CN110643590B - 一种真菌来源β螺旋桨型重组植酸酶r-AoPhytase及其表达菌株和应用 - Google Patents
一种真菌来源β螺旋桨型重组植酸酶r-AoPhytase及其表达菌株和应用 Download PDFInfo
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- CN110643590B CN110643590B CN201911074264.0A CN201911074264A CN110643590B CN 110643590 B CN110643590 B CN 110643590B CN 201911074264 A CN201911074264 A CN 201911074264A CN 110643590 B CN110643590 B CN 110643590B
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Abstract
本发明公开了一种真菌来源β螺旋桨型重组植酸酶r‑AoPhytase及其表达菌株和应用，所述β螺旋桨型重组植酸酶r‑AoPhytase的原始来源为捕食线虫性真菌Arthrobotry oligosopra，其氨基酸序列为如下氨基酸序列中的一种：(1)序列表中的SEQ ID No：1；(2)序列表中的SEQ ID No：1经取代、缺失或添加一个或几个氨基酸残基且编码相同功能蛋白质的氨基酸序列；(3)与序列表中的SEQ ID No：1具有80％以上同源性的突变氨基酸序列。本发明重组植酸酶r‑AoPhytase具有较高的比酶活，接近中性的最适pH和较高的最适温度，具有促进不同饲料样品中无机磷、可溶性矿物质的释放能力，适用于饲料加工与生产中。
Description
技术领域
本发明属于分子生物学和基因工程领域，具体涉及一种真菌来源β螺旋桨型重组植酸酶r-AoPhytase及其表达菌株和应用。
背景技术
植酸酶制剂在动物和人类营养品中有广泛的应用。它可水解植酸盐(肌醇-1，2，3，4，5，6-六磷酸盐)，生成低级肌醇磷酸衍生物和无机磷酸(Mullaney,Daly,&Ullah,2000)。植酸盐被认为是植物中磷酸盐和肌醇的主要储存形式，植酸衍生物占植物源饲料中磷的60-80％。同时植酸也是一种聚阴离子螯合剂，它可与几种主要的有营养价值的二价阳离子，如Ca2+，Mg2+，Zn2+，Cu2+，Fe2+和Mn2+形成复合物(Chen&Headquarters,2000)。植酸酶被广泛应用于商业化的家禽、猪和鱼类饲料中，以提高磷、矿物质、氨基酸等的利用率。肌醇六磷酸盐分子以及与其结合的营养物质不会被消化道吸收，但可以被植酸酶酶促降解，促进其吸收。在饲料中添加植酸酶也可以明显提高矿质元素钙、镁及微量元素锌、铜、铁、锰等的利用率(Lonnerdal,2000)。除此以外，在饲料中添加植酸酶还能够提高动物对蛋白质和氨基酸的消化吸收，这是因为植酸酶在水解植酸释放磷的同时，可以将与植酸络合的蛋白质释放出来，便于消化道分泌的各种蛋白酶作用；同时还可以释放与植酸结合的内源性蛋白酶、淀粉酶、脂肪酶等，从而提高消化利用率。植酸酶也可以用于食品的加工过程，在食品中添加植酸酶既可以提高磷及其他矿质元素的利用率，也可以提高蛋白质、淀粉和脂肪等营养物质的利用率(Haefner et al.,2005)。
植酸酶广泛存在于动植物中，如水稻、小麦、玉米(Ullah&Gibson,1988)、大豆等一些植物种子中均含有植酸酶，哺乳动物的小肠及脊椎动物的红细胞中含有植酸酶，但含量很少且活性很低。在牛等反刍动物的瘤胃中植酸酶的活性较高。也存在于微生物(细菌、真菌和酵母)中，如霉菌中的曲霉属就存在植酸酶。目前研究最多的是通过微生物生产植酸酶，蛋白分离纯化和理化性质分析结果表明，它们是植酸酶工业化的最佳来源(Lei,Porres,Mullaney,&Brinch-Pedersen,2007)。
BPP型植酸酶是最近发现的一类全新的碱性磷酸酶酶，是在自然界中最丰富的和结构多样性最广的一类植酸酶。迄今为止，所有的已知的BPP型植酸酶均来源于细菌，特别是芽孢杆菌(Singh&Satyanarayana,2015)。例如，从枯草芽孢杆菌和解淀粉芽孢杆菌中分别分离出的植酸酶phyC和TS-Phy就属于BPPs(Ha et al.,2000)。目前细菌来源的BPP型植酸酶的比酶活偏低，且最适pH和最适温度不适宜于饲料加工工艺的应用。
针对这一技术需求，本发明开发了一种来源于真菌的BPP型植酸酶。我们首次以捕食线虫性真菌Arthrobotry oligosopra为出发材料，研发了真菌来源的β螺旋桨型植酸酶Aophytase及其重组制备方法，发现该真菌来源的BPP型重组植酸酶具有较高的比酶活性，接近中性的最适pH和较高的最适温度，其具有促进不同饲料样品中无机磷、可溶性矿物质的释放能力，适用于饲料加工与生产中的应用。
发明内容
本发明针对上述现有技术的不足，提供了一种真菌来源β螺旋桨型重组植酸酶r-AoPhytase及其表达菌株和应用。本发明重组植酸酶r-AoPhytase具有较高的比酶活，接近中性的最适pH和较高的最适温度，具有促进不同饲料样品中无机磷、可溶性矿物质的释放能力，适用于饲料加工与生产中的应用。
本发明真菌来源β螺旋桨型重组植酸酶r-AoPhytase，其原始来源是捕食线虫性真菌Arthrobotry oligosopra，由782aa氨基酸组成，分子量为84.2KDa，等电点为5.58，其氨基酸序列为如下氨基酸序列中的一种：
(1)序列表中的SEQ ID No：1；
(2)序列表中的SEQ ID No：1经取代、缺失或添加一个或几个氨基酸残基且编码相同功能蛋白质的氨基酸序列；
(3)与序列表中的SEQ ID No：1具有80％以上同源性的突变氨基酸序列。
编码所述β螺旋桨型重组植酸酶的编码基因，为具有如下核苷酸序列中的一种：
(1)序列表中的SEQ ID No：2或SEQ ID No：3；
(2)序列表中的SEQ ID No：2或SEQ ID No：3经取代、缺失或添加一个或几个核苷酸残基且编码相同功能蛋白质的核苷酸序列；
(3)编码序列表中SEQ ID No：1蛋白质序列的多核苷酸序列；
(4)与序列表中的SEQ ID No：2或SEQ ID No：3具有80％以上同源性的突变核苷酸序列。
本发明真菌来源β螺旋桨型重组植酸酶r-AoPhytase的重组表达菌株，其分类命名为：毕赤酵母GS115(5′PAOX1::pPICαZA-Aophytase)(Pichia pastoris GS115(5′PAOX1::pPICαZA-Aophytase))，保藏单位：中国典型培养物保藏中心(CCTCC)，地址：中国.武汉.武汉大学，保藏日期：2019年10月8日，保藏编号：CCTCC NO：M 2019775。
所述重组表达菌株的制备方法，首先通过化学合成所述r-AoPhytase基因，利用EcoRⅠ和SalⅠ双酶切并与表达质粒载体连接，将连接产物转化感受态细胞，获得表达载体；然后通过SacⅠ酶使表达载体线性化，并将线性化的质粒电转化表达宿主细胞，利用引物P1和P2进行菌落PCR鉴定，获得可重组表达r-AoPhytase的重组工程菌株。
所述引物P1的核苷酸序列如SEQ ID No：4所示，所述引物P2的核苷酸序列如SEQID No：5所示。
