CN113699134A - 植酸酶突变体 - Google Patents

植酸酶突变体 Download PDF

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CN113699134A
CN113699134A CN202011596375.0A CN202011596375A CN113699134A CN 113699134 A CN113699134 A CN 113699134A CN 202011596375 A CN202011596375 A CN 202011596375A CN 113699134 A CN113699134 A CN 113699134A
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吴秀秀
李馨培
李�瑞
宋清清
黄亦钧
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Weifang Kdn Biotech Co ltd
Qingdao Vland Biotech Group Co Ltd
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Abstract

本发明涉及生物技术领域，特别涉及一种植酸酶突变体、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。本发明提供的突变体在选自下组中的至少一个位置上包含氨基酸的取代：36、126、211、253、258、266。所述突变体的耐热性均得到显著提高，从而有利于植酸酶在饲料中的广泛应用。

Description

植酸酶突变体
技术领域
本发明涉及生物技术领域，特别涉及一种植酸酶突变体、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。
背景技术
植酸酶是一种能水解植酸的磷酸酶类。它能将植酸磷（六磷酸肌醇）降解为肌醇和无机磷酸。此酶分为两类: 3 -植酸酶（EC. 3. 1. 3. 8）和 6 -植酸酶（EC. 3. 1. 2. 6）。植酸酶广泛存在于植物、动物和微生物中, 如玉米、小麦等高等植物, 枯草芽孢杆菌、假单孢杆菌、乳酸杆菌、大肠杆菌等原核微生物及酵母、根霉、曲霉等真核微生物中。
在谷物、豆类和油料等作物籽实中，磷的基本贮存形式是植酸磷，其含量高达 1%～3%，它占植物中总磷的 60%～80%。但是以植酸磷形式存在的磷却因单胃动物体内缺乏能分解植酸的酶而难以被利用，其利用率仅在 0%～40%，从而造成了许多问题：首先是造成磷源浪费，一方面饲料中的磷源不能得到有效利用，另一方面为了满足动物对磷的需求，又必须在饲料中添加无机磷, 提高了饲料成本；其次是形成高磷粪便污染环境。饲料中 85%左右的植酸磷会被动物直接排出体外，粪便中大量的植酸磷使水和土壤受到严重污染。另外，植酸磷还是一种抗营养因子，它在动物胃肠道的消化吸收过程中会与多种金属离子如 Zn2 +、Ca2+、Cu2+、Fe2+等以及蛋白质螯合成相应的不溶性复合物，降低了动物对这些营养物质的有效利用。
植酸酶可作为一种单胃动物的饲料添加剂，它的饲喂效果已在世界范围内得到了确证。它可使植物性饲料中磷的利用率提60%，粪便中磷排泄量减少40%，同时还可降低植酸的抗营养作用。因此在饲料中添加植酸酶对提高畜禽业生产效益及降低植酸磷对环境的污染有重要意义。
现工业化生产的植酸酶主要有来源于黑曲霉的真菌植酸酶和来源于大肠杆菌的细菌植酸酶两种。其中来源于大肠杆菌的植酸酶APPA具有高比活性及良好的消化道稳定性等特点。目前主要通过在粉末饲料直接添加或颗粒饲料后喷涂的方法应用在饲料行业。
因为目前在颗粒饲料生产过程中有一个短暂的80-90℃的高温阶段。细菌植酸酶APPA热稳定性较差，其水溶液在70℃下保温5分钟剩余酶活性低于30％，直接添加到动物饲料中进行制粒后存留酶活一般低于20%，使APPA植酸酶在颗粒饲料的应用受到限制。采用饲料制粒后植酸酶液喷涂到饲料上的方法不仅增加设备投入，而且对酶制剂的稳定性、饲料中分布均一性都无法很好的保证。因此，提高热稳定性对目前饲料用植酸酶具有重要的现实意义。
发明内容
有鉴于此，本发明提供一种植酸酶突变体，获得突变体蛋白，提高其耐热性，从而有利于植酸酶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：
本发明涉及一种植酸酶突变体，其包含与SEQ ID NO:3具有至少90%同一性的氨基酸序列，且与SEQ ID NO:3相比在选自下组中的至少一个位置上包含氨基酸的取代：36，126，211，253，258，266。
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:3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:3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：A36P，N126E，V211W，Q253Y，Q258E，S266P。
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：A36P，N126E，V211W，Q253Y，Q258E，S266P，A36P/V211W，A36P/Q253Y，V211W/Q253Y，A36P/V211W/Q253Y，A36P/N126E /V211W/Q253Y。
本发明还涉及编码上述植酸酶突变体的DNA分子。
本发明还涉及包含上述DNA分子的重组表达载体。
本发明还涉及一种宿主细胞，包含上述重组表达载体。
将上述的质粒转入宿主细胞中，重组表达的植酸酶突变体的耐热性得到显著提升。
在本发明的一些实施例中，宿主细胞为毕赤酵母（Pichia pastoris）。
在本发明的一些实施例中，宿主细胞为里氏木霉（Trichoderma reesei）。
本发明还提供了上述植酸酶突变体的制备方法，包括：
步骤1：获取编码植酸酶突变体的DNA分子，所述植酸酶突变体包含与SEQ ID NO:3具有至少90%同一性的氨基酸序列，且与SEQ ID NO:3相比在选自下组中的至少一个位置上包含至少一种氨基酸的取代：36，126，211，253，258，266；
步骤2：将步骤1获得的所述DNA分子与表达载体融合，构建重组表达载体，转化宿主细胞；
步骤3：诱导含重组表达载体的宿主细胞表达融合蛋白，分离纯化表达的融合蛋白。
在本发明的一些实施例中，步骤1所述的植酸酶突变体包含下组中至少一个氨基酸的取代：A36P，N126E，V211W，Q253Y，Q258E，S266P。
在本发明的一些实施例中，步骤2所述的宿主细胞为毕赤酵母（Pichia pastoris）。
在本发明的一些实施例中，步骤2所述的宿主细胞为里氏木霉（Trichoderma reesei）。
本发明还提供了上述植酸酶突变体在饲料中的应用。
本发明以植酸酶APPA-M0为基础，提供了包含A36P、N126E、V211W、Q253Y、Q258E、S266P中至少一个突变位点的突变体。与APPA-M0相比，所述突变体在 80℃条件下处理5min后，酶活残留率普遍提高了8.9%-121.2%，耐热性得到显著提高。其中，突变体PHY-M2、PHY-M3、PHY-M7、PHY-M9、PHY-M10和PHY-M11在85℃条件下处理5min后，酶活残留率仍能达到50.98-74.60%，比APPA-M0普遍提高了17.2%-71.5%，耐热性更强。本发明提供的突变体耐热性得到显著提高，有利于植酸酶在饲料中的广泛应用。
具体实施方式
本发明公开了一种植酸酶突变体、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。
本发明中，用于限定氨基酸位置的命名法基于以编号ABF60232保藏于Genbank的大肠杆菌的植酸酶的氨基酸序列，其作为SEQ ID NO:1在序列表中给出（SEQ ID NO:1的氨基酸1-410）。因此，在本上下文中，用于位置编号的基础SEQ ID NO：1，始于Q1（Gln1）并且止于L410（Leu410）。SEQ ID NO：1作为位置编号的标准，并因此作为命名的基础。
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、毕赤酵母GS115、载体pPIC9k、Amp、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；
上层培养基：0.1%MgSO4，1%KH2PO4，0.6%(NH4)2SO4，1%葡萄糖，18.3%山梨醇，0.35%琼脂糖；
下层培养基平板：2%葡萄糖，0.5%(NH4)2SO4，1.5%KH2PO4，0.06%MgSO4，0.06%CaCl2，1.5%琼脂。
下面结合实施例，进一步阐述本发明：
实施例1 耐热突变体的筛选
申请人对野生型植酸酶 APPA(氨基酸序列为SEQ ID NO:1，其编码核苷酸序列为SEQ ID NO:2)的10个位点进行突变(W46E，Q62W，G70E，A73P，T114H，N137V，D142R，S146E，R159Y，Y255D)，得到植酸酶突变体APPA-M0，其氨基酸序列为SEQ ID NO:3，参照该序列合成一个编码核苷酸序列为SEQ ID NO:4。与植酸酶APPA相比，突变体APPA-M0的耐热性得到显著提升，经75℃处理5min后，植酸酶APPA残余酶活不足10%，而突变体APPA-M0的残余酶活高于85%。
为了进一步提高植酸酶突变体APPA-M0的耐热性，申请人对其基因进行蛋白结构分析，该蛋白有两个结构域：N端的134个氨基酸残基与C端的152个氨基酸残基共同组成结构域1，剩余中间124氨基酸残基组成结构域2，保守序列和活性中心均位于结构域1中，在不破坏蛋白二级结构与活性中心的前提下，进一步对该基因进行突变。
1.1设计PCR引物M0-F1、M0-R1：
M0-F1：GGCGAATTC CAGTCAGAACCAGAGTTGAAGTT（下划线为限制性内切酶EcoRI识别位点）；
M0-R1：ATAGCGGCCGC TTACAAGGAACAAGCAGGGAT（下划线为限制性内切酶NotI识别位点）。
以APPA-M0基因（SEQ ID NO：4）为模板，利用上述引物用GeneMorph II随机突变PCR试剂盒（Stratagene）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150ul含有0.1mM IPTG的LB+Amp培养基，37℃ 220rpm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40ul裂解液至两块新的96孔板，将其中一块96孔板于75℃处理5min；然后向两块96孔板中各加入80ul底物，于37℃反应30min后加入80ul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-M0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-M0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APPA-M0耐热性，又不会影响其酶活及原有酶学性质的突变位点：A36P，N126E，V211W，Q253Y，Q258E，S266P。
在植酸酶APPA-M0的基础上，本发明提供了分别包含A36P、N126E、V211W、Q253Y、Q258E、S266P单个突变位点的单点突变体，分别命名为PHY-M1、PHY-M2、PHY-M3、PHY-M4、PHY-M5、PHY-M6，其氨基酸序列分别为SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ IDNO:11、SEQ ID NO:13、SEQ ID NO:15，其编码核苷酸序列为SEQ ID NO:6、SEQ ID NO:8、SEQID NO:10、SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16。
本发明进一步提供了包含A36P/V211W、A36P/Q253Y、V211W/Q253Y两个突变位点组合的突变体，分别命名为PHY-M7、PHY-M8、PHY-M9，其氨基酸序列分别为SEQ ID NO:17、SEQID NO:19、SEQ ID NO:21，其编码核苷酸序列为SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22。
本发明还提供了包含A36P/V211W/Q253Y三个突变位点组合的突变体，命名为PHY-M10，其氨基酸序列分别为SEQ ID NO:23，其编码核苷酸序列为SEQ ID NO:24。
本发明还提供了包含A36P/N126E/V211W/Q253Y四个突变位点组合的突变体，命名为PHY-M11，其氨基酸序列分别为SEQ ID NO:25，其编码核苷酸序列为SEQ ID NO:26。
实施例2 植酸酶突变体在毕赤酵母中的表达
依照毕赤酵母的密码偏爱性分别对APPA-M0的基因序列SEQ ID NO：4，以及突变体的基因序列进行优化合成，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。
2.1表达载体的构建
将合成的APPA-M0和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 0.5ul，10×Buffer 2.0μL，dNTPs(2.5mM) 2.0μL，5’AOX引物（10M）：0.5μL，3’AOX引物：0.5μL，ddH2O 14.5μL，反应程序：95℃预变性5min，30个循环： 94℃ 30sec，55℃ 30sec，72℃2min，72℃ 10min）。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。
2.2毕赤酵母工程菌株的构建
2.2.1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30mL 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4mL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1mL灭菌水洗一遍后，4℃、9000 rpm离心2 min，轻轻弃上清，预冷的1mL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100-150μl山梨醇(1 mol/L)轻柔重悬菌体。
2.2.2转化和筛选
