CN114736886A - 植酸酶突变体及其制备方法 - Google Patents
植酸酶突变体及其制备方法 Download PDFInfo
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- CN114736886A CN114736886A CN202210433393.XA CN202210433393A CN114736886A CN 114736886 A CN114736886 A CN 114736886A CN 202210433393 A CN202210433393 A CN 202210433393A CN 114736886 A CN114736886 A CN 114736886A
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- phytase
- mutant
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- phyan
- thr
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- 229940085127 phytase Drugs 0.000 title claims abstract description 128
- 238000002360 preparation method Methods 0.000 title abstract description 6
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Abstract
本发明为一种植酸酶突变体及其制备方法,植酸酶突变体的氨基酸序列如SEQ ID No.2所示,所述植酸酶突变体是在野生植酸酶PhyAn的氨基酸序列如SEQ ID NO.1所示基础上进行的突变,所述的突变体的突变位点及序列为A63G+Y65H,即63位的丙氨酸和65位的酪氨酸同时被甘氨酸和组氨酸所取代。本发明与野生植酸酶PhyAn相比,突变体的催化效率得到提升,意味着63位点处的甘氨酸残基的替代减小了植酸酶的空间位阻,有利于酶与底物的结合进行有利于提升其催化效率;且突变体的pH耐受性同时提升,意味着65位点处的组氨酸改变了催化区域中带电氨基酸的数量进行有利于增强其pH耐受性。
Description
技术领域
本发明涉及基因工程和酶工程领域,具体为一种植酸酶突变体及其制备方法。
背景技术
植酸(肌醇六磷酸)广泛存在于自然界的植物中,带有六个磷酸基团,可作为磷酸的储存库。植酸与钙、镁、铁、锌等矿物质元素和蛋白质等营养物质结合易形成蛋白-植酸-矿物元素复合物,大大降低了营养物质的吸收利用及某些植物性食物和一些植物蛋白分离物中矿物质的营养效价。且猪、鱼等动物体内缺乏植酸酶,无法很好的水解植酸,且磷又作为其生长必须的矿物质元素,这使得动物对植酸中磷元素的生物利用度非常低且其生长需要摄入大量无机磷。因此,在饲料中添加植酸酶目前被认为是解决上述问题的重要方法。
植酸酶是一种胞外酶,可以水解植酸上的磷酸基团。其在自然界中广泛存在,在动物、植物和微生物中均有发现。其中在植物组织如谷物、豆类、蔬菜,特别是萌发的种子和花粉中都发现了植酸酶;且细菌、真菌、霉菌等很多微生物均能产生具有优良特性的植酸酶。不同来源的植酸酶的性能也有所差异,由于某些微生物自身能够耐受相对极端的自然环境,因此其产生的胞外植酸酶对酸性和高温等环境也凸显了一定优势。来源于曲霉Aspergillus neoniger的植酸酶PhyAn具有热稳定性和pH稳定性较好的固有优势,其在70℃的温度下的酶活残留率可达90%以上,且其在pH为3的条件下相对酶活达到100%,这对于想要应用为工业饲料的植酸酶来说,来源于该曲霉Aspergillus neoniger的野生型植酸酶PhyAn的原有优良特性使其更具优势。在饲料中添加植酸酶可以有效水解植酸,破坏植酸与矿物质元素的亲和力进而有效增加营养物质的吸收利用及矿物质元素的营养效价,还能补充动物体内的磷元素、减少无机磷的释放以及缓解环境污染。植酸酶已经被公认为第三大饲用酶,可以通过微生物合成进而作为饲料添加剂应用于饲料行业。