所述表达质粒载体选自pPICZαA、pPICZαB、pPICZαC、pPIC6αA、pPIC6αB、pPIC6αC、pPICZA、pPICZB、pPICZC、pPIC9K或pPIC3.5K等。
所述表达宿主选自Pichia pastoris SMD1168(H)、SMD1165、SMD1163、GS115、X-33、MP-36、MC100-3或KM71(H)等。
所述表达载体优选为pPICZαA-Aophytase，所述表达宿主细胞优选为毕赤酵母GS115。
以所述重组表达菌株制备β螺旋桨型重组植酸酶r-AoPhytase的方法，包括如下步骤：
步骤1：将所述重组表达菌株以1:1000接种至10mL-1L BMGY培养基中，220rpm摇床30℃培养约48h，测得OD600＝2-8；
步骤2：将培养物以1500rpm离心10min，弃去上清，利用100mL-5L BMMY培养基，重悬沉淀，测得OD600＝0.4-4.0，220rpm摇床30℃诱导72h；
步骤3：将上述培养液于4℃，8000rpm离心10min，收集上清，制备得粗酶液；
步骤4：在上述粗酶液中加入5mL Ni NTA Beads 6FF，4℃温孵2h，将温孵产物加入空柱管，收集流穿液。
步骤5：利用20mM咪唑缓冲液清洗填料，洗脱杂质；然后用40-500mmol/L咪唑缓冲液分级洗脱目的蛋白，获得纯化的r-AoPhytase蛋白。
下面以表达质粒载体pPICZαA、表达宿主Pichia pastoris GS115为例，对真菌β螺旋桨型重组植酸酶r-AoPhytase的制备方法进行说明，具体包括如下步骤：
步骤1：根据AoPhytase氨基酸序列SEQ ID NO.1和核苷酸序列SEQ ID NO.2(Genbank accession NO.22896793)，针对巴斯德毕赤酵母表达***进行密码子优化，获得AoPhytase基因序列(序列表中的SEQ ID NO.3)，或与SEQ ID NO.2或SEQ ID NO.3核苷酸序列80％以上同源性的突变序列；
步骤2：EcoRⅠ和SalⅠ双酶切上述核苷酸片段，16℃下与相同双酶切的载体pPICZαA过夜连接，并将连接产物转化感受态DH5α细胞，获得表达载体pPICZαA-Aophytase；
步骤3：采用SacⅠ酶使表达载体pPICZαA-Aophytase线性化；
步骤4：将线性化的质粒pPICZαA-Aophytase电转化毕赤酵母GS115；
步骤5：在转化平板YPDSZ上，利用引物P1和P2进行菌落PCR鉴定，获得阳性转化子，其引物P1的核苷酸序列如SEQ ID NO.4所示，引物P1的核苷酸序列如SEQ ID NO.5所示；
步骤6：在毕赤酵母中进行重组蛋白的诱导表达：接种阳性克隆至1mL BMGY培养基中，于30℃培养，至OD600为2-6(约48h)；将上述培养物1500rpm离心10min，弃上清，加入2mLBMMY培养基重悬菌体沉淀，于30℃培养72h，进行诱导表达；
步骤7：取1mL诱导菌体悬液，离心后将上清移至超滤管中浓缩至40μl，进行SDS-PAGE分析。
进一步地，真菌β螺旋桨型植酸酶r-AoPhytase的重组表达的放大培养和分离纯化过程，包括如下步骤：
1、将重组毕赤酵母工程菌以1:1000接种至10mL-1L BMGY培养基中，优选50mLBMGY培养基，220rpm摇床30℃培养约48h，测得OD600＝2-8，优选OD600＝4.6；
2、将培养物以1500rpm离心10min，弃去上清，利用100mL-5L BMMY培养基，优选200mL BMMY培养基，重悬沉淀，测得OD600＝0.4-4.0，优选OD600＝1.1，220rpm摇床30℃诱导72h；
3、将上述培养液于4℃，8000rpm离心10min，收集上清，制备得粗酶液；
4、在上述粗酶液中加入5mL Ni NTA Beads 6FF，4℃温孵2h，将温孵产物加入空柱管，收集流穿液。
5、利用20mM咪唑缓冲液(10mM Na2HPO4，2mM KH2PO4，0.8％NaCl，0.02％KCl，5％Glycerol，20mM Imidazole，pH6.0)清洗填料，洗脱杂质；
6、用40-500mmol/L咪唑缓冲液分级洗脱目的蛋白，优选地用100mM或250mM咪唑洗脱液(含10mM Na2HPO4，2mM KH2PO4，0.8％NaCl，0.02％KCl，5％Glycerol，pH6.0)洗脱目的蛋白，获得纯化的r-AoPhytase蛋白。
7、将100mM或250mM咪唑洗脱液制备的纯化的r-AoPhytase蛋白，或合并100与250mM咪唑洗脱液洗脱下的纯化r-AoPhytase蛋白样品，经SDS-PAGE检测以及WesternBlotting分析，确定其重组蛋白纯度，并采用BCA法测定蛋白浓度。
本发明β螺旋桨型重组植酸酶r-AoPhytase的应用，是将其作为植酸酶制剂用于饲料的加工和生产过程中，可以显著促进大豆粉中无机磷的释放，显著促进硬质小麦粉和龙爪粟粉中Ca、Fe、Zn和Mg等矿物质离子的可溶性释放。
所述β螺旋桨型重组植酸酶r-AoPhytase的最适pH为7.5，最适温度为50℃；Ca2+，Na+，K+对其酶活性有增强作用；Li+，Mg2+对酶活性有轻微的抑制或几乎没有影响；Fe3+，Ni2+，Cu2+，Zn2+，Mn2+，SDS和EDTA对酶活性有不同程度的强抑制作用，其中EDTA、Zn2+、Mn2+对其活性的抑制作用最显著。
所述β螺旋桨型重组植酸酶r-AoPhytase的动力学参数如下：Vmax＝40-200μmolmin-1mg-1，优选地Vmax＝72.46μmol min-1mg-1；Km＝0.1-2.5mM，优选地Km＝1.015mM；Kcat＝2-50s-1，优选地Kcat＝13.57s-1。
所述β螺旋桨型重组植酸酶r-AoPhytase的比酶活远远高于其他细菌，例如芽孢杆菌、紫色杆菌、土地杆菌，沙雷氏菌等来源的BPP型植酸酶，比酶活性在50U-100U/mg，优选为74.71U/mg。
与现有技术相比，本发明的有益效果体现在：
1、本发明提供的真菌β螺旋桨型重组植酸酶r-AoPhytase比酶活为74.71U/mg，远远高于细菌来源，例如芽孢杆菌、紫色杆菌、土地杆菌，沙雷氏菌等来源的BPP型植酸酶的比酶活性。
2、本发明通过密码子优化的方法，克服了毕赤酵母的密码子偏爱性，使重组蛋白获得较高分泌表达；重组蛋白分泌在培养液中，降低了下游分离纯化的成本。
3、本发明提供的真菌β螺旋桨型重组植酸酶r-AoPhytase及其植酸酶制剂，其最适pH在7.5左右，更接近中性；其最适温度较高，为50℃；可以有效应用与在饲料工业加工与生产工艺中，有效促进饲料样品中的对无机磷、可溶性矿物质的释放。
附图说明
图1是pPICZαA-phytase质粒及其酶切线性化结果。将构建好的质粒pPICZαA-phytase用SacI酶切后，利用琼脂糖凝胶电泳检测线性化效果。M：DL5000 DNA Marker；1：pPICZαA-phytase质粒；2：线性化质粒pPICZαA-phytase在凝胶上的位置在5000bp以上，与目的片段的5767bp大小相符。