分别将2.1构建得到的表达质粒用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，然后在含不同浓度遗传霉素的YPD平板（0.5mg/mL-8mg/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1d；再转入BMMY培养基中，30℃、250rpm振荡培养；每天添加0.5%的甲醇，诱导表达4 d；9000rpm离心10min去除菌体，即得到分别含植酸酶APPA-M0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5.0mmol/L植酸钠中释放1μmol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25mL比色管，各加入1.8mL乙酸缓冲液（pH 5.0）、0.2mL样品反应液，混匀，37℃预热5min。在甲管中加入4mL底物溶液，乙管中加入4mL终止液，混匀，37℃反应30min，反应结束后甲管中加入4mL终止液，乙管中加入4mL底物溶液，混匀。静置10min，分别在415nm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。
实施例3 植酸酶突变体在里氏木霉中的表达
依照木霉的密码子偏爱性，分别对APPA-M0的基因序列SEQ ID NO：4，以及突变体的基因序列进行优化合成，并且在合成序列5’和3’两端分别加上KpnI和MluI两个酶切位点。
3.1表达载体的构建
将合成后的植酸酶基因片段与pSC1G载体分别用限制性内切酶KpnI和MluI（Fermentas）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）分别将上述植酸酶基因与pSC1G载体的酶切产物连接并转化大肠杆菌Trans5α(Transgen)，用氨苄青霉素进行选择，并对克隆进行测序(Invitrogen)验证。测序正确后，即得到含有植酸酶基因的重组质粒。
3.2 里氏木霉重组菌株的构建
（1）原生质体制备
取宿主菌里氏木霉（Trichoderma reesei）UE孢子悬液，接种于PDA平板上，30℃培养6 天；待其产孢丰富后，切取约1cm×1cm的菌落置于含120 mL YEG+U（0.5%酵母粉、1%葡萄糖、0.1%尿苷）的液体培养基中，30℃，220 rpm振荡培养14~16 h；
用无菌纱布过滤收集菌丝体，并用无菌水清洗一次；将菌丝体置于含有20 mL10mg/mL裂解酶液（Sigma L1412）的三角瓶中，30℃，90 rpm作用1-2 h；用显微镜观察检测原生质体转化进展；
将预冷的20 mL 1.2 M山梨醇（1.2 M山梨醇，50 mM Tris-Cl，50 mM CaCl2）加入上述三角瓶中，轻轻摇匀，用无菌Miracloth滤布过滤收集滤液，3000 rpm，4℃离心10 min；弃上清，加入预冷的5 mL 1.2 M山梨醇溶液悬浮菌体，3000 rpm，4℃离心10 min；弃上清，加入适量预冷的1.2 M山梨醇悬浮分装（200 μL/管，原生质体浓度为108个/mL）。
（2）表达载体转化
以下操作均在冰上进行，分别取10 μg上述构建的到的重组质粒加入到含有200 μL原生质体溶液的7 mL无菌离心管中，然后加入50 μL 25% PEG（25% PEG，50 mM Tris-Cl，50 mM CaCl2），轻弹管底混匀，冰上放置20 min；加入2 mL 25% PEG，混匀后室温放置5min；加入4 mL 1.2 M山梨醇，轻轻混匀后倒入熔化并保持在55℃的上层培养基中；轻轻混匀后铺在制备好的下层培养基平板上，30℃培养5~7 d至有转化子长出，将生长出的转化子挑至下层培养基平板进行复筛，菌落边缘形态较光滑的菌株为阳性转化子。
按照上述方法，申请人分别构建得到重组表达APPA-M0和上述植酸酶突变体的里氏木霉工程菌株。
（3）发酵验证和酶活测定
将上述构建得到的里氏木霉工程菌株分别接种至PDA固体平板，在30℃恒温培养箱倒置培养6-7天，待孢子丰富后，分别取两块直径1cm的菌丝块接种于含有50mL发酵培养基（1.5%葡萄糖，1.7%乳糖，2.5%玉米浆，0.44%(NH4)2SO4，0.09%MgSO4，2%KH2PO4，0.04%CaCl2，0.018%吐温-80，0.018%微量元素）的250mL三角瓶中，30℃培养48小时，然后25℃培养48小时。将发酵液离心，即得到分别含植酸酶APPA和上述植酸酶突变体的发酵上清液。
采用实施例2所述方法分别对构建得到的里氏木霉重组菌株发酵上清液进行植酸酶酶活测定。
实施例4 热稳定性分析
用预热10min、pH5.0 的0.25M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5min，85℃处理5min，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。具体结果见表1和表2。
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 植酸酶突变体在80℃条件下的耐热性分析
植酸酶突变体 80℃处理5min后酶活残留率
APPA-M0 45.05%
PHY-M1 49.07%
PHY-M2 60.00%
PHY-M3 81.91%
PHY-M4 70.51%
PHY-M5 52.95%
PHY-M6 52.33%
PHY-M7 84.95%
PHY-M8 72.57%
PHY-M9 88.42%
PHY-M10 95.22%
PHY-M11 99.63%
从表1的结果可以看出，与植酸酶APPA-M0相比，本发明提供的包含A36P、N126E、V211W、Q253Y、Q258E、S266P单点突变的植酸酶突变体PHY-M1、PHY-M2、PHY-M3、PHY-M4、PHY-M5、PHY-M6在80℃条件下处理5min后，酶活残留率普遍提高了8.9%-121.2%。从而说明，本发明提供的突变位点A36P、N126E、V211W、Q253Y、Q258E、S266P能显著提高植酸酶的耐热性。
与对应的单点突变体相比，分别包含A36P/V211W、A36P/Q253Y、V211W/Q253Y两个突变位点组合的植酸酶突变体PHY-M7、PHY-M8、PHY-M9，包含A36P/V211W/Q253Y三个突变位点组合的植酸酶突变体PHY-M10，以及包含A36P/N126E/V211W/Q253Y四个突变位点组合的植酸酶突变体PHY-M11的耐热性进一步得到提高，取得了意料不到的技术效果。
表2 植酸酶突变体在85℃条件下的耐热性分析
植酸酶突变体 85℃处理5min后酶活残留率
APPA-M0 43.49%
PHY-M2 50.98%
PHY-M3 52.87%
PHY-M7 55.20%
PHY-M9 62.26%
PHY-M10 69.51%
PHY-M11 74.60%
其中，包含N126E、V211W单点突变的植酸酶突变体PHY-M2和PHY-M3，包含A36P/V211W、V211W/Q253Y两个突变位点组合的植酸酶突变体PHY-M7和PHY-M9，包含A36P/V211W/Q253Y三个突变位点组合的植酸酶突变体PHY-M10，以及包含A36P/N126E/V211W/Q253Y四个突变位点组合的植酸酶突变体PHY-M11，在85℃条件下处理5min后，酶活残留率仍能达到50.98-74.60%，比APPA-M0普遍提高了17.2%-71.5%，耐热性更强。
综上所述，本发明提供的植酸酶突变体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。
序列表
<110> 青岛蔚蓝生物集团有限公司
<120> 植酸酶突变体
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Leu Gln Arg Thr Pro Glu Val Ala Arg Ser Arg Ala Thr Pro Leu Leu
260 265 270
Asp Leu Ile Lys Thr Ala Leu Thr Pro His Pro Pro Gln Lys Gln Ala
275 280 285
Tyr Gly Val Thr Leu Pro Thr Ser Val Leu Phe Ile Ala Gly His Asp
290 295 300
Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu Glu Leu Asn Trp Thr Leu
305 310 315 320
Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Glu Leu Val Phe Glu
325 330 335
Arg Trp Arg Arg Leu Ser Asp Asn Ser Gln Trp Ile Gln Val Ser Leu
340 345 350
Val Phe Gln Thr Leu Gln Gln Met Arg Asp Lys Thr Pro Leu Ser Leu
355 360 365
Asn Thr Pro Pro Gly Glu Val Lys Leu Thr Leu Ala Gly Cys Glu Glu
370 375 380
Arg Asn Ala Gln Gly Met Cys Ser Leu Ala Gly Phe Thr Gln Ile Val
385 390 395 400
Asn Glu Ala Arg Ile Pro Ala Cys Ser Leu
405 410
<210> 4
<211> 1230
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
caatctgaac cagaattgaa gttggaatct gttgttattg tttcccgtca cggtgttaga 60
gccccaacta aggctactca attgatgcaa gatgttactc cagatgcttg gccaacttgg 120
ccagttaagt tgggtgaatt gactccaaga ggtggtgaat tgattgctta cttgggtcat 180
tactggagac aaagattggt tgctgatgaa ttgttgccaa agaagggttg tccacaatct 240
ggtcaagttg ctattattgc tgatgttgat gaacgcacta gaaagaccgg tgaggctttt 300
gctgctggtt tggctccaga ttgtgctatt actgttcatc atcaagctga tacttcttcc 360
ccagatccat tgtttaaccc attgaagact ggtgtttgtc aattggatgt tgctaacgtt 420
actagagcta ttttggaaag agctggtggt tctattgctg attttactgg tcattaccaa 480
accgcctttc gtgaattgga aagagttttg aactttccac aatccaactt gtgtttgaag 540
agagaaaagc aagatgagtc ctgttccttg acccaagctc ttccatctga attgaaggtt 600
tctgctgata acgtttcttt gactggtgct gtttctttgg cttctatgtt gactgaaatt 660
ttcttgttgc agcaggctca aggtatgcca gaaccaggtt ggggtagaat tactgattct 720
catcaatgga acactttgtt gtctttgcat aacgctcaat ttgacttgtt gcaaagaact 780
ccagaagttg ctagatctag agctactcca ttgttggatt tgattaagac tgctttgact 840
ccacatccac cacaaaagca ggcttacggt gttactttgc caacttctgt tttgtttatt 900
gccggtcatg ataccaactt ggctaacttg ggtggtgctt tggaattgaa ctggactttg 960
ccaggtcaac cagataacac tccaccaggt ggtgaattgg tttttgaaag atggagaaga 1020
ttgtccgata actctcaatg gattcaagtt tctttggtct ttcagacctt gcagcaaatg 1080
agagataaga ctccattgtc tttgaacact ccaccaggtg aagttaagtt gactttggct 1140
ggttgtgaag aaagaaacgc tcaaggtatg tgttctttgg ctggttttac tcaaattgtc 1200
aacgaggcta gaatcccagc ttgttctttg 1230
<210> 5
<211> 410
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Gln Ser Glu Pro Glu Leu Lys Leu Glu Ser Val Val Ile Val Ser Arg
1 5 10 15
His Gly Val Arg Ala Pro Thr Lys Ala Thr Gln Leu Met Gln Asp Val
20 25 30
Thr Pro Asp Pro Trp Pro Thr Trp Pro Val Lys Leu Gly Glu Leu Thr
35 40 45
Pro Arg Gly Gly Glu Leu Ile Ala Tyr Leu Gly His Tyr Trp Arg Gln
50 55 60
Arg Leu Val Ala Asp Glu Leu Leu Pro Lys Lys Gly Cys Pro Gln Ser
65 70 75 80
Gly Gln Val Ala Ile Ile Ala Asp Val Asp Glu Arg Thr Arg Lys Thr
85 90 95
Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro Asp Cys Ala Ile Thr Val
100 105 110
His His Gln Ala Asp Thr Ser Ser Pro Asp Pro Leu Phe Asn Pro Leu
115 120 125
Lys Thr Gly Val Cys Gln Leu Asp Val Ala Asn Val Thr Arg Ala Ile
130 135 140
Leu Glu Arg Ala Gly Gly Ser Ile Ala Asp Phe Thr Gly His Tyr Gln
145 150 155 160
Thr Ala Phe Arg Glu Leu Glu Arg Val Leu Asn Phe Pro Gln Ser Asn
165 170 175
