总稀释倍数;
C——根据实际样液的吸光值由直线回归方程计算出的酶活性,U;
m——试样质量或体积,g/mL;
30——反应时间。
采用上述方法分别对构建得到的毕赤酵母重组菌株发酵上清液进行植酸酶酶活测定。
(3)测定结果
按照上述方法进行酶活检测,结果显示:上述构建得到的重组表达植酸酶AN及其突变体的毕赤酵母重组菌株发酵上清液的酶活为208-385 U/mL。
实施例4 热稳定性分析
用预热10 min、pH 5.0的0.25 M乙酸钠缓冲液将上述获得的表达植酸酶突变体的重组菌株发酵上清液各稀释10倍;然后将稀释后的样品分别进行如下处理:80 ℃处理3min,85℃处理3 min,90℃处理3 min,结束时取样并冷却至室温;分别测定热处理后样品的植酸酶酶活,以未处理样品的酶活计100%,计算酶活残留率。
酶活残留率(%)=未处理样品的酶活/热处理后样品的酶活×100%。
表1 为中性植酸酶突变体在80℃条件下的耐热性分析。
结果显示,与野生型中性植酸酶AN相比,本发明提供的包含G35E,E169R,T248R单点、两点和三点突变的中性植酸酶突变体在80℃条件下处理5min后,酶活残留率均得到显著提高,从而说明,本发明提供的突变位点G35E,E169R,T248R能显著提高中性植酸酶的耐热性。
综上所述,本发明提供的中性植酸酶突变体的耐热性得到显著提高,从而有利于其在饲料中的广泛应用。
Claims (10)
1.一种中性植酸酶突变体,其特征在于,所述的中性植酸酶突变体包含与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:35,169,248。
2.如权利要求1所述的中性植酸酶突变体,其特征在于,所述的中性植酸酶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所述的中性植酸酶突变体,其特征在于,所述的中性植酸酶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任一项所述的中性植酸酶突变体,其特征在于,所述的中性植酸酶突变体包含下组中至少一个氨基酸的取代:G35E,E169R,T248R。
5.如权利要求4所述的中性植酸酶突变体,其特征在于,所述的中性植酸酶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:G35E+E169R,G35E+T248R,E169R+T248R,G35E+E169R+T248R。
6.编码权利要求1-5任一项所述的中性植酸酶突变体的DNA分子。
7.包含权利要求6所述的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,所述的宿主细胞为毕赤酵母。
10.权利要求1-5任一项所述的中性植酸酶突变体在饲料中的应用。
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