图2是AoPhytase在毕赤酵母基因组中的整合及其诱导表达。(A)线性化的pPICZαA-phytase在酵母基因组中的整合示意图；(B)在博来霉素筛选平板上，对阳性转化子的PCR鉴定；(C)阳性转化子诱导表达后，发酵液里重组蛋白的SDS-PAGE分析。
图3是Ni NTA agarose亲和层析纯化r-AoPhytase及其SDS-PAGE和WesternBlotting分析。(A)不同浓度的咪唑洗脱液经Ni NTA agrose亲和层析柱获得r-AoPhytase组分；(B)纯化r-AoPhytase的SDS-PAGE分析结果；(C)纯化r-AoPhytase的WesternBlotting分析结果。
图4是定磷标准曲线。在1ml磷标准测定用液加入1ml AMES显色液，50℃水浴20min后，冷却至室温，在700nm处测定OD值。以OD值为纵坐标，磷浓度为横坐标绘制OD-磷浓度标准曲线。经线性拟合和回归，获得线性方程为y＝6.2417x+0.0723，R2＝0.9971。
图5是pH值、温度、表面活性剂、螯合剂及不同金属离子对重组AoPhytase酶活的影响。(A)pH值对重组AoPhytase酶活；(B)温度对重组AoPhytase酶活；(C)表面活性剂、螯合剂及不同金属离子对重组AoPhytase酶活的影响。
图6是重组AoPhytase酶动力学参数。(A)在酶含量一定时，随着底物植酸浓度的增加，其无机磷的释放量也逐渐增加；(B)以反应速度V的倒数对底物浓度[S]的倒数进行L-B作图，获得拟合曲线为y＝0.014+0.0138。由此计算，得Aophytase的Vmax＝72.46μmol min- 1mg-1，Km＝1.015mM Kcat＝13.57s-1。
图7是重组AoPhytase对饲料样品中无机磷或矿物质释放的影响。(A)随着重组植酸酶量的增加，大豆粉中无机磷的释放量会相应增多；(B)1g硬质小麦粉或龙爪粟粉中总矿物质的量；(C)重组植酸酶的加入促进硬质小麦粉中矿物质显著释放；(D)重组植酸酶的加入促进龙爪粟粉中矿物质显著释放。
具体实施方式
下面通过实施例来对本发明的技术方案进一步解释，但本发明的保护范围不受实施例的任何形式上的限制。实施例中的实施方法，如无特别说明，均为常规方法。
实施例1：AoPhytase密码子优化及其在毕赤酵母中的重组表达
根据AoPhytase氨基酸序列SEQ ID NO.1和核苷酸序列SEQ ID NO.2(GenbankaccessionNO.22896793)，针对巴斯德毕赤酵母表达***进行密码子优化，获得AoPhytase基因序列(序列表中的SEQ ID NO.3)。然后利用EcoRⅠ和SalⅠ双酶切优化的基因片段，将其与相同双酶切的载体pPICZαA于16℃过夜连接，并将连接产物转化感受态DH5α细胞，在含有Zeocin抗性的LB固体培养基平板上筛选阳性转化子。如图1所示，获得表达载体pPICZαA-Aophytase。
取20μg质粒pPICZαA-Aophytase，加入2μl限制性内切酶Fast DigestSacⅠ酶和10×FastDigest Buffer50μl，补加ddH2O至500μl，37℃反应过夜，使其线性化。酶切反应结束后，在反应体系中添加500μl异丙醇剧烈振荡，12000rpm离心10min，弃去上清，沉淀加1mL75％乙醇颠倒混匀。12000rpm离心10min后，弃去上清，沉淀加20μl无菌水重溶线性化质粒。利用琼脂糖凝胶电泳检测线性化效果(图1)。
将线性化的质粒pPICZαA-Aophytase转化毕赤酵母GS115，利用单一重组(singlecrossover)机制将Aophytase基因及其筛选基因ble表达盒整合到毕赤酵母GS115基因组上AOX 1基因的5’启动子区(5’PAOX1)(图2A)。取线性化后的10μL质粒加入到100μL酵母感受态细胞GS115中，混匀后转至0.2cm冰预冷的电转化杯中。冰浴5min后，设定电压1.5kV；电容25μF；电阻200Ω，进行电击转化。立即转入500μL 1M山梨醇溶液中，室温静置20min，再加入500μL YPD培养基，30℃，220rpm振荡孵育1h。取100μL上述溶液涂布于YPDSZ平板，30℃倒置培养约2-3天。通过菌落PCR鉴定，利用引物5'AOX
(5'-GACTGGTTCCAATTGACAAGC-3')和3'AOX(5'-GCAAATGGCATTCTGACATCC-3')从YPDSZ平板上挑选20个克隆进行PCR验证阳性克隆。PCR反应条件如下：95℃预变性3min；95℃变性20s，55℃退火20s，72℃延伸1min，共30个循环；最后72℃延伸10min。如图2B所示，在博来霉素筛选平板上，挑选20个转化子克隆进行PCR验证，结果均为阳性转化子。
最后在毕赤酵母中进行重组蛋白的诱导表达。挑选10个阳性克隆接种至1mLBMGY培养基中，于30℃培养，至OD600约为2-6(约48h)。将上述培养物1500rpm离心10min，弃上清，加入2mL BMMY培养基重悬菌体沉淀，于30℃培养72h，进行诱导表达。诱导结束后，取1mL诱导菌体悬液，12000rpm离心5min后，将上清取至超滤管中浓缩至40μl。将浓缩后的上清取至1.5mL离心管，并在管内添加Loading Buffer，沸水浴10min。最终将制备的样品进行SDS-PAGE分析。如图2C所示，随机挑选10个阳性转化子，用甲醇诱导表达72小时，取发酵上清液，浓缩后经SDS-PAGE分析后，发现在66.2-116KD位置有目的条带，说明工程菌经甲醇可以有效诱导表达重组植酸酶，并将其分泌到培养基中。
实施例2：重组AoPhytase的纯化
1、粗酶液的制备：以1:1000接种重组的毕赤酵母工程菌至50mL BMGY培养基中，220rpm30℃培养约48h，测得OD600＝4.6。然后将培养物以1500rpm离心10min，弃去上清，利用200mL BMMY培养基重悬沉淀，测得OD600＝1.1，220rpm，30℃诱导72h。将上述培养液于4℃，8000rpm离心10min，上清即为粗酶液。
2、植酸酶的分离纯化：在上清中加入5mL Ni NTA Beads 6FF，4℃温孵2h。将温孵产物加入空柱管，收集流出液。利用20mM咪唑缓冲液(10mM Na2HPO4，2mM KH2PO4，0.8％NaCl，0.02％KCl，5％Glycerol，20mM Imidazole，pH6.0)清洗填料，收集清洗液。再用100和250mM咪唑缓冲液洗脱，收集洗脱液。最后将洗脱液全部以1:100的比例透析至无咪唑的缓冲液(10mM Na2HPO4，2mM KH2PO4，0.8％NaCl，0.02％KCl，5％Glycerol，pH6.0)。如图3A所示，在100-250mM咪唑洗脱液的洗脱下，可以获得电泳纯的植酸酶蛋白。图3B-C分别显示了SDS-PAGE和Western Blotting分析结果，表明重组Aophytase达到了SDS-PAGE纯度，分子量为95-116kD之间。
实施例3：重组AoPhytase酶活分析
1、定磷标准曲线的绘制：量取0、2、4、6、8和10ml的磷标准储备液于100ml的容量瓶中，稀释至刻度，制成磷含量为0、0.1、0.2、0.3、0.4和0.5mmol/L的标准曲线测定用液。取六个15ml离心管，分别加入上述浓度的标准测定用液1ml后，再加入1ml AMES显色液，50℃水浴20min后，冷却至室温，在700nm处测定OD值，绘制OD-磷浓度标准曲线，如图4所示。