Leu Cys Leu Lys Arg Glu Lys Gln Asp Glu Ser Cys Ser Leu Thr Gln
180 185 190
Ala Leu Pro Ser Glu Leu Lys Val Ser Ala Asp Asn Val Ser Leu Thr
195 200 205
Gly Ala Val Ser Leu Ala Ser Met Leu Thr Glu Ile Phe Leu Leu Gln
210 215 220
Gln Ala Gln Gly Met Pro Glu Pro Gly Trp Gly Arg Ile Thr Asp Ser
225 230 235 240
His Gln Trp Asn Thr Leu Leu Ser Leu His Asn Ala Gln Phe Asp Leu
245 250 255
Leu Gln Arg Thr Pro Glu Val Ala Arg Ser Arg Ala Thr Pro Leu Leu
260 265 270
Asp Leu Ile Lys Thr Ala Leu Thr Pro His Pro Pro Gln Lys Gln Ala
275 280 285
Tyr Gly Val Thr Leu Pro Thr Ser Val Leu Phe Ile Ala Gly His Asp
290 295 300
Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu Glu Leu Asn Trp Thr Leu
305 310 315 320
Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Glu Leu Val Phe Glu
325 330 335
Arg Trp Arg Arg Leu Ser Asp Asn Ser Gln Trp Ile Gln Val Ser Leu
340 345 350
Val Phe Gln Thr Leu Gln Gln Met Arg Asp Lys Thr Pro Leu Ser Leu
355 360 365
Asn Thr Pro Pro Gly Glu Val Lys Leu Thr Leu Ala Gly Cys Glu Glu
370 375 380
Arg Asn Ala Gln Gly Met Cys Ser Leu Ala Gly Phe Thr Gln Ile Val
385 390 395 400
Asn Glu Ala Arg Ile Pro Ala Cys Ser Leu
405 410
<210> 6
<211> 1230
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
caatctgaac cagaattgaa gttggaatct gttgttattg tttcccgtca cggtgttaga 60
gccccaacta aggctactca attgatgcaa gatgttactc cagatccatg gccaacttgg 120
ccagttaagt tgggtgaatt gactccaaga ggtggtgaat tgattgctta cttgggtcat 180
tactggagac aaagattggt tgctgatgaa ttgttgccaa agaagggttg tccacaatct 240
ggtcaagttg ctattattgc tgatgttgat gaacgcacta gaaagaccgg tgaggctttt 300
gctgctggtt tggctccaga ttgtgctatt actgttcatc atcaagctga tacttcttcc 360
ccagatccat tgtttaaccc attgaagact ggtgtttgtc aattggatgt tgctaacgtt 420
actagagcta ttttggaaag agctggtggt tctattgctg attttactgg tcattaccaa 480
accgcctttc gtgaattgga aagagttttg aactttccac aatccaactt gtgtttgaag 540
agagaaaagc aagatgagtc ctgttccttg acccaagctc ttccatctga attgaaggtt 600
tctgctgata acgtttcttt gactggtgct gtttctttgg cttctatgtt gactgaaatt 660
ttcttgttgc agcaggctca aggtatgcca gaaccaggtt ggggtagaat tactgattct 720
catcaatgga acactttgtt gtctttgcat aacgctcaat ttgacttgtt gcaaagaact 780
ccagaagttg ctagatctag agctactcca ttgttggatt tgattaagac tgctttgact 840
ccacatccac cacaaaagca ggcttacggt gttactttgc caacttctgt tttgtttatt 900
gccggtcatg ataccaactt ggctaacttg ggtggtgctt tggaattgaa ctggactttg 960
ccaggtcaac cagataacac tccaccaggt ggtgaattgg tttttgaaag atggagaaga 1020
ttgtccgata actctcaatg gattcaagtt tctttggtct ttcagacctt gcagcaaatg 1080
agagataaga ctccattgtc tttgaacact ccaccaggtg aagttaagtt gactttggct 1140
ggttgtgaag aaagaaacgc tcaaggtatg tgttctttgg ctggttttac tcaaattgtc 1200
aacgaggcta gaatcccagc ttgttctttg 1230
<210> 7
<211> 410
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Gln Ser Glu Pro Glu Leu Lys Leu Glu Ser Val Val Ile Val Ser Arg
1 5 10 15
His Gly Val Arg Ala Pro Thr Lys Ala Thr Gln Leu Met Gln Asp Val
20 25 30
Thr Pro Asp Ala Trp Pro Thr Trp Pro Val Lys Leu Gly Glu Leu Thr
35 40 45
Pro Arg Gly Gly Glu Leu Ile Ala Tyr Leu Gly His Tyr Trp Arg Gln
50 55 60
Arg Leu Val Ala Asp Glu Leu Leu Pro Lys Lys Gly Cys Pro Gln Ser
65 70 75 80
Gly Gln Val Ala Ile Ile Ala Asp Val Asp Glu Arg Thr Arg Lys Thr
85 90 95
Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro Asp Cys Ala Ile Thr Val
100 105 110
His His Gln Ala Asp Thr Ser Ser Pro Asp Pro Leu Phe Glu Pro Leu
115 120 125
Lys Thr Gly Val Cys Gln Leu Asp Val Ala Asn Val Thr Arg Ala Ile
130 135 140
Leu Glu Arg Ala Gly Gly Ser Ile Ala Asp Phe Thr Gly His Tyr Gln
145 150 155 160
Thr Ala Phe Arg Glu Leu Glu Arg Val Leu Asn Phe Pro Gln Ser Asn
165 170 175
Leu Cys Leu Lys Arg Glu Lys Gln Asp Glu Ser Cys Ser Leu Thr Gln
180 185 190
Ala Leu Pro Ser Glu Leu Lys Val Ser Ala Asp Asn Val Ser Leu Thr
195 200 205
Gly Ala Val Ser Leu Ala Ser Met Leu Thr Glu Ile Phe Leu Leu Gln
210 215 220
Gln Ala Gln Gly Met Pro Glu Pro Gly Trp Gly Arg Ile Thr Asp Ser
225 230 235 240
His Gln Trp Asn Thr Leu Leu Ser Leu His Asn Ala Gln Phe Asp Leu
245 250 255
Leu Gln Arg Thr Pro Glu Val Ala Arg Ser Arg Ala Thr Pro Leu Leu
260 265 270
Asp Leu Ile Lys Thr Ala Leu Thr Pro His Pro Pro Gln Lys Gln Ala
275 280 285
Tyr Gly Val Thr Leu Pro Thr Ser Val Leu Phe Ile Ala Gly His Asp
290 295 300
Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu Glu Leu Asn Trp Thr Leu
305 310 315 320
Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Glu Leu Val Phe Glu
325 330 335
Arg Trp Arg Arg Leu Ser Asp Asn Ser Gln Trp Ile Gln Val Ser Leu
340 345 350
Val Phe Gln Thr Leu Gln Gln Met Arg Asp Lys Thr Pro Leu Ser Leu
355 360 365
Asn Thr Pro Pro Gly Glu Val Lys Leu Thr Leu Ala Gly Cys Glu Glu
370 375 380
Arg Asn Ala Gln Gly Met Cys Ser Leu Ala Gly Phe Thr Gln Ile Val
385 390 395 400
Asn Glu Ala Arg Ile Pro Ala Cys Ser Leu
405 410
<210> 8
<211> 1230
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
caatctgaac cagaattgaa gttggaatct gttgttattg tttcccgtca cggtgttaga 60
gccccaacta aggctactca attgatgcaa gatgttactc cagatgcttg gccaacttgg 120
ccagttaagt tgggtgaatt gactccaaga ggtggtgaat tgattgctta cttgggtcat 180
tactggagac aaagattggt tgctgatgaa ttgttgccaa agaagggttg tccacaatct 240
ggtcaagttg ctattattgc tgatgttgat gaacgcacta gaaagaccgg tgaggctttt 300
gctgctggtt tggctccaga ttgtgctatt actgttcatc atcaagctga tacttcttcc 360
ccagatccat tgtttgagcc attgaagact ggtgtttgtc aattggatgt tgctaacgtt 420
actagagcta ttttggaaag agctggtggt tctattgctg attttactgg tcattaccaa 480
accgcctttc gtgaattgga aagagttttg aactttccac aatccaactt gtgtttgaag 540
agagaaaagc aagatgagtc ctgttccttg acccaagctc ttccatctga attgaaggtt 600
tctgctgata acgtttcttt gactggtgct gtttctttgg cttctatgtt gactgaaatt 660
ttcttgttgc agcaggctca aggtatgcca gaaccaggtt ggggtagaat tactgattct 720
catcaatgga acactttgtt gtctttgcat aacgctcaat ttgacttgtt gcaaagaact 780
ccagaagttg ctagatctag agctactcca ttgttggatt tgattaagac tgctttgact 840
ccacatccac cacaaaagca ggcttacggt gttactttgc caacttctgt tttgtttatt 900
gccggtcatg ataccaactt ggctaacttg ggtggtgctt tggaattgaa ctggactttg 960
ccaggtcaac cagataacac tccaccaggt ggtgaattgg tttttgaaag atggagaaga 1020
ttgtccgata actctcaatg gattcaagtt tctttggtct ttcagacctt gcagcaaatg 1080
agagataaga ctccattgtc tttgaacact ccaccaggtg aagttaagtt gactttggct 1140
ggttgtgaag aaagaaacgc tcaaggtatg tgttctttgg ctggttttac tcaaattgtc 1200
aacgaggcta gaatcccagc ttgttctttg 1230
<210> 9
<211> 410
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Gln Ser Glu Pro Glu Leu Lys Leu Glu Ser Val Val Ile Val Ser Arg
1 5 10 15
His Gly Val Arg Ala Pro Thr Lys Ala Thr Gln Leu Met Gln Asp Val
20 25 30
Thr Pro Asp Ala Trp Pro Thr Trp Pro Val Lys Leu Gly Glu Leu Thr