申请号CN201610421861.6, 申请(专利权)人昆明爱科特生物科技有限公司公开了如下技术,突变体由编码无花果曲霉植酸酶氨基酸序列发生改变而获得;本发明使用烟曲霉植酸酶的部分氨基酸序列来替代无花果曲霉植酸酶氨基酸序列,使其成为耐热性高的植酸酶;以无花果曲霉植酸酶基因序列作为模版,通过基因点突变技术使无花果植酸酶基因发生突变,获得了新的植酸酶基因的突变体SEQ ID NO.2,该植酸酶基因的突变体与质粒pPIC9K等相连构建重复质粒,转化宿主GS115等获得基因工程菌株,通过基因工程菌株发酵,获得新的植酸酶突变体;能有较宽的pH作用范围和较理想耐热特性,适合耐高温制粒,较好地满足饲料工业的要求。
上述专利描述的发明内容是对植酸酶进行了单点突变使得突变体的耐热、酶活等性能改善,并没有采用了一种对其催化氨基酸部分的氨基酸进行改造的策略,设计并构建突变体。
在饲料行业中,饲料添加剂的制备过程需要饲用酶自身具有良好的耐热及耐酸的特性,且酶的催化效率也是决定酶能否较好发挥活性的重要依据。因此,亟待进一步通过理性设计和利用工程菌株异源表达而改善植酸酶的酶学特性,进而提升作为饲料添加剂的植酸酶的应用价值具有重要作用。
发明内容
本发明目的在于提供一种植酸酶突变体及其制备方法,对比野生型植酸酶,该突变体可以保持原有温度特性,且其催化效率和pH耐受性均得到提升,在饲料领域具有更好的应用潜力和价值。
为达成上述目的,本发明提出如下技术方案:一种植酸酶突变体,所述植酸酶突变体的氨基酸序列如SEQ ID NO.2所示。
本发明进一步揭示,所述植酸酶突变体是在野生植酸酶PhyAn的氨基酸序列如SEQID NO.1所示基础上进行的突变,所述的突变体的突变位点及序列为A63G+Y65H,即63位的丙氨酸和65位的酪氨酸同时被甘氨酸和组氨酸所取代。
本发明进一步揭示一种核苷酸,编码上述的植酸酶突变体。
核苷酸序列为SEQ ID NO.3。
本发明进一步揭示一种重组载体,其包含上述的核苷酸。
本发明进一步揭示一种重组细胞,其包含上述的重组载体,所述的重组细胞,选择为毕赤酵母GS115。
本发明进一步揭示制备上述的植酸酶突变体的方法,以野生植酸酶PhyAn催化区域的氨基酸序列RHGX1RX2P为突变体对象,设计其突变体A63G+Y65H;
通过PCR反应对植酸酶PhyAn的DNA片段进行突变,插入线性化载体pPIC9K中重组连接,构建突变体的重组表达载体;
将测序验证的重组表达载体转入表达宿主毕赤酵母GS115获得阳性重组菌株,重组菌株分泌表达,发酵制备植酸酶突变体A63G+Y65H。
本发明还给出上述的植酸酶突变体在水解植酸领域的应用。
本发明还给出上述植酸酶突变体在饲料或饲料添加剂中的应用。
有益效果,本申请的技术方案具备如下技术效果:
1、本发明通过改变野生植酸酶PhyAn催化区域的空间位阻和可变氨基酸电荷构建突变体A63G+Y65H,与野生植酸酶PhyAn相比,突变体的催化效率得到提升,意味着63位点处的甘氨酸残基的替代减小了植酸酶的空间位阻,有利于酶与底物的结合进行有利于提升其催化效率;且突变体的pH耐受性同时提升,意味着65位点处的组氨酸改变了催化区域中带电氨基酸的数量进行有利于增强其pH耐受性。
应当理解,前述构思以及在下面更加详细地描述的额外构思的所有组合只要在这样的构思不相互矛盾的情况下都可以被视为本公开的发明主题的一部分。