2、酶活测定：将浓度为0.088mg/ml纯酶溶液稀释10倍后，取100μl稀释酶液与900μl底物溶液(2mM植酸钠溶解在100mM Tris-HCl缓冲液中，pH 7.0)混合，对照组加入1ml10％三氯乙酸，在37℃下温育30分钟。在反应样品加入1ml 10％三氯乙酸终止反应，对照组样品加入950μl底物溶液。冷却后加入2mLAMES显色液，于A700测量吸光度。实验重复三次。植酸酶酶活性单位(U)定义为：在37℃条件下，每分钟从植酸钠中释放出1μmol无机磷所需要的酶量为一个酶活单位。酶活性按如下公式计算：
酶活性＝[(OD-OD0)×N]/(K×T)；比酶活按如下公式计算：比酶活＝酶活性(U)/加入测试体系中的酶量(mg)。
注：酶活性单位(U)为μmol P/min；
OD：测定样品在700nm下的吸光度；OD0：对照样品在700nm下的吸光度；
N，样品的稀释倍数；K，标准曲线斜率；T，酶促反应时间。
依据上面的公式计算，测得的重组植酸酶的比酶活为74.71U/mg(表1)。表1也列出了其他研究组报道的不同细菌来源的BPP型重组植酸酶比酶活性。通过比较，我们发现真菌来源的重组AoPhytase的比酶活远远高于其他细菌，例如芽孢杆菌、紫色杆菌、土地杆菌，沙雷氏菌等来源的BPP型植酸酶。
表1重组AoPhytase的比酶活及其与不同重组植酸酶(BPP型)比活性的比较
实施例4：pH值、温度、金属离子、表面活性剂与络合剂对重组AoPhytase酶活的影响
1、最适pH测定：采用下列pH缓冲液：甘氨酸-HCl(100mM，pH2.0-3.5)，乙酸钠-乙酸(100mM，pH3.5-6.5)，Tris-HCl(100mM，pH6.5-9.0)和甘氨酸-NaOH(100mM，pH9.0-10)，制备2mM植酸钠溶液，作为底物溶液。取不同pH的底物溶液4份，以一份先加10％的三氯乙酸后加酶液的混合液作为对照，按上述酶活测定方法进行酶活性测定，共做三次重复，测定其在A700处吸光值的变化，以pH与酶的相对活性作图，求最适pH。如图5A所示，在PH5-8之间表现出了明显的植酸酶活性，其最适PH为7.5，这一性质与BPP型植酸酶为碱性植酸酶相符。
2、最适温度测定：将植酸钠溶解在pH 7.0的100mM Tris-HCl缓冲液中，制备2mM植酸钠作为底物溶液。从20至70℃范围内，通过以10℃间隔设置温度变化，测定其在700nm处吸光值的变化，按上述酶活测定方法计算植酸酶活性。以温度与酶的相对活性作图，求得最适温度。由图5B可知在20-80℃范围内，重组植酸酶活性的变化，可知其最适温度为50℃。
3、不同金属离子、表面活性剂与络合剂对酶活的影响：利用100mM Tris-HCl(pH7.0)缓冲液制备2mM植酸钠底物溶液，分别在其中加入1mM Ca2+，Na+，K+，Li+，Mg2+，Fe3+，Ni2+，Cu2+，Zn2+，Mn2+，SDS和EDTA。然后将100μl稀释10倍后的酶溶液与900μl底物溶液混合，测定植酸酶活性，分析金属离子、表面活性剂与化学络合剂对纯化酶活性的影响。以空白Tris-HCl缓冲液作对照样品。将混合后的样品在37℃下温育30分钟。待测样品加入1ml10％三氯乙酸终止反应，对照组样品加入950μl底物溶液。冷却后加入2mL显色液，在A700测量吸光度。每组实验重复三次。图5C显示了表面活性剂SDS、螯合剂EDTA及不同金属离子对重组AoPhytase酶活的影响。我们发现Ca2+，Na+，K+对酶活性有轻微的增强，Li+，Mg2+对酶活性有轻微的抑制或几乎没有影响，Fe3+，Ni2+，Cu2+，Zn2+，Mn2+，SDS和EDTA对酶活性有不同程度的强抑制作用。其中EDTA，Zn2+，Mn2+对其活性的抑制作用最明显，抑制率分别达到了47.7％,68.2％,97.7％。
实施例5：组AoPytase动力学参数的测定
在最佳反应条件下(最佳pH和最适温度，pH7.5，50℃)，测定重组植酸酶比酶活及其动力学参数。配制0.125、0.25、0.5、1.0、2.0和4.0mM等不同浓度的植酸钠反应液(100mMTris-HCl,pH7.5)，分别将100μl酶溶液(0.5U)与上述底物溶液900μl混合。在50℃下温育10分钟后，加入1ml 10％三氯乙酸终止反应。对照组是将100μl酶溶液后，再加入1ml10％三氯乙酸，50℃下温育10分钟后，最终加入950μl底物溶液。最终将样品冷却后，加入2mL显色液，在A700测量吸光度从而定量反应释放的无机磷，实验重复三次。使用Lineweaver-Burk法作图计算动力学常数。如图6A所示，在酶含量一定时，随着底物植酸浓度的增加，其无机磷的释放量也逐渐增加。如图6B所示，以反应速度V的倒数为纵坐标，底物浓度[S]的倒数为横坐标，进行L-B作图，获得拟合曲线为y＝0.014+0.0138。令x＝0，则1/Vmax＝0.0138，Vmax＝72.46μmol min-1mg-1，令y＝0，则-1/Km＝-0.9857，Km＝1.015mM；根据植酸酶的理论分子量84.195kD，计算得出Kcat＝13.57s-1。
实施例6：重组AoPytase促进大豆粉中植酸磷的水解
测定加入不同酶量下(0.5U，1U，1.5U，2U，2.5U)大豆粉中磷的释放量。将1g大豆粉加入50ml离心管中，然后加入9ml缓冲液(100mM Tris-HCl，pH 7.5)，共15管。将离心管在37℃下振荡(150rpm)孵育60分钟，然后分别在大豆粉溶液加入1ml酶制剂，使其终酶量为0.5-2.5U/g大豆粉。采用10ml15％三氯乙酸作为对照，每份样品重复3管。反应结束后，在待测样品管中加入10ml 15％三氯乙酸终止反应，在对照管中加入1ml酶制剂。待冷却至常温后，5000rpm离心反应液5min。各管取2ml上清，加入2ml显色液，在A700测量吸光度。如图7A所示，重组植酸酶可以显著促进大豆粉中无机磷的释放。随着酶量的增加，磷的释放量会相应增多。
实施例7：重组AoPytase促进硬质小麦粉和龙爪粟粉中矿物质的释放
1、矿物质总量测定：分别称取硬质小麦粉和龙爪稷粉各1g，溶解在5mlddH2O中，将混合物在50℃孵育2h。往各管中加入6ml酸混合液(硝酸：硫酸：高氯酸的体积比是10：1：4)，在通风橱内200℃干燥，干燥后的样品在马弗炉内以450℃处理至白色粉末，将粉末溶于1ml35％浓盐酸中，用ddH2O定容至10ml。将样品过滤并做100倍稀释处理。每个样品重复三次测试。图7B显示了硬质小麦粉和龙爪稷粉中矿物质的总含量。
2、释放矿物质可溶率测定：分别称取硬质小麦粉和龙爪稷粉各1g，重悬在6ml缓冲液(140mM NaCl，5mM KCl)中，往其中加入植酸酶(20U)，用ddH2O将溶液体积补齐至10ml，50℃孵育2h，5000rpm离心20min。取上清稀释50倍后，进行ICP-MS测定。以不加植酸酶的样品作为对照，每个样品重复三次。如图7C所示，重组植酸酶能显著促进硬质小麦粉中Ca、Fe、Zn和Mg离子的释放，其释放率与对照比较，分别增加13.2％，4.3％，10.5％，和20.5％。如图7D所示，重组植酸酶也能显著促进龙爪粟粉中Ca、Fe、Zn和Mg离子的释放，其释放率与对照比较，分别增加32.1％，1.8％，4.3％，和1.6％。