35 40 45
Pro Arg Gly Gly Glu Leu Ile Ala Tyr Leu Gly His Tyr Trp Arg Gln
50 55 60
Arg Leu Val Ala Asp Glu Leu Leu Pro Lys Lys Gly Cys Pro Gln Ser
65 70 75 80
Gly Gln Val Ala Ile Ile Ala Asp Val Asp Glu Arg Thr Arg Lys Thr
85 90 95
Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro Asp Cys Ala Ile Thr Val
100 105 110
His His Gln Ala Asp Thr Ser Ser Pro Asp Pro Leu Phe Asn Pro Leu
115 120 125
Lys Thr Gly Val Cys Gln Leu Asp Val Ala Asn Val Thr Arg Ala Ile
130 135 140
Leu Glu Arg Ala Gly Gly Ser Ile Ala Asp Phe Thr Gly His Tyr Gln
145 150 155 160
Thr Ala Phe Arg Glu Leu Glu Arg Val Leu Asn Phe Pro Gln Ser Asn
165 170 175
Leu Cys Leu Lys Arg Glu Lys Gln Asp Glu Ser Cys Ser Leu Thr Gln
180 185 190
Ala Leu Pro Ser Glu Leu Lys Val Ser Ala Asp Asn Val Ser Leu Thr
195 200 205
Gly Ala Trp Ser Leu Ala Ser Met Leu Thr Glu Ile Phe Leu Leu Gln
210 215 220
Gln Ala Gln Gly Met Pro Glu Pro Gly Trp Gly Arg Ile Thr Asp Ser
225 230 235 240
His Gln Trp Asn Thr Leu Leu Ser Leu His Asn Ala Gln Phe Asp Leu
245 250 255
Leu Gln Arg Thr Pro Glu Val Ala Arg Ser Arg Ala Thr Pro Leu Leu
260 265 270
Asp Leu Ile Lys Thr Ala Leu Thr Pro His Pro Pro Gln Lys Gln Ala
275 280 285
Tyr Gly Val Thr Leu Pro Thr Ser Val Leu Phe Ile Ala Gly His Asp
290 295 300
Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu Glu Leu Asn Trp Thr Leu
305 310 315 320
Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Glu Leu Val Phe Glu
325 330 335
Arg Trp Arg Arg Leu Ser Asp Asn Ser Gln Trp Ile Gln Val Ser Leu
340 345 350
Val Phe Gln Thr Leu Gln Gln Met Arg Asp Lys Thr Pro Leu Ser Leu
355 360 365
Asn Thr Pro Pro Gly Glu Val Lys Leu Thr Leu Ala Gly Cys Glu Glu
370 375 380
Arg Asn Ala Gln Gly Met Cys Ser Leu Ala Gly Phe Thr Gln Ile Val
385 390 395 400
Asn Glu Ala Arg Ile Pro Ala Cys Ser Leu
405 410
<210> 10
<211> 1230
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
caatctgaac cagaattgaa gttggaatct gttgttattg tttcccgtca cggtgttaga 60
gccccaacta aggctactca attgatgcaa gatgttactc cagatgcttg gccaacttgg 120
ccagttaagt tgggtgaatt gactccaaga ggtggtgaat tgattgctta cttgggtcat 180
tactggagac aaagattggt tgctgatgaa ttgttgccaa agaagggttg tccacaatct 240
ggtcaagttg ctattattgc tgatgttgat gaacgcacta gaaagaccgg tgaggctttt 300
gctgctggtt tggctccaga ttgtgctatt actgttcatc atcaagctga tacttcttcc 360
ccagatccat tgtttaaccc attgaagact ggtgtttgtc aattggatgt tgctaacgtt 420
actagagcta ttttggaaag agctggtggt tctattgctg attttactgg tcattaccaa 480
accgcctttc gtgaattgga aagagttttg aactttccac aatccaactt gtgtttgaag 540
agagaaaagc aagatgagtc ctgttccttg acccaagctc ttccatctga attgaaggtt 600
tctgctgata acgtttcttt gactggtgct tggtctttgg cttctatgtt gactgaaatt 660
ttcttgttgc agcaggctca aggtatgcca gaaccaggtt ggggtagaat tactgattct 720
catcaatgga acactttgtt gtctttgcat aacgctcaat ttgacttgtt gcaaagaact 780
ccagaagttg ctagatctag agctactcca ttgttggatt tgattaagac tgctttgact 840
ccacatccac cacaaaagca ggcttacggt gttactttgc caacttctgt tttgtttatt 900
gccggtcatg ataccaactt ggctaacttg ggtggtgctt tggaattgaa ctggactttg 960
ccaggtcaac cagataacac tccaccaggt ggtgaattgg tttttgaaag atggagaaga 1020
ttgtccgata actctcaatg gattcaagtt tctttggtct ttcagacctt gcagcaaatg 1080
agagataaga ctccattgtc tttgaacact ccaccaggtg aagttaagtt gactttggct 1140
ggttgtgaag aaagaaacgc tcaaggtatg tgttctttgg ctggttttac tcaaattgtc 1200
aacgaggcta gaatcccagc ttgttctttg 1230
<210> 11
<211> 410
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Gln Ser Glu Pro Glu Leu Lys Leu Glu Ser Val Val Ile Val Ser Arg
1 5 10 15
His Gly Val Arg Ala Pro Thr Lys Ala Thr Gln Leu Met Gln Asp Val
20 25 30
Thr Pro Asp Ala Trp Pro Thr Trp Pro Val Lys Leu Gly Glu Leu Thr
35 40 45
Pro Arg Gly Gly Glu Leu Ile Ala Tyr Leu Gly His Tyr Trp Arg Gln
50 55 60
Arg Leu Val Ala Asp Glu Leu Leu Pro Lys Lys Gly Cys Pro Gln Ser
65 70 75 80
Gly Gln Val Ala Ile Ile Ala Asp Val Asp Glu Arg Thr Arg Lys Thr
85 90 95
Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro Asp Cys Ala Ile Thr Val
100 105 110
His His Gln Ala Asp Thr Ser Ser Pro Asp Pro Leu Phe Asn Pro Leu
115 120 125
Lys Thr Gly Val Cys Gln Leu Asp Val Ala Asn Val Thr Arg Ala Ile
130 135 140
Leu Glu Arg Ala Gly Gly Ser Ile Ala Asp Phe Thr Gly His Tyr Gln
145 150 155 160
Thr Ala Phe Arg Glu Leu Glu Arg Val Leu Asn Phe Pro Gln Ser Asn
165 170 175
Leu Cys Leu Lys Arg Glu Lys Gln Asp Glu Ser Cys Ser Leu Thr Gln
180 185 190
Ala Leu Pro Ser Glu Leu Lys Val Ser Ala Asp Asn Val Ser Leu Thr
195 200 205
Gly Ala Val Ser Leu Ala Ser Met Leu Thr Glu Ile Phe Leu Leu Gln
210 215 220
Gln Ala Gln Gly Met Pro Glu Pro Gly Trp Gly Arg Ile Thr Asp Ser
225 230 235 240
His Gln Trp Asn Thr Leu Leu Ser Leu His Asn Ala Tyr Phe Asp Leu
245 250 255
Leu Gln Arg Thr Pro Glu Val Ala Arg Ser Arg Ala Thr Pro Leu Leu
260 265 270
Asp Leu Ile Lys Thr Ala Leu Thr Pro His Pro Pro Gln Lys Gln Ala
275 280 285
Tyr Gly Val Thr Leu Pro Thr Ser Val Leu Phe Ile Ala Gly His Asp
290 295 300
Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu Glu Leu Asn Trp Thr Leu
305 310 315 320
Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Glu Leu Val Phe Glu
325 330 335
Arg Trp Arg Arg Leu Ser Asp Asn Ser Gln Trp Ile Gln Val Ser Leu
340 345 350
Val Phe Gln Thr Leu Gln Gln Met Arg Asp Lys Thr Pro Leu Ser Leu
355 360 365
Asn Thr Pro Pro Gly Glu Val Lys Leu Thr Leu Ala Gly Cys Glu Glu
370 375 380
Arg Asn Ala Gln Gly Met Cys Ser Leu Ala Gly Phe Thr Gln Ile Val
385 390 395 400
Asn Glu Ala Arg Ile Pro Ala Cys Ser Leu
405 410
<210> 12
<211> 1230
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
caatctgaac cagaattgaa gttggaatct gttgttattg tttcccgtca cggtgttaga 60
gccccaacta aggctactca attgatgcaa gatgttactc cagatgcttg gccaacttgg 120
ccagttaagt tgggtgaatt gactccaaga ggtggtgaat tgattgctta cttgggtcat 180
tactggagac aaagattggt tgctgatgaa ttgttgccaa agaagggttg tccacaatct 240
ggtcaagttg ctattattgc tgatgttgat gaacgcacta gaaagaccgg tgaggctttt 300
gctgctggtt tggctccaga ttgtgctatt actgttcatc atcaagctga tacttcttcc 360
ccagatccat tgtttaaccc attgaagact ggtgtttgtc aattggatgt tgctaacgtt 420
actagagcta ttttggaaag agctggtggt tctattgctg attttactgg tcattaccaa 480
accgcctttc gtgaattgga aagagttttg aactttccac aatccaactt gtgtttgaag 540
agagaaaagc aagatgagtc ctgttccttg acccaagctc ttccatctga attgaaggtt 600
tctgctgata acgtttcttt gactggtgct gtttctttgg cttctatgtt gactgaaatt 660
ttcttgttgc agcaggctca aggtatgcca gaaccaggtt ggggtagaat tactgattct 720
catcaatgga acactttgtt gtctttgcat aacgcttact ttgacttgtt gcaaagaact 780
ccagaagttg ctagatctag agctactcca ttgttggatt tgattaagac tgctttgact 840
ccacatccac cacaaaagca ggcttacggt gttactttgc caacttctgt tttgtttatt 900