结合附图从下面的描述中可以更加全面地理解本发明教导的前述和其他方面、实施例和特征。本发明的其他附加方面例如示例性实施方式的特征和/或有益效果将在下面的描述中显见,或通过根据本发明教导的具体实施方式的实践中得知。
附图说明
附图不意在按比例绘制。在附图中,在各个图中示出的每个相同或近似相同的组成部分可以用相同的标号表示。为了清晰起见,在每个图中,并非每个组成部分均被标记。现在,将通过例子并参考附图来描述本发明的各个方面的实施例,其中:
图1为植酸酶PhyAn和本发明揭示的植酸酶突变体的最适温度图。
图2为植酸酶PhyAn和本发明揭示的植酸酶突变体的温度耐受性图。
图3为植酸酶PhyAn和本发明揭示的植酸酶突变体的最适pH图。
图4为植酸酶PhyAn和本发明揭示的植酸酶突变体的pH耐受性图。
具体实施方式
为了更了解本发明的技术内容,特举具体实施例并配合所附图式说明如下。在本公开中参照附图来描述本发明的各方面,附图中示出了许多说明的实施例。本公开的实施例不必定义在包括本发明的所有方面。应当理解,上面介绍的多种构思和实施例,以及下面更加详细地描述的那些构思和实施方式可以以很多方式中任意一种来实施,这是因为本发明所公开的构思和实施例并不限于任何实施方式。另外,本发明公开的一些方面可以单独使用,或者与本发明公开的其他方面的任何适当组合来使用。
实施例一:
野生植酸酶PhyAn来源于Aspergillus neoniger,属于HAP家族,该家族的植酸酶催化区域的保守性氨基酸序列为RHGX1RX2P,其中X1和X2为可变氨基酸残基,即拥有相同保守催化位点的 HAP 家族的植酸酶在这两个氨基酸上会产生差异,植酸酶PhyAn的催化区域序列为RHGARYP。
本发明以提升野生植酸酶PhyAn的催化效率和pH稳定性为目的,对其氨基酸序列进行理性设计,构建突变体。
催化效率与酶与底物结合的亲和力有关,进而与催化区域中氨基酸侧链的空间位阻密切相关;其次,酶的pH耐受性与氨基酸的电荷情况密不可分,酶发挥活性关键部位的带电氨基酸的改变很可能会影响酶对酸性环境的耐受性。因此,为减少野生植酸酶PhyAn催化区域的空间位阻及改变氨基酸带电性,本发明将催化区域RHGX1RX2P中(分别为A和Y)X1和X2两个可变氨基酸残基同时进行突变,分别为A突变为G,Y突变为H,命名为A63G+Y65H。野生植酸酶PhyAn的氨基酸序列如SEQ ID NO.1所示,突变体A63G+Y65H的氨基酸序列如SEQ IDNO.2所示。
进一步的说,将野生植酸酶PhyAn的氨基酸序列翻译为核苷酸序列,如SEQ IDNO.3,根据表达宿主毕赤酵母GS115的密码子偏好性进行优化,并根据基因序列设计突变体的PCR扩增引物,引物序列如下表1所示。
以野生植酸酶PhyAn的DNA片段为模板,使用PCR高保真酶进行植酸酶突变体基因片段的扩增,经琼脂糖凝胶电泳检验和片段的纯化处理后与线性化的毕赤酵母GS115表达载体pPIC9K进行重组连接,转化至大肠杆菌DH5α感受态细胞中筛选阳性克隆转化子。将转化子的质粒提取并进行测序验证,完成植酸酶PhyAn的基因突变及表达载体的构建。
表1 植酸酶突变体A63G+Y65H的扩增引物序列
引物名称 | 序列 |
(A63G+Y65H)-f | 5’CTTACCCTTAGAGTCACCTGGGTGTCTAGCACCGTGTCTGGACAAAAC-3’ |
(A63G+Y65H)-r | 5’GTCCAGACACGGTGCTAGACACCCAGGTGACTCTAAGGGTAAGAAG-3’ |
实施例二:植酸酶PhyAn及其突变体A63G+Y65H在毕赤酵母GS115中的表达。
(1)植酸酶PhyAn及其突变体A63G+Y65H转化毕赤酵母GS115;