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Singh,B.,&Satyanarayana,T.(2015).Fungal phytases:characteristics andamelioration of nutritional quality and growth of non-ruminants.J AnimPhysiol Anim Nutr(Berl),99(4),646-660.doi:10.1111/jpn.12236
Ullah,A.H.,&Gibson,D.M.(1988).Purification and characterization ofacid phosphatase fromcotyledons of germinating soybean seeds.Archives ofBiochemistry&Biophysics,260(2),503-513.Viader-Salvadó,J.M.,A Gallegos-López,J.,Gerardo
J.,Castillo-Galván,M.,Rojo,A.,&Guerrero-Olazaran,M.(2010).Design of Thermostable Beta-Propeller Phytases withActivity over aBroad Range of pHs and Their Overproduction by Pichia pastoris(Vol.76).Yao,M.-Z.,Lu,W.-L.,Chen,T.-G.,Wang,W.,Fu,Y.-J.,Yang,B.-S.,&Liang,A.-H.(2013).Effect of metals ions on thermostable alkaline phytase from Bacillussubtilis YCJS isolated from soybean rhizosphere soil.Annals of Microbiology,64(3),1123-1131.doi:10.1007/s13213-013-0751-5
Zhang,R.,Yang,P.,Huang,H.,Shi,P.,Yuan,T.,&Yao,B.(2011).Two types ofphytases(histidine acid phytase and beta-propeller phytase)in Serratiasp.TN49 from the gut of Batocera horsfieldi(coleoptera)larvae.Curr Microbiol,63(5),408-415.doi:10.1007/s00284-011-9995-0
Zhang,R.,Yang,P.,Huang,H.,Yuan,T.,Shi,P.,Meng,K.,&Yao,B.(2011).Molecular and biochemical characterization of a new alkaline beta-propellerphytase from the insect symbiotic bacterium Janthinobacterium sp.TN115.ApplMicrobiol Biotechnol,92(2),317-325.doi:10.1007/s00253-011-3309-0
序列表
SEQ ID NO.1：
MKSSHNLSGLILLCLMGYIHPTSAADKFSITLPITARTSSVESDSAAVYYPSKSKYSPIFIGNDGSAETGGFHVYELYGKRSDALVKELGAYKTGRSKLVEVVYGEDRDFVVTLSMSDGMFRVFEVDGKNGVRGLKAEKLVRGDFSAMCTWKSKVGEYVYVLGKRWGYRFLVREKKGGRGVEVVQTQEFGIPIEPNSCTVSPEGKVFLAGDSGKVFSFLAVDETAAPKIVEVGELGGGDEVKGLKIYHGKKDTYLLVGLEDGIEVFDIKKLGSSLGKIQFDDEELEVGDFAVHQTSAKGYEDGSIVFAGEDGEGKFFGVSSLTPLFKALGKGKLNTKYDPRDCTDNHARPKKCANLSDCNGYGYCPKDSRDKKATCDCFPGLTGKTCNKITCPSNCTSPSHGTCTGPNICTCIPPFTGENCATLAVPARYETEESGGADGDDPAIWIHPTDKTKSRIITTVKSEVGSGLGVYDLKGKRTGGVSGGEPNNVDVLYGVEFAGRKVDLAVAACRADDTICIYEITPTGDLVTIPGGVQPLPPAVKELEKKFKVYGSCVYHSPKTGAYHIFVNSKSSLYLQFQLSATTDGKLNTTLVRHFYAGNRGQVEGCVVDDENSSLFLGEEPYGIWSYDAEPDQPAVGTLVDNTVVDGGKLHADVEGVTLVYGKTKKEGYIIVSCQGYSEYNIYQRYPPHEFVMSFSIPDNKEKGVDRVTNTDGITAVGANLGKEWPYGMVVVHDDVNEAAGGGVRADATFKIVGLGDILGNKAVKELGLLKGVDENWDPRK
SEQ ID NO.2：
SEQ ID NO.3：
gaattcGCTGACAAGTTCTCCATCACTTTGCCAATTACTGCCAGAACCTCTTCCGTTGAATCTGACTCTGCTGCCGTTTACTACCCATCCAAGTCTAAGTACTCCCCAATCTTCATCGGTAACGACGGTTCTGCTGAAACTGGTGGTTTTCACGTCTACGAGTTGTACGGTAAGAGATCCGACGCCTTGGTCAAAGAATTGGGTGCTTACAAGACCGGTAGATCCAAGTTGGTTGAGGTTGTTTACGGTGAGGACAGAGACTTCGTTGTCACTTTGTCTATGTCCGACGGTATGTTCAGAGTGTTCGAGGTCGATGGTAAGAACGGTGTCAGAGGTTTGAAGGCCGAGAAGTTGGTCAGAGGTGATTTCTCCGCTATGTGTACCTGGAAGTCTAAGGTTGGTGAGTACGTCTACGTCCTGGGTAAAAGATGGGGTTACAGATTCTTGGTCCGTGAGAAGAAAGGTGGTAGAGGTGTTGAGGTCGTTCAGACTCAAGAGTTCGGTATTCCAATCGAGCCAAACTCCTGTACTGTTTCCCCAGAAGGTAAGGTTTTCTTGGCTGGTGACTCCGGTAAGGTGTTTTCTTTTTTGGCCGTTGACGAGACTGCTGCCCCAAAGATAGTTGAAGTTGGTGAACTTGGTGGTGGTGACGAGGTTAAGGGTTTGAAGATCTACCACGGTAAGAAGGACACCTACTTGTTGGTTGGTTTGGAGGACGGTATCGAGGTGTTCGACATTAAGAAGTTGGGATCCTCCTTGGGTAAGATCCAATTCGACGACGAAGAGTTGGAAGTCGGTGATTTCGCTGTTCATCAGACTTCCGCTAAGGGTTACGAAGATGGTTCCATCGTTTTCGCTGGTGAAGATGGTGAGGGAAAGTTCTTCGGTGTTTCCTCCTTGACTCCTCTGTTCAAGGCTCTTGGTAAGGGTAAGCTGAACACCAAGTACGACCCAAGAGACTGTACTGACAACCACGCTAGACCAAAGAAGTGTGCTAACTTGTCCGACTGCAACGGTTACGGTTACTGTCCAAAGGACTCCAGAGACAAGAAGGCTACTTGCGACTGTTTTCCAGGTTTGACCGGTAAGACCTGCAACAAGATTACCTGTCCATCCAACTGCACTTCCCCATCTCATGGTACTTGTACCGGTCCAAACATCTGCACTTGTATCCCACCATTCACTGGTGAGAACTGTGCTACTTTGGCTGTTCCAGCTAGATACGAGACTGAAGAATCTGGTGGTGCTGATGGTGATGACCCAGCTATTTGGATTCACCCAACTGACAAGACCAAGTCCAGAATCATCACCACCGTTAAGTCCGAAGTTGGTTCCGGTTTGGGTGTCTACGATCTGAAGGGTAAGAGAACCGGTGGTGTTTCAGGTGGTGAACCTAACAACGTTGACGTCTTGTACGGTGTTGAGTTCGCCGGTAGAAAGGTTGACTTGGCTGTTGCTGCTTGTAGAGCTGACGACACCATCTGTATCTACGAGATCACTCCAACCGGTGACTTGGTTACTATTCCAGGTGGTGTTCAACCATTGCCACCAGCTGTCAAAGAGCTGGAAAAGAAGTTCAAGGTCTACGGTTCCTGCGTCTACCACTCTCCAAAGACTGGTGCTTACCACATCTTCGTCAACTCCAAGTCCTCCTTGTACTTGCAGTTCCAGTTGTCCGCTACTACTGACGGAAAGTTGAACACTACCCTGGTCAGACACTTCTACGCTGGTAACAGAGGTCAAGTTGAGGGTTGTGTTGTTGACGACGAAAACTCTTCCCTGTTCTTGGGTGAAGAACCATACGGTATTTGGTCCTACGATGCTGAACCAGACCAACCTGCTGTTGGTACTTTGGTTGACAACACTGTCGTTGACGGTGGTAAGTTGCACGCTGATGTTGAGGGTGTTACCTTGGTTTACGGAAAGACCAAGAAAGAGGGTTACATCATCGTTTCTTGCCAGGGTTACTCCGAGTACAACATCTACCAAAGATACCCACCACACGAGTTCGTCATGTCCTTCTCTATCCCTGACAACAAAGAGAAGGGCGTTGACAGAGTTACCAACACTGACGGTATTACTGCCGTTGGTGCCAACTTGGGTAAAGAATGGCCATACGGAATGGTTGTTGTCCACGACGATGTTAACGAAGCTGCTGGTGGTGGCGTTAGAGCTGATGCTACTTTTAAGATTGTCGGTCTGGGTGACATCTTGGGTAACAAGGCTGTGAAAGAGTTGGGTTTGCTGAAGGGTGTTGATGAGAACTGGGACCCAAGAAAGgtcgacgaattc:EcoRI；gtcgac:SacI
SEQ ID NO.4：
5'-GACTGGTTCCAATTGACAAGC-3'
SEQ ID NO.5：
5'-GCAAATGGCATTCTGACATCC-3'
SEQUENCE LISTING
<110> 安徽大学
<120> 一种真菌来源β螺旋桨型重组植酸酶r-AoPhytase及其制备方法和应用
<130>
<160> 5
<170> PatentIn version 3.1
<210> 1
<211> 782
<212> PRT
<213> Arthrobotrys oligospora
<400> 1
Met Lys Ser Ser His Asn Leu Ser Gly Leu Ile Leu Leu Cys Leu Met
1 5 10 15
Gly Tyr Ile His Pro Thr Ser Ala Ala Asp Lys Phe Ser Ile Thr Leu
20 25 30
Pro Ile Thr Ala Arg Thr Ser Ser Val Glu Ser Asp Ser Ala Ala Val
35 40 45
Tyr Tyr Pro Ser Lys Ser Lys Tyr Ser Pro Ile Phe Ile Gly Asn Asp
50 55 60
Gly Ser Ala Glu Thr Gly Gly Phe His Val Tyr Glu Leu Tyr Gly Lys
65 70 75 80
Arg Ser Asp Ala Leu Val Lys Glu Leu Gly Ala Tyr Lys Thr Gly Arg
85 90 95
Ser Lys Leu Val Glu Val Val Tyr Gly Glu Asp Arg Asp Phe Val Val
100 105 110
Thr Leu Ser Met Ser Asp Gly Met Phe Arg Val Phe Glu Val Asp Gly
115 120 125
Lys Asn Gly Val Arg Gly Leu Lys Ala Glu Lys Leu Val Arg Gly Asp
130 135 140
Phe Ser Ala Met Cys Thr Trp Lys Ser Lys Val Gly Glu Tyr Val Tyr
145 150 155 160
Val Leu Gly Lys Arg Trp Gly Tyr Arg Phe Leu Val Arg Glu Lys Lys
165 170 175
Gly Gly Arg Gly Val Glu Val Val Gln Thr Gln Glu Phe Gly Ile Pro
180 185 190
Ile Glu Pro Asn Ser Cys Thr Val Ser Pro Glu Gly Lys Val Phe Leu
195 200 205
Ala Gly Asp Ser Gly Lys Val Phe Ser Phe Leu Ala Val Asp Glu Thr
210 215 220
Ala Ala Pro Lys Ile Val Glu Val Gly Glu Leu Gly Gly Gly Asp Glu
225 230 235 240
Val Lys Gly Leu Lys Ile Tyr His Gly Lys Lys Asp Thr Tyr Leu Leu
245 250 255
Val Gly Leu Glu Asp Gly Ile Glu Val Phe Asp Ile Lys Lys Leu Gly
260 265 270
Ser Ser Leu Gly Lys Ile Gln Phe Asp Asp Glu Glu Leu Glu Val Gly
275 280 285