gccggtcatg ataccaactt ggctaacttg ggtggtgctt tggaattgaa ctggactttg 960
ccaggtcaac cagataacac tccaccaggt ggtgaattgg tttttgaaag atggagaaga 1020
ttgtccgata actctcaatg gattcaagtt tctttggtct ttcagacctt gcagcaaatg 1080
agagataaga ctccattgtc tttgaacact ccaccaggtg aagttaagtt gactttggct 1140
ggttgtgaag aaagaaacgc tcaaggtatg tgttctttgg ctggttttac tcaaattgtc 1200
aacgaggcta gaatcccagc ttgttctttg 1230
<210> 13
<211> 410
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Gln Ser Glu Pro Glu Leu Lys Leu Glu Ser Val Val Ile Val Ser Arg
1 5 10 15
His Gly Val Arg Ala Pro Thr Lys Ala Thr Gln Leu Met Gln Asp Val
20 25 30
Thr Pro Asp Ala Trp Pro Thr Trp Pro Val Lys Leu Gly Glu Leu Thr
35 40 45
Pro Arg Gly Gly Glu Leu Ile Ala Tyr Leu Gly His Tyr Trp Arg Gln
50 55 60
Arg Leu Val Ala Asp Glu Leu Leu Pro Lys Lys Gly Cys Pro Gln Ser
65 70 75 80
Gly Gln Val Ala Ile Ile Ala Asp Val Asp Glu Arg Thr Arg Lys Thr
85 90 95
Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro Asp Cys Ala Ile Thr Val
100 105 110
His His Gln Ala Asp Thr Ser Ser Pro Asp Pro Leu Phe Asn Pro Leu
115 120 125
Lys Thr Gly Val Cys Gln Leu Asp Val Ala Asn Val Thr Arg Ala Ile
130 135 140
Leu Glu Arg Ala Gly Gly Ser Ile Ala Asp Phe Thr Gly His Tyr Gln
145 150 155 160
Thr Ala Phe Arg Glu Leu Glu Arg Val Leu Asn Phe Pro Gln Ser Asn
165 170 175
Leu Cys Leu Lys Arg Glu Lys Gln Asp Glu Ser Cys Ser Leu Thr Gln
180 185 190
Ala Leu Pro Ser Glu Leu Lys Val Ser Ala Asp Asn Val Ser Leu Thr
195 200 205
Gly Ala Val Ser Leu Ala Ser Met Leu Thr Glu Ile Phe Leu Leu Gln
210 215 220
Gln Ala Gln Gly Met Pro Glu Pro Gly Trp Gly Arg Ile Thr Asp Ser
225 230 235 240
His Gln Trp Asn Thr Leu Leu Ser Leu His Asn Ala Gln Phe Asp Leu
245 250 255
Leu Glu Arg Thr Pro Glu Val Ala Arg Ser Arg Ala Thr Pro Leu Leu
260 265 270
Asp Leu Ile Lys Thr Ala Leu Thr Pro His Pro Pro Gln Lys Gln Ala
275 280 285
Tyr Gly Val Thr Leu Pro Thr Ser Val Leu Phe Ile Ala Gly His Asp
290 295 300
Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu Glu Leu Asn Trp Thr Leu
305 310 315 320
Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Glu Leu Val Phe Glu
325 330 335
Arg Trp Arg Arg Leu Ser Asp Asn Ser Gln Trp Ile Gln Val Ser Leu
340 345 350
Val Phe Gln Thr Leu Gln Gln Met Arg Asp Lys Thr Pro Leu Ser Leu
355 360 365
Asn Thr Pro Pro Gly Glu Val Lys Leu Thr Leu Ala Gly Cys Glu Glu
370 375 380
Arg Asn Ala Gln Gly Met Cys Ser Leu Ala Gly Phe Thr Gln Ile Val
385 390 395 400
Asn Glu Ala Arg Ile Pro Ala Cys Ser Leu
405 410
<210> 14
<211> 1230
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
caatctgaac cagaattgaa gttggaatct gttgttattg tttcccgtca cggtgttaga 60
gccccaacta aggctactca attgatgcaa gatgttactc cagatgcttg gccaacttgg 120
ccagttaagt tgggtgaatt gactccaaga ggtggtgaat tgattgctta cttgggtcat 180
tactggagac aaagattggt tgctgatgaa ttgttgccaa agaagggttg tccacaatct 240
ggtcaagttg ctattattgc tgatgttgat gaacgcacta gaaagaccgg tgaggctttt 300
gctgctggtt tggctccaga ttgtgctatt actgttcatc atcaagctga tacttcttcc 360
ccagatccat tgtttaaccc attgaagact ggtgtttgtc aattggatgt tgctaacgtt 420
actagagcta ttttggaaag agctggtggt tctattgctg attttactgg tcattaccaa 480
accgcctttc gtgaattgga aagagttttg aactttccac aatccaactt gtgtttgaag 540
agagaaaagc aagatgagtc ctgttccttg acccaagctc ttccatctga attgaaggtt 600
tctgctgata acgtttcttt gactggtgct gtttctttgg cttctatgtt gactgaaatt 660
ttcttgttgc agcaggctca aggtatgcca gaaccaggtt ggggtagaat tactgattct 720
catcaatgga acactttgtt gtctttgcat aacgctcaat ttgacttgtt ggagagaact 780
ccagaagttg ctagatctag agctactcca ttgttggatt tgattaagac tgctttgact 840
ccacatccac cacaaaagca ggcttacggt gttactttgc caacttctgt tttgtttatt 900
gccggtcatg ataccaactt ggctaacttg ggtggtgctt tggaattgaa ctggactttg 960
ccaggtcaac cagataacac tccaccaggt ggtgaattgg tttttgaaag atggagaaga 1020
ttgtccgata actctcaatg gattcaagtt tctttggtct ttcagacctt gcagcaaatg 1080
agagataaga ctccattgtc tttgaacact ccaccaggtg aagttaagtt gactttggct 1140
ggttgtgaag aaagaaacgc tcaaggtatg tgttctttgg ctggttttac tcaaattgtc 1200
aacgaggcta gaatcccagc ttgttctttg 1230
<210> 15
<211> 410
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Gln Ser Glu Pro Glu Leu Lys Leu Glu Ser Val Val Ile Val Ser Arg
1 5 10 15
His Gly Val Arg Ala Pro Thr Lys Ala Thr Gln Leu Met Gln Asp Val
20 25 30
Thr Pro Asp Ala Trp Pro Thr Trp Pro Val Lys Leu Gly Glu Leu Thr
35 40 45
Pro Arg Gly Gly Glu Leu Ile Ala Tyr Leu Gly His Tyr Trp Arg Gln
50 55 60
Arg Leu Val Ala Asp Glu Leu Leu Pro Lys Lys Gly Cys Pro Gln Ser
65 70 75 80
Gly Gln Val Ala Ile Ile Ala Asp Val Asp Glu Arg Thr Arg Lys Thr
85 90 95
Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro Asp Cys Ala Ile Thr Val
100 105 110
His His Gln Ala Asp Thr Ser Ser Pro Asp Pro Leu Phe Asn Pro Leu
115 120 125
Lys Thr Gly Val Cys Gln Leu Asp Val Ala Asn Val Thr Arg Ala Ile
130 135 140
Leu Glu Arg Ala Gly Gly Ser Ile Ala Asp Phe Thr Gly His Tyr Gln
145 150 155 160
Thr Ala Phe Arg Glu Leu Glu Arg Val Leu Asn Phe Pro Gln Ser Asn
165 170 175
Leu Cys Leu Lys Arg Glu Lys Gln Asp Glu Ser Cys Ser Leu Thr Gln
180 185 190
Ala Leu Pro Ser Glu Leu Lys Val Ser Ala Asp Asn Val Ser Leu Thr
195 200 205
Gly Ala Val Ser Leu Ala Ser Met Leu Thr Glu Ile Phe Leu Leu Gln
210 215 220
Gln Ala Gln Gly Met Pro Glu Pro Gly Trp Gly Arg Ile Thr Asp Ser
225 230 235 240
His Gln Trp Asn Thr Leu Leu Ser Leu His Asn Ala Gln Phe Asp Leu
245 250 255
Leu Gln Arg Thr Pro Glu Val Ala Arg Pro Arg Ala Thr Pro Leu Leu
260 265 270
Asp Leu Ile Lys Thr Ala Leu Thr Pro His Pro Pro Gln Lys Gln Ala
275 280 285
Tyr Gly Val Thr Leu Pro Thr Ser Val Leu Phe Ile Ala Gly His Asp
290 295 300
Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu Glu Leu Asn Trp Thr Leu
305 310 315 320
Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Glu Leu Val Phe Glu
325 330 335
Arg Trp Arg Arg Leu Ser Asp Asn Ser Gln Trp Ile Gln Val Ser Leu
340 345 350
Val Phe Gln Thr Leu Gln Gln Met Arg Asp Lys Thr Pro Leu Ser Leu
355 360 365
Asn Thr Pro Pro Gly Glu Val Lys Leu Thr Leu Ala Gly Cys Glu Glu
370 375 380
Arg Asn Ala Gln Gly Met Cys Ser Leu Ala Gly Phe Thr Gln Ile Val
385 390 395 400
Asn Glu Ala Arg Ile Pro Ala Cys Ser Leu
405 410
<210> 16
<211> 1230
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
caatctgaac cagaattgaa gttggaatct gttgttattg tttcccgtca cggtgttaga 60
gccccaacta aggctactca attgatgcaa gatgttactc cagatgcttg gccaacttgg 120
ccagttaagt tgggtgaatt gactccaaga ggtggtgaat tgattgctta cttgggtcat 180
tactggagac aaagattggt tgctgatgaa ttgttgccaa agaagggttg tccacaatct 240
ggtcaagttg ctattattgc tgatgttgat gaacgcacta gaaagaccgg tgaggctttt 300
gctgctggtt tggctccaga ttgtgctatt actgttcatc atcaagctga tacttcttcc 360