将植酸酶PhyAn及其突变体A63G+Y65H的表达载体使用Plasmid DNA Kit试剂盒进行质粒提取,并使用Sac1限制性内切酶对其进行线性化酶切处理,纯化处理后的产物将进行转化。将10 μL需要转化的酶切产物转移至100 μL毕赤酵母GS115感受态细胞中,并将上述混合样品转移至2 mm电转杯中于冰上孵育15 min,在工作电压为2 kV、脉冲时间约为0.5ms的条件下进行电击,结束后立即加入预冷的1 mL 1 M 山梨醇溶液,吸打混匀后转移至新的1.5 mL离心管中,置于30 °C培养箱中孵育1 h,离心后涂布于YPD培养基中,于30 °C培养3-5天。
(2)植酸酶PhyAn及其突变体A63G+Y65H的发酵表达;
将YPD转化板上的转化子全部接于48孔板中发酵,条件为30 °C、180 rpm,每隔24h用0.5%甲醇诱导一次,共诱导三次。培养完成后对上清液简单测定其酶活,在野生植酸酶PhyAn和突变体A63G+Y65H各挑选1株酶活较高的转化子进行摇瓶发酵表达。
将植酸酶PhyAn和突变体A63G+Y65H转化子接种至20 mL BMGY培养基中发酵表达,发酵条件为30 °C、180 rpm,从培养的第24 h开始,每间隔24 h添加0.5%的甲醇进行诱导表达,诱导三次后离心并收集上清液。
(3)植酸酶PhyAn及其突变体A63G+Y65H的分离纯化;
发酵液离心收集的上清液为粗酶液,使用截留分子量50 kDa的超滤离心管对粗酶液进行浓缩,并用超纯水洗涤1-2次进行脱盐处理。处理后的溶液使用阴离子交换柱QFFcolumn(5 mL,GE Healthcare)进行植酸酶的柱纯化,纯化仪器为AKTA蛋白纯化系统,纯化回收的样品作为野生植酸酶PhyAn及其突变体A63G+Y65H的酶液,用来进行进一步酶学性质的探究。
实施例三
重组植酸酶PhyAn及其突变体A63G+Y65H酶学性质分析
植酸酶的活性检测根据中华人民共和国国家标准《GB/T 18634-2009》给出的钼钒法进行。具体测定方法为:将植酸酶酶液使用0.25 mol/L pH 5.5的乙酸缓冲液稀释至适宜倍数,取0.2 mL稀释酶液于25 mL试管中,再加入1.8 mL乙酸缓冲液,涡旋混匀后将试管置于37 ℃水浴锅中预热5 min;之后向试管中加入4 mL 7.5 mmol/L植酸钠溶液,混匀后将混合样品置于37 ℃水浴锅反应30 min,再向试管中加入4 mL终止液,涡旋混匀并室温静置10min,测定其在415 nm波长处的吸光值。
植酸酶的酶活定义为,在37 °C、pH 5.5的条件下,植酸酶每分钟从浓度为5 mmol/L植酸钠溶液中释放出1 μmol无机磷,即为一个植酸酶活性单位,单位为U。
植酸酶的酶活力计算公式为:每毫升样品的植酸酶活力X(U/mL)=Y(根据实际样品吸光值由标准曲线计算出的无机磷的量,单位μmol)/(反应时间*样品体积)*稀释倍数
(1)植酸酶PhyAn及其突变体A63G+Y65H的最适温度及温度稳定性;
测定植酸酶样品在pH 5.5和不同温度(30、35、40、45、50、55、60、65、70、75、80 °C)条件下的活性来确定其最适温度。以最高酶活力为100%,计算植酸酶样品在各温度下的相对酶活。热稳定性定义为植酸酶样品经不同温度(70、75、80、85、90、95 °C)孵育处理10分钟后的残留酶活。植酸酶样品在最适温度孵育的酶的残留酶活为100%,以此计算经其他温度处理后的残留酶活。
植酸酶PhyAn及其突变体A63G+Y65H的最适温度如图1所示,二者的最适温度均为45 °C,在此温度下具有最大的相对酶活力。温度稳定性如图2所示,野生植酸酶PhyAn及其突变体A63G+Y65H的稳定稳定性相差不大,经过85 °C和90 °C处理10 min后突变体A63G+Y65H的残留酶活略高于野生植酸酶PhyAn的残留酶活。上述最适温度和温度稳定性的结果表明本发明提供的植酸酶突变体A63G+Y65H不会改变其温度特性,经过较高温度的处理仍能保持较好的酶活水平。