Asp Phe Ala Val His Gln Thr Ser Ala Lys Gly Tyr Glu Asp Gly Ser
290 295 300
Ile Val Phe Ala Gly Glu Asp Gly Glu Gly Lys Phe Phe Gly Val Ser
305 310 315 320
Ser Leu Thr Pro Leu Phe Lys Ala Leu Gly Lys Gly Lys Leu Asn Thr
325 330 335
Lys Tyr Asp Pro Arg Asp Cys Thr Asp Asn His Ala Arg Pro Lys Lys
340 345 350
Cys Ala Asn Leu Ser Asp Cys Asn Gly Tyr Gly Tyr Cys Pro Lys Asp
355 360 365
Ser Arg Asp Lys Lys Ala Thr Cys Asp Cys Phe Pro Gly Leu Thr Gly
370 375 380
Lys Thr Cys Asn Lys Ile Thr Cys Pro Ser Asn Cys Thr Ser Pro Ser
385 390 395 400
His Gly Thr Cys Thr Gly Pro Asn Ile Cys Thr Cys Ile Pro Pro Phe
405 410 415
Thr Gly Glu Asn Cys Ala Thr Leu Ala Val Pro Ala Arg Tyr Glu Thr
420 425 430
Glu Glu Ser Gly Gly Ala Asp Gly Asp Asp Pro Ala Ile Trp Ile His
435 440 445
Pro Thr Asp Lys Thr Lys Ser Arg Ile Ile Thr Thr Val Lys Ser Glu
450 455 460
Val Gly Ser Gly Leu Gly Val Tyr Asp Leu Lys Gly Lys Arg Thr Gly
465 470 475 480
Gly Val Ser Gly Gly Glu Pro Asn Asn Val Asp Val Leu Tyr Gly Val
485 490 495
Glu Phe Ala Gly Arg Lys Val Asp Leu Ala Val Ala Ala Cys Arg Ala
500 505 510
Asp Asp Thr Ile Cys Ile Tyr Glu Ile Thr Pro Thr Gly Asp Leu Val
515 520 525
Thr Ile Pro Gly Gly Val Gln Pro Leu Pro Pro Ala Val Lys Glu Leu
530 535 540
Glu Lys Lys Phe Lys Val Tyr Gly Ser Cys Val Tyr His Ser Pro Lys
545 550 555 560
Thr Gly Ala Tyr His Ile Phe Val Asn Ser Lys Ser Ser Leu Tyr Leu
565 570 575
Gln Phe Gln Leu Ser Ala Thr Thr Asp Gly Lys Leu Asn Thr Thr Leu
580 585 590
Val Arg His Phe Tyr Ala Gly Asn Arg Gly Gln Val Glu Gly Cys Val
595 600 605
Val Asp Asp Glu Asn Ser Ser Leu Phe Leu Gly Glu Glu Pro Tyr Gly
610 615 620
Ile Trp Ser Tyr Asp Ala Glu Pro Asp Gln Pro Ala Val Gly Thr Leu
625 630 635 640
Val Asp Asn Thr Val Val Asp Gly Gly Lys Leu His Ala Asp Val Glu
645 650 655
Gly Val Thr Leu Val Tyr Gly Lys Thr Lys Lys Glu Gly Tyr Ile Ile
660 665 670
Val Ser Cys Gln Gly Tyr Ser Glu Tyr Asn Ile Tyr Gln Arg Tyr Pro
675 680 685
Pro His Glu Phe Val Met Ser Phe Ser Ile Pro Asp Asn Lys Glu Lys
690 695 700
Gly Val Asp Arg Val Thr Asn Thr Asp Gly Ile Thr Ala Val Gly Ala
705 710 715 720
Asn Leu Gly Lys Glu Trp Pro Tyr Gly Met Val Val Val His Asp Asp
725 730 735
Val Asn Glu Ala Ala Gly Gly Gly Val Arg Ala Asp Ala Thr Phe Lys
740 745 750
Ile Val Gly Leu Gly Asp Ile Leu Gly Asn Lys Ala Val Lys Glu Leu
755 760 765
Gly Leu Leu Lys Gly Val Asp Glu Asn Trp Asp Pro Arg Lys
770 775 780
<210> 2
<211> 2349
<212> DNA
<213> Arthrobotrys oligospora
<400> 2
atgaagtcct cacataacct ctcggggtta atcctccttt gtttgatggg gtacatccat 60
cccaccagcg ctgccgataa attctcaatc acactcccaa tcacagctcg gacttcatcc 120
gtcgaatccg atagcgcagc agtctactac ccttctaaat ccaagtactc accaatattc 180
atcggtaacg acgggtctgc cgaaaccggc ggattccatg tttatgaact atacggcaaa 240
cgaagtgatg ccttggtgaa agagcttgga gcgtacaaaa ctgggaggag taagttggtg 300
gaggtggttt atggtgaaga tagggacttt gtggttacgc tgtccatgtc ggatgggatg 360
tttagggtct ttgaggttga tggaaagaat ggagtgaggg gtcttaaggc ggaaaagctc 420
gttaggggtg atttttcggc gatgtgtacg tggaagagta aggttggaga gtacgtgtat 480
gttttgggga agagatgggg gtataggttc ttggttaggg agaagaaggg cgggagggga 540
gtcgaggttg tccaaacaca agaatttgga attcctattg aaccgaattc gtgtactgtt 600