ccagatccat tgtttaaccc attgaagact ggtgtttgtc aattggatgt tgctaacgtt 420
actagagcta ttttggaaag agctggtggt tctattgctg attttactgg tcattaccaa 480
accgcctttc gtgaattgga aagagttttg aactttccac aatccaactt gtgtttgaag 540
agagaaaagc aagatgagtc ctgttccttg acccaagctc ttccatctga attgaaggtt 600
tctgctgata acgtttcttt gactggtgct gtttctttgg cttctatgtt gactgaaatt 660
ttcttgttgc agcaggctca aggtatgcca gaaccaggtt ggggtagaat tactgattct 720
catcaatgga acactttgtt gtctttgcat aacgctcaat ttgacttgtt gcaaagaact 780
ccagaagttg ctagaccaag agctactcca ttgttggatt tgattaagac tgctttgact 840
ccacatccac cacaaaagca ggcttacggt gttactttgc caacttctgt tttgtttatt 900
gccggtcatg ataccaactt ggctaacttg ggtggtgctt tggaattgaa ctggactttg 960
ccaggtcaac cagataacac tccaccaggt ggtgaattgg tttttgaaag atggagaaga 1020
ttgtccgata actctcaatg gattcaagtt tctttggtct ttcagacctt gcagcaaatg 1080
agagataaga ctccattgtc tttgaacact ccaccaggtg aagttaagtt gactttggct 1140
ggttgtgaag aaagaaacgc tcaaggtatg tgttctttgg ctggttttac tcaaattgtc 1200
aacgaggcta gaatcccagc ttgttctttg 1230
<210> 17
<211> 410
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 17
Gln Ser Glu Pro Glu Leu Lys Leu Glu Ser Val Val Ile Val Ser Arg
1 5 10 15
His Gly Val Arg Ala Pro Thr Lys Ala Thr Gln Leu Met Gln Asp Val
20 25 30
Thr Pro Asp Pro Trp Pro Thr Trp Pro Val Lys Leu Gly Glu Leu Thr
35 40 45
Pro Arg Gly Gly Glu Leu Ile Ala Tyr Leu Gly His Tyr Trp Arg Gln
50 55 60
Arg Leu Val Ala Asp Glu Leu Leu Pro Lys Lys Gly Cys Pro Gln Ser
65 70 75 80
Gly Gln Val Ala Ile Ile Ala Asp Val Asp Glu Arg Thr Arg Lys Thr
85 90 95
Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro Asp Cys Ala Ile Thr Val
100 105 110
His His Gln Ala Asp Thr Ser Ser Pro Asp Pro Leu Phe Asn Pro Leu
115 120 125
Lys Thr Gly Val Cys Gln Leu Asp Val Ala Asn Val Thr Arg Ala Ile
130 135 140
Leu Glu Arg Ala Gly Gly Ser Ile Ala Asp Phe Thr Gly His Tyr Gln
145 150 155 160
Thr Ala Phe Arg Glu Leu Glu Arg Val Leu Asn Phe Pro Gln Ser Asn
165 170 175
Leu Cys Leu Lys Arg Glu Lys Gln Asp Glu Ser Cys Ser Leu Thr Gln
180 185 190
Ala Leu Pro Ser Glu Leu Lys Val Ser Ala Asp Asn Val Ser Leu Thr
195 200 205
Gly Ala Trp Ser Leu Ala Ser Met Leu Thr Glu Ile Phe Leu Leu Gln
210 215 220
Gln Ala Gln Gly Met Pro Glu Pro Gly Trp Gly Arg Ile Thr Asp Ser
225 230 235 240
His Gln Trp Asn Thr Leu Leu Ser Leu His Asn Ala Gln Phe Asp Leu
245 250 255
Leu Gln Arg Thr Pro Glu Val Ala Arg Ser Arg Ala Thr Pro Leu Leu
260 265 270
Asp Leu Ile Lys Thr Ala Leu Thr Pro His Pro Pro Gln Lys Gln Ala
275 280 285
Tyr Gly Val Thr Leu Pro Thr Ser Val Leu Phe Ile Ala Gly His Asp
290 295 300
Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu Glu Leu Asn Trp Thr Leu
305 310 315 320
Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Glu Leu Val Phe Glu
325 330 335
Arg Trp Arg Arg Leu Ser Asp Asn Ser Gln Trp Ile Gln Val Ser Leu
340 345 350
Val Phe Gln Thr Leu Gln Gln Met Arg Asp Lys Thr Pro Leu Ser Leu
355 360 365
Asn Thr Pro Pro Gly Glu Val Lys Leu Thr Leu Ala Gly Cys Glu Glu
370 375 380
Arg Asn Ala Gln Gly Met Cys Ser Leu Ala Gly Phe Thr Gln Ile Val
385 390 395 400
Asn Glu Ala Arg Ile Pro Ala Cys Ser Leu
405 410
<210> 18
<211> 1230
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
caatctgaac cagaattgaa gttggaatct gttgttattg tttcccgtca cggtgttaga 60
gccccaacta aggctactca attgatgcaa gatgttactc cagatccatg gccaacttgg 120
ccagttaagt tgggtgaatt gactccaaga ggtggtgaat tgattgctta cttgggtcat 180
tactggagac aaagattggt tgctgatgaa ttgttgccaa agaagggttg tccacaatct 240
ggtcaagttg ctattattgc tgatgttgat gaacgcacta gaaagaccgg tgaggctttt 300
gctgctggtt tggctccaga ttgtgctatt actgttcatc atcaagctga tacttcttcc 360
ccagatccat tgtttaaccc attgaagact ggtgtttgtc aattggatgt tgctaacgtt 420
actagagcta ttttggaaag agctggtggt tctattgctg attttactgg tcattaccaa 480
accgcctttc gtgaattgga aagagttttg aactttccac aatccaactt gtgtttgaag 540
agagaaaagc aagatgagtc ctgttccttg acccaagctc ttccatctga attgaaggtt 600
tctgctgata acgtttcttt gactggtgct tggtctttgg cttctatgtt gactgaaatt 660
ttcttgttgc agcaggctca aggtatgcca gaaccaggtt ggggtagaat tactgattct 720
catcaatgga acactttgtt gtctttgcat aacgctcaat ttgacttgtt gcaaagaact 780
ccagaagttg ctagatctag agctactcca ttgttggatt tgattaagac tgctttgact 840
ccacatccac cacaaaagca ggcttacggt gttactttgc caacttctgt tttgtttatt 900
gccggtcatg ataccaactt ggctaacttg ggtggtgctt tggaattgaa ctggactttg 960
ccaggtcaac cagataacac tccaccaggt ggtgaattgg tttttgaaag atggagaaga 1020
ttgtccgata actctcaatg gattcaagtt tctttggtct ttcagacctt gcagcaaatg 1080
agagataaga ctccattgtc tttgaacact ccaccaggtg aagttaagtt gactttggct 1140
ggttgtgaag aaagaaacgc tcaaggtatg tgttctttgg ctggttttac tcaaattgtc 1200
aacgaggcta gaatcccagc ttgttctttg 1230
<210> 19
<211> 410
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 19
Gln Ser Glu Pro Glu Leu Lys Leu Glu Ser Val Val Ile Val Ser Arg
1 5 10 15
His Gly Val Arg Ala Pro Thr Lys Ala Thr Gln Leu Met Gln Asp Val
20 25 30
Thr Pro Asp Pro Trp Pro Thr Trp Pro Val Lys Leu Gly Glu Leu Thr
35 40 45
Pro Arg Gly Gly Glu Leu Ile Ala Tyr Leu Gly His Tyr Trp Arg Gln
50 55 60
Arg Leu Val Ala Asp Glu Leu Leu Pro Lys Lys Gly Cys Pro Gln Ser
65 70 75 80
Gly Gln Val Ala Ile Ile Ala Asp Val Asp Glu Arg Thr Arg Lys Thr
85 90 95
Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro Asp Cys Ala Ile Thr Val
100 105 110
His His Gln Ala Asp Thr Ser Ser Pro Asp Pro Leu Phe Asn Pro Leu
115 120 125
Lys Thr Gly Val Cys Gln Leu Asp Val Ala Asn Val Thr Arg Ala Ile
130 135 140
Leu Glu Arg Ala Gly Gly Ser Ile Ala Asp Phe Thr Gly His Tyr Gln
145 150 155 160
Thr Ala Phe Arg Glu Leu Glu Arg Val Leu Asn Phe Pro Gln Ser Asn
165 170 175
Leu Cys Leu Lys Arg Glu Lys Gln Asp Glu Ser Cys Ser Leu Thr Gln
180 185 190
Ala Leu Pro Ser Glu Leu Lys Val Ser Ala Asp Asn Val Ser Leu Thr
195 200 205
Gly Ala Val Ser Leu Ala Ser Met Leu Thr Glu Ile Phe Leu Leu Gln
210 215 220
Gln Ala Gln Gly Met Pro Glu Pro Gly Trp Gly Arg Ile Thr Asp Ser
225 230 235 240
His Gln Trp Asn Thr Leu Leu Ser Leu His Asn Ala Tyr Phe Asp Leu
245 250 255
Leu Gln Arg Thr Pro Glu Val Ala Arg Ser Arg Ala Thr Pro Leu Leu
260 265 270
Asp Leu Ile Lys Thr Ala Leu Thr Pro His Pro Pro Gln Lys Gln Ala
275 280 285
Tyr Gly Val Thr Leu Pro Thr Ser Val Leu Phe Ile Ala Gly His Asp
290 295 300
Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu Glu Leu Asn Trp Thr Leu
305 310 315 320
Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Glu Leu Val Phe Glu
325 330 335
Arg Trp Arg Arg Leu Ser Asp Asn Ser Gln Trp Ile Gln Val Ser Leu
340 345 350
Val Phe Gln Thr Leu Gln Gln Met Arg Asp Lys Thr Pro Leu Ser Leu
355 360 365
Asn Thr Pro Pro Gly Glu Val Lys Leu Thr Leu Ala Gly Cys Glu Glu