(2)植酸酶PhyAn及其突变体A63G+Y65H的最适pH及pH稳定性;
将植酸酶样品使用不同pH值的缓冲液进行稀释,并将底物植酸钠使用不同pH值的缓冲液溶解,使酶与底物在不同pH值的环境中进行相对酶活的测定,得出植酸酶PhyAn及其突变体A63G+Y65H的最适pH。各pH值的缓冲液分别为:pH 2.0-3.5 (0.05 M Gly-HCl)、pH4.0-6.0 (0.05 M NaAc-HAc)、pH 6.5-8.0 (0.05 M Tris-HCl)。将植酸酶样品在上述相同的各pH值缓冲液中于37 °C孵育2 h并立即预冷后作为经过处理的待测酶液,并与底物植酸钠在标准酶活反应条件(pH 5.5,37 °C,30 min)下反应并计算得出不同处理酶液样品的残留酶活。
最适pH如图3所示,野生植酸酶PhyAn的最适pH值为5.5,突变体A63G+Y65H在pH3.0的条件下相对酶活达到100%,在pH 5.5条件下的相对酶活也可达90%,在图3所示的pH曲线中呈现两个峰度,这说明本发明提供的突变体A63G+Y65H的最适pH更广,在酸性条件下的相对酶活升高,比野生植酸酶PhyAn更能在酸性条件下发挥更好的活性。如图4所示,突变体A63G+Y65H的pH稳定性较野生植酸酶PhyAn而言得到提升,经过不同pH值的缓冲液处理后的残留酶活均更好,这意味着本发明提供的突变体A63G+Y65H具有更优越的pH特性。
(3)植酸酶PhyAn及其突变体A63G+Y65H的动力学参数;
使用浓度梯度为0.125-5.0 mM的植酸钠底物测定植酸酶PhyAn及其突变体A63G+Y65H的动力学参数。将植酸酶样品与不同浓度的底物在pH 5.5、37 °C的条件下反应7 min,根据植酸酶酶活方法计算得出酶活,使用Origin 8.0 软件计算Michaelis-Menten 参数、Km、kcat值。
植酸酶PhyAn及其突变体A63G+Y65H的动力学参数如表2所示,突变体A63G+Y65H的Km值降低,意味着酶与底物的亲和力增强。二者的Vmax相差不大,保持在相同的水平。kcat/Km值显示突变体的催化效率较野生植酸酶PhyAn得到大幅度的提升。因此,本发明提供的突变体A63G+Y65H可以使其催化效率提升。
表2 为植酸酶PhyAn及其突变体A63G+Y65H的动力学参数
综上所述,本发明在野生植酸酶PhyAn的基础上,提供了一个突变体A63G+Y65H,其pH耐受性和催化效率均得到提升。本发明所采用的理性设计思路为改变没催化区域可变氨基酸的空间位阻及带电性来改善酶的酶学性质,这为后续工业酶改造与开发提供了一种有价值的思路,同时也为更进一步推动植酸酶的工业化利用奠定基础。
虽然本发明已以较佳实施例揭露如上,然其并非用以限定本发明。本发明所属技术领域中具有通常知识者,在不脱离本发明的精神和范围内,当可作各种的更动与润饰。因此,本发明的保护范围当视权利要求书所界定者为准。
SEQUENCE LISTING
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taccagcgat acgaatcgct cacaagaaac atcattcctt tcatccgatc ctctggctcc 600
agccgcgtga tcgcctccgg caagaaattc atcgagggct tccagagcac caagctgaag 660
gatcctcgtg cccagcccgg ccaatcgtcg cccaagatcg acgtggtcat ttccgaggcc 720
agctcatcca acaacactct cgacccaggc acctgcactg tctttgaaga cagcgaattg 780