tcacctgaag ggaaggtatt cttggcgggt gatagtggga aggttttctc cttcttagca 660
gttgacgaaa ctgctgcgcc aaagatcgta gaagttgggg agttgggcgg tggtgatgag 720
gttaagggcc tcaagatata tcatggaaag aaagatactt atcttctcgt tggattggaa 780
gatggaatcg aagtctttga tatcaagaaa ctgggaagtt cattaggaaa gatccagttc 840
gacgatgaag agctcgaggt tggagatttt gcagtacacc agacaagtgc gaagggatat 900
gaggatggga gtattgtttt tgcaggggag gatggggaag ggaagttctt tggagtcagt 960
tctttgactc ctttgtttaa agcattaggg aagggaaagc tgaacacgaa gtacgaccca 1020
cgagactgta ccgacaacca tgcgaggccg aagaagtgcg ccaatctttc tgattgcaat 1080
ggatacggat actgcccgaa agattctcga gataagaagg ccacctgcga ttgtttcccc 1140
gggcttacag gaaagacatg taataaaatt acctgcccat cgaactgtac atccccatcc 1200
catggaactt gtactggtcc aaacatctgt acttgcatcc cacccttcac cggcgaaaac 1260
tgtgcaaccc tagctgttcc agctagatac gaaacagaag agagtggcgg agcagatgga 1320
gatgatcccg ccatatggat tcatccaacc gacaaaacaa agtcgaggat tattacaaca 1380
gtaaagagtg aggttggaag tggacttggg gtatacgatt tgaagggcaa gagaacggga 1440
ggtgttagcg gcggtgaacc gaataatgtg gatgttttgt atggagttga atttgcaggg 1500
aggaaggttg atttggcagt ggcggcttgt agagctgatg atactatctg tatatacgaa 1560
atcaccccca ccggcgattt agtcaccatc ccaggcggcg ttcaacctct cccgccagcc 1620
gtcaaagaac tagagaagaa attcaaagtt tacggctcct gcgtctacca ttctcccaaa 1680
acaggagcat atcatatttt cgtcaactcc aaatcctcgc tgtatcttca attccagctt 1740
tctgcgacta cagacggaaa attgaatact actttggtgc gacactttta tgcaggaaat 1800
agaggacagg tagaaggctg tgtggtggat gacgagaact ctagcctttt ccttggtgaa 1860
gagccgtatg gaatttggag ttatgatgcc gaacctgatc aaccggcggt tggaacacta 1920
gtcgataaca cagtcgttga cggtggaaaa cttcatgccg atgtcgaggg agtcacacta 1980
gtctatggaa agactaaaaa agaaggatac atcatcgttt cctgtcaagg atacagtgaa 2040
tacaatattt accaacgata tccaccccat gagtttgtca tgagctttag cattccggat 2100
aataaagaaa agggcgtgga tagagtcacg aatacagatg ggattactgc tgttggagcg 2160
aacttgggga aggaatggcc ctatggaatg gtggttgtac atgatgatgt taatgaagct 2220
gcgggtggag gggttagagc cgacgccaca tttaagatcg taggattggg ggatattttg 2280
ggcaataagg cagtgaagga actgggtctt ttgaaagggg tggatgagaa ctgggatcct 2340
agaaagtag 2349
<210> 3
<211> 2286
<212> DNA
<213> 人工序列（Artificial sequence)
<400> 3
gaattcgctg acaagttctc catcactttg ccaattactg ccagaacctc ttccgttgaa 60
tctgactctg ctgccgttta ctacccatcc aagtctaagt actccccaat cttcatcggt 120
aacgacggtt ctgctgaaac tggtggtttt cacgtctacg agttgtacgg taagagatcc 180
gacgccttgg tcaaagaatt gggtgcttac aagaccggta gatccaagtt ggttgaggtt 240
gtttacggtg aggacagaga cttcgttgtc actttgtcta tgtccgacgg tatgttcaga 300
gtgttcgagg tcgatggtaa gaacggtgtc agaggtttga aggccgagaa gttggtcaga 360
ggtgatttct ccgctatgtg tacctggaag tctaaggttg gtgagtacgt ctacgtcctg 420
ggtaaaagat ggggttacag attcttggtc cgtgagaaga aaggtggtag aggtgttgag 480
gtcgttcaga ctcaagagtt cggtattcca atcgagccaa actcctgtac tgtttcccca 540
gaaggtaagg ttttcttggc tggtgactcc ggtaaggtgt tttctttttt ggccgttgac 600
gagactgctg ccccaaagat agttgaagtt ggtgaacttg gtggtggtga cgaggttaag 660
ggtttgaaga tctaccacgg taagaaggac acctacttgt tggttggttt ggaggacggt 720
atcgaggtgt tcgacattaa gaagttggga tcctccttgg gtaagatcca attcgacgac 780
gaagagttgg aagtcggtga tttcgctgtt catcagactt ccgctaaggg ttacgaagat 840
ggttccatcg ttttcgctgg