370 375 380
Arg Asn Ala Gln Gly Met Cys Ser Leu Ala Gly Phe Thr Gln Ile Val
385 390 395 400
Asn Glu Ala Arg Ile Pro Ala Cys Ser Leu
405 410
<210> 20
<211> 1230
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
caatctgaac cagaattgaa gttggaatct gttgttattg tttcccgtca cggtgttaga 60
gccccaacta aggctactca attgatgcaa gatgttactc cagatccatg gccaacttgg 120
ccagttaagt tgggtgaatt gactccaaga ggtggtgaat tgattgctta cttgggtcat 180
tactggagac aaagattggt tgctgatgaa ttgttgccaa agaagggttg tccacaatct 240
ggtcaagttg ctattattgc tgatgttgat gaacgcacta gaaagaccgg tgaggctttt 300
gctgctggtt tggctccaga ttgtgctatt actgttcatc atcaagctga tacttcttcc 360
ccagatccat tgtttaaccc attgaagact ggtgtttgtc aattggatgt tgctaacgtt 420
actagagcta ttttggaaag agctggtggt tctattgctg attttactgg tcattaccaa 480
accgcctttc gtgaattgga aagagttttg aactttccac aatccaactt gtgtttgaag 540
agagaaaagc aagatgagtc ctgttccttg acccaagctc ttccatctga attgaaggtt 600
tctgctgata acgtttcttt gactggtgct gtttctttgg cttctatgtt gactgaaatt 660
ttcttgttgc agcaggctca aggtatgcca gaaccaggtt ggggtagaat tactgattct 720
catcaatgga acactttgtt gtctttgcat aacgcttact ttgacttgtt gcaaagaact 780
ccagaagttg ctagatctag agctactcca ttgttggatt tgattaagac tgctttgact 840
ccacatccac cacaaaagca ggcttacggt gttactttgc caacttctgt tttgtttatt 900
gccggtcatg ataccaactt ggctaacttg ggtggtgctt tggaattgaa ctggactttg 960
ccaggtcaac cagataacac tccaccaggt ggtgaattgg tttttgaaag atggagaaga 1020
ttgtccgata actctcaatg gattcaagtt tctttggtct ttcagacctt gcagcaaatg 1080
agagataaga ctccattgtc tttgaacact ccaccaggtg aagttaagtt gactttggct 1140
ggttgtgaag aaagaaacgc tcaaggtatg tgttctttgg ctggttttac tcaaattgtc 1200
aacgaggcta gaatcccagc ttgttctttg 1230
<210> 21
<211> 410
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 21
Gln Ser Glu Pro Glu Leu Lys Leu Glu Ser Val Val Ile Val Ser Arg
1 5 10 15
His Gly Val Arg Ala Pro Thr Lys Ala Thr Gln Leu Met Gln Asp Val
20 25 30
Thr Pro Asp Ala Trp Pro Thr Trp Pro Val Lys Leu Gly Glu Leu Thr
35 40 45
Pro Arg Gly Gly Glu Leu Ile Ala Tyr Leu Gly His Tyr Trp Arg Gln
50 55 60
Arg Leu Val Ala Asp Glu Leu Leu Pro Lys Lys Gly Cys Pro Gln Ser
65 70 75 80
Gly Gln Val Ala Ile Ile Ala Asp Val Asp Glu Arg Thr Arg Lys Thr
85 90 95
Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro Asp Cys Ala Ile Thr Val
100 105 110
His His Gln Ala Asp Thr Ser Ser Pro Asp Pro Leu Phe Asn Pro Leu
115 120 125
Lys Thr Gly Val Cys Gln Leu Asp Val Ala Asn Val Thr Arg Ala Ile
130 135 140
Leu Glu Arg Ala Gly Gly Ser Ile Ala Asp Phe Thr Gly His Tyr Gln
145 150 155 160
Thr Ala Phe Arg Glu Leu Glu Arg Val Leu Asn Phe Pro Gln Ser Asn
165 170 175
Leu Cys Leu Lys Arg Glu Lys Gln Asp Glu Ser Cys Ser Leu Thr Gln
180 185 190
Ala Leu Pro Ser Glu Leu Lys Val Ser Ala Asp Asn Val Ser Leu Thr
195 200 205
Gly Ala Trp Ser Leu Ala Ser Met Leu Thr Glu Ile Phe Leu Leu Gln
210 215 220
Gln Ala Gln Gly Met Pro Glu Pro Gly Trp Gly Arg Ile Thr Asp Ser
225 230 235 240
His Gln Trp Asn Thr Leu Leu Ser Leu His Asn Ala Tyr Phe Asp Leu
245 250 255
Leu Gln Arg Thr Pro Glu Val Ala Arg Ser Arg Ala Thr Pro Leu Leu
260 265 270
Asp Leu Ile Lys Thr Ala Leu Thr Pro His Pro Pro Gln Lys Gln Ala
275 280 285
Tyr Gly Val Thr Leu Pro Thr Ser Val Leu Phe Ile Ala Gly His Asp
290 295 300
Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu Glu Leu Asn Trp Thr Leu
305 310 315 320
Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Glu Leu Val Phe Glu
325 330 335
Arg Trp Arg Arg Leu Ser Asp Asn Ser Gln Trp Ile Gln Val Ser Leu
340 345 350
Val Phe Gln Thr Leu Gln Gln Met Arg Asp Lys Thr Pro Leu Ser Leu
355 360 365
Asn Thr Pro Pro Gly Glu Val Lys Leu Thr Leu Ala Gly Cys Glu Glu
370 375 380
Arg Asn Ala Gln Gly Met Cys Ser Leu Ala Gly Phe Thr Gln Ile Val
385 390 395 400
Asn Glu Ala Arg Ile Pro Ala Cys Ser Leu
405 410
<210> 22
<211> 1230
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
caatctgaac cagaattgaa gttggaatct gttgttattg tttcccgtca cggtgttaga 60
gccccaacta aggctactca attgatgcaa gatgttactc cagatgcttg gccaacttgg 120
ccagttaagt tgggtgaatt gactccaaga ggtggtgaat tgattgctta cttgggtcat 180
tactggagac aaagattggt tgctgatgaa ttgttgccaa agaagggttg tccacaatct 240
ggtcaagttg ctattattgc tgatgttgat gaacgcacta gaaagaccgg tgaggctttt 300
gctgctggtt tggctccaga ttgtgctatt actgttcatc atcaagctga tacttcttcc 360
ccagatccat tgtttaaccc attgaagact ggtgtttgtc aattggatgt tgctaacgtt 420
actagagcta ttttggaaag agctggtggt tctattgctg attttactgg tcattaccaa 480
accgcctttc gtgaattgga aagagttttg aactttccac aatccaactt gtgtttgaag 540
agagaaaagc aagatgagtc ctgttccttg acccaagctc ttccatctga attgaaggtt 600
tctgctgata acgtttcttt gactggtgct tggtctttgg cttctatgtt gactgaaatt 660
ttcttgttgc agcaggctca aggtatgcca gaaccaggtt ggggtagaat tactgattct 720
catcaatgga acactttgtt gtctttgcat aacgcttact ttgacttgtt gcaaagaact 780
ccagaagttg ctagatctag agctactcca ttgttggatt tgattaagac tgctttgact 840
ccacatccac cacaaaagca ggcttacggt gttactttgc caacttctgt tttgtttatt 900
gccggtcatg ataccaactt ggctaacttg ggtggtgctt tggaattgaa ctggactttg 960
ccaggtcaac cagataacac tccaccaggt ggtgaattgg tttttgaaag atggagaaga 1020
ttgtccgata actctcaatg gattcaagtt tctttggtct ttcagacctt gcagcaaatg 1080
agagataaga ctccattgtc tttgaacact ccaccaggtg aagttaagtt gactttggct 1140
ggttgtgaag aaagaaacgc tcaaggtatg tgttctttgg ctggttttac tcaaattgtc 1200
aacgaggcta gaatcccagc ttgttctttg 1230
<210> 23
<211> 410
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 23
Gln Ser Glu Pro Glu Leu Lys Leu Glu Ser Val Val Ile Val Ser Arg
1 5 10 15
His Gly Val Arg Ala Pro Thr Lys Ala Thr Gln Leu Met Gln Asp Val
20 25 30
Thr Pro Asp Pro Trp Pro Thr Trp Pro Val Lys Leu Gly Glu Leu Thr
35 40 45
Pro Arg Gly Gly Glu Leu Ile Ala Tyr Leu Gly His Tyr Trp Arg Gln
50 55 60
Arg Leu Val Ala Asp Glu Leu Leu Pro Lys Lys Gly Cys Pro Gln Ser
65 70 75 80
Gly Gln Val Ala Ile Ile Ala Asp Val Asp Glu Arg Thr Arg Lys Thr
85 90 95
Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro Asp Cys Ala Ile Thr Val
100 105 110
His His Gln Ala Asp Thr Ser Ser Pro Asp Pro Leu Phe Asn Pro Leu
115 120 125
Lys Thr Gly Val Cys Gln Leu Asp Val Ala Asn Val Thr Arg Ala Ile
130 135 140
Leu Glu Arg Ala Gly Gly Ser Ile Ala Asp Phe Thr Gly His Tyr Gln
145 150 155 160
Thr Ala Phe Arg Glu Leu Glu Arg Val Leu Asn Phe Pro Gln Ser Asn
165 170 175
Leu Cys Leu Lys Arg Glu Lys Gln Asp Glu Ser Cys Ser Leu Thr Gln
180 185 190
Ala Leu Pro Ser Glu Leu Lys Val Ser Ala Asp Asn Val Ser Leu Thr
195 200 205
Gly Ala Trp Ser Leu Ala Ser Met Leu Thr Glu Ile Phe Leu Leu Gln
210 215 220
Gln Ala Gln Gly Met Pro Glu Pro Gly Trp Gly Arg Ile Thr Asp Ser
225 230 235 240
His Gln Trp Asn Thr Leu Leu Ser Leu His Asn Ala Tyr Phe Asp Leu