gccgatgccg tcgaagccaa tttcaccgcc acgttcgtcc cctccattcg tcaacgtctg 840
gagaacgacc tgtctggcgt gactctcaca gacacagagg tgacctacct catggacatg 900
tgctccttcg acaccatctc caccagcacc gtcgacacca agctgtcccc cttctgtgac 960
ctgttcactc atgacgaatg gatcaactac gactacctcc agtccctgaa aaagtactac 1020
ggccatggcg cgggtaaccc gctcggcccg acccagggcg tcggctacgc taacgagctc 1080
atcgcccgtc tcacccactc gcctgtccac gatgacacca gctccaacca cactttggac 1140
tcgaacccgg ctacttttcc gctcaactct actctctatg cggacttttc ccatgataac 1200
ggcatcatct ctattctctt tgctttgggt ctgtacaacg gcactaagcc gctgtctacc 1260
acgaccgtgg agaatatcac ccagacagat ggattttcgt ctgcttggac ggttccgttt 1320
gcttcgcgtc tgtacgttga gatgatgcag tgtcaggccg agcaggagcc gctggtccgt 1380
gttttggtta atgatcgcgt tgtcccgctg catggttgtc cggttgatgc tttggggaga 1440
tgtacccggg atagctttgt gagggggttg agctttgcta gatctggggg tgattgggcg 1500
gagtgctttg cttagctgaa ctaccttgat ggatggtatg tatcaatcgg agtacatatc 1560
attacttcat gtatgtgttt acgaagatgt acatattgaa actatcgatg ataactacct 1620
cg 1622
Claims (9)
1.一种植酸酶突变体,其特征在于,所述植酸酶突变体的氨基酸序列如SEQ ID No.2所示。
2.根据权利要求1所述的一种植酸酶突变体,其特征在于:所述植酸酶突变体是在野生植酸酶PhyAn的氨基酸序列如SEQ ID NO.1所示基础上进行的突变,所述的突变体的突变位点及序列为A63G+Y65H,即63位的丙氨酸和65位的酪氨酸同时被甘氨酸和组氨酸所取代。
3.一种核苷酸,其特征在于,编码权利要求1或2所述的植酸酶突变体。
4.根据权利要求3所述的核苷酸,其特征在于,核苷酸序列为SEQ ID NO. 3。
5.重组载体,其特征在于,包含权利要求3或4所述的核苷酸。
6.重组细胞,其特征在于,包含权利要求5所述的重组载体,所述的重组细胞,选择为毕赤酵母GS115。
7.制备权利要求1或2所述的植酸酶突变体的方法,其特征在于:以野生植酸酶PhyAn催化区域的氨基酸序列RHGX1RX2P为突变体对象,设计其突变体A63G+Y65H;
通过PCR反应对植酸酶PhyAn的DNA片段进行突变,插入线性化载体pPIC9K中重组连接,构建突变体的重组表达载体;
将测序验证的重组表达载体转入表达宿主毕赤酵母GS115获得阳性重组菌株,重组菌株分泌表达,发酵制备植酸酶突变体A63G+Y65H。
8.如权利要求1或2所述的植酸酶突变体在水解植酸领域的应用。
9.如权利要求1或2所述植酸酶突变体在饲料或饲料添加剂中的应用。
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