245 250 255
Leu Gln Arg Thr Pro Glu Val Ala Arg Ser Arg Ala Thr Pro Leu Leu
260 265 270
Asp Leu Ile Lys Thr Ala Leu Thr Pro His Pro Pro Gln Lys Gln Ala
275 280 285
Tyr Gly Val Thr Leu Pro Thr Ser Val Leu Phe Ile Ala Gly His Asp
290 295 300
Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu Glu Leu Asn Trp Thr Leu
305 310 315 320
Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Glu Leu Val Phe Glu
325 330 335
Arg Trp Arg Arg Leu Ser Asp Asn Ser Gln Trp Ile Gln Val Ser Leu
340 345 350
Val Phe Gln Thr Leu Gln Gln Met Arg Asp Lys Thr Pro Leu Ser Leu
355 360 365
Asn Thr Pro Pro Gly Glu Val Lys Leu Thr Leu Ala Gly Cys Glu Glu
370 375 380
Arg Asn Ala Gln Gly Met Cys Ser Leu Ala Gly Phe Thr Gln Ile Val
385 390 395 400
Asn Glu Ala Arg Ile Pro Ala Cys Ser Leu
405 410
<210> 24
<211> 1230
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
caatctgaac cagaattgaa gttggaatct gttgttattg tttcccgtca cggtgttaga 60
gccccaacta aggctactca attgatgcaa gatgttactc cagatccatg gccaacttgg 120
ccagttaagt tgggtgaatt gactccaaga ggtggtgaat tgattgctta cttgggtcat 180
tactggagac aaagattggt tgctgatgaa ttgttgccaa agaagggttg tccacaatct 240
ggtcaagttg ctattattgc tgatgttgat gaacgcacta gaaagaccgg tgaggctttt 300
gctgctggtt tggctccaga ttgtgctatt actgttcatc atcaagctga tacttcttcc 360
ccagatccat tgtttaaccc attgaagact ggtgtttgtc aattggatgt tgctaacgtt 420
actagagcta ttttggaaag agctggtggt tctattgctg attttactgg tcattaccaa 480
accgcctttc gtgaattgga aagagttttg aactttccac aatccaactt gtgtttgaag 540
agagaaaagc aagatgagtc ctgttccttg acccaagctc ttccatctga attgaaggtt 600
tctgctgata acgtttcttt gactggtgct tggtctttgg cttctatgtt gactgaaatt 660
ttcttgttgc agcaggctca aggtatgcca gaaccaggtt ggggtagaat tactgattct 720
catcaatgga acactttgtt gtctttgcat aacgcttact ttgacttgtt gcaaagaact 780
ccagaagttg ctagatctag agctactcca ttgttggatt tgattaagac tgctttgact 840
ccacatccac cacaaaagca ggcttacggt gttactttgc caacttctgt tttgtttatt 900
gccggtcatg ataccaactt ggctaacttg ggtggtgctt tggaattgaa ctggactttg 960
ccaggtcaac cagataacac tccaccaggt ggtgaattgg tttttgaaag atggagaaga 1020
ttgtccgata actctcaatg gattcaagtt tctttggtct ttcagacctt gcagcaaatg 1080
agagataaga ctccattgtc tttgaacact ccaccaggtg aagttaagtt gactttggct 1140
ggttgtgaag aaagaaacgc tcaaggtatg tgttctttgg ctggttttac tcaaattgtc 1200
aacgaggcta gaatcccagc ttgttctttg 1230
<210> 25
<211> 410
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 25
Gln Ser Glu Pro Glu Leu Lys Leu Glu Ser Val Val Ile Val Ser Arg
1 5 10 15
His Gly Val Arg Ala Pro Thr Lys Ala Thr Gln Leu Met Gln Asp Val
20 25 30
Thr Pro Asp Pro Trp Pro Thr Trp Pro Val Lys Leu Gly Glu Leu Thr
35 40 45
Pro Arg Gly Gly Glu Leu Ile Ala Tyr Leu Gly His Tyr Trp Arg Gln
50 55 60
Arg Leu Val Ala Asp Glu Leu Leu Pro Lys Lys Gly Cys Pro Gln Ser
65 70 75 80
Gly Gln Val Ala Ile Ile Ala Asp Val Asp Glu Arg Thr Arg Lys Thr
85 90 95
Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro Asp Cys Ala Ile Thr Val
100 105 110
His His Gln Ala Asp Thr Ser Ser Pro Asp Pro Leu Phe Glu Pro Leu
115 120 125
Lys Thr Gly Val Cys Gln Leu Asp Val Ala Asn Val Thr Arg Ala Ile
130 135 140
Leu Glu Arg Ala Gly Gly Ser Ile Ala Asp Phe Thr Gly His Tyr Gln
145 150 155 160
Thr Ala Phe Arg Glu Leu Glu Arg Val Leu Asn Phe Pro Gln Ser Asn
165 170 175
Leu Cys Leu Lys Arg Glu Lys Gln Asp Glu Ser Cys Ser Leu Thr Gln
180 185 190
Ala Leu Pro Ser Glu Leu Lys Val Ser Ala Asp Asn Val Ser Leu Thr
195 200 205
Gly Ala Trp Ser Leu Ala Ser Met Leu Thr Glu Ile Phe Leu Leu Gln
210 215 220
Gln Ala Gln Gly Met Pro Glu Pro Gly Trp Gly Arg Ile Thr Asp Ser
225 230 235 240
His Gln Trp Asn Thr Leu Leu Ser Leu His Asn Ala Tyr Phe Asp Leu
245 250 255
Leu Gln Arg Thr Pro Glu Val Ala Arg Ser Arg Ala Thr Pro Leu Leu
260 265 270
Asp Leu Ile Lys Thr Ala Leu Thr Pro His Pro Pro Gln Lys Gln Ala
275 280 285
Tyr Gly Val Thr Leu Pro Thr Ser Val Leu Phe Ile Ala Gly His Asp
290 295 300
Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu Glu Leu Asn Trp Thr Leu
305 310 315 320
Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Glu Leu Val Phe Glu
325 330 335
Arg Trp Arg Arg Leu Ser Asp Asn Ser Gln Trp Ile Gln Val Ser Leu
340 345 350
Val Phe Gln Thr Leu Gln Gln Met Arg Asp Lys Thr Pro Leu Ser Leu
355 360 365
Asn Thr Pro Pro Gly Glu Val Lys Leu Thr Leu Ala Gly Cys Glu Glu
370 375 380
Arg Asn Ala Gln Gly Met Cys Ser Leu Ala Gly Phe Thr Gln Ile Val
385 390 395 400
Asn Glu Ala Arg Ile Pro Ala Cys Ser Leu
405 410
<210> 26
<211> 1230
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
caatctgaac cagaattgaa gttggaatct gttgttattg tttcccgtca cggtgttaga 60
gccccaacta aggctactca attgatgcaa gatgttactc cagatccatg gccaacttgg 120
ccagttaagt tgggtgaatt gactccaaga ggtggtgaat tgattgctta cttgggtcat 180
tactggagac aaagattggt tgctgatgaa ttgttgccaa agaagggttg tccacaatct 240
ggtcaagttg ctattattgc tgatgttgat gaacgcacta gaaagaccgg tgaggctttt 300
gctgctggtt tggctccaga ttgtgctatt actgttcatc atcaagctga tacttcttcc 360
ccagatccat tgtttgagcc attgaagact ggtgtttgtc aattggatgt tgctaacgtt 420
actagagcta ttttggaaag agctggtggt tctattgctg attttactgg tcattaccaa 480
accgcctttc gtgaattgga aagagttttg aactttccac aatccaactt gtgtttgaag 540
agagaaaagc aagatgagtc ctgttccttg acccaagctc ttccatctga attgaaggtt 600
tctgctgata acgtttcttt gactggtgct tggtctttgg cttctatgtt gactgaaatt 660
ttcttgttgc agcaggctca aggtatgcca gaaccaggtt ggggtagaat tactgattct 720
catcaatgga acactttgtt gtctttgcat aacgcttact ttgacttgtt gcaaagaact 780
ccagaagttg ctagatctag agctactcca ttgttggatt tgattaagac tgctttgact 840
ccacatccac cacaaaagca ggcttacggt gttactttgc caacttctgt tttgtttatt 900
gccggtcatg ataccaactt ggctaacttg ggtggtgctt tggaattgaa ctggactttg 960
ccaggtcaac cagataacac tccaccaggt ggtgaattgg tttttgaaag atggagaaga 1020
ttgtccgata actctcaatg gattcaagtt tctttggtct ttcagacctt gcagcaaatg 1080
agagataaga ctccattgtc tttgaacact ccaccaggtg aagttaagtt gactttggct 1140
ggttgtgaag aaagaaacgc tcaaggtatg tgttctttgg ctggttttac tcaaattgtc 1200
aacgaggcta gaatcccagc ttgttctttg 1230

Claims (8)

1.一种植酸酶突变体，其特征在于，所述的突变体包含与SEQ ID NO:3具有至少90%同一性的氨基酸序列，且与SEQ ID NO:3相比在选自下组中的至少一个位置上包含氨基酸的取代：36，126，211，253，258，266。
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:3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:3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：A36P，N126E，V211W，Q253Y，Q258E，S266P。
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：A36P，N126E，V211W，Q253Y，Q258E，S266P，A36P/V211W，A36P/Q253Y，V211W/Q253Y，A36P/V211W/Q253Y，A36P/ N126E /V211W/Q253Y。
6.编码权利要求1-5任一所述植酸酶突变体的DNA分子。
7.具有如权利要求6所述DNA分子的载体。
8.一种宿主细胞，其特征在于，所述宿主细胞包含如权利要求7所述的载体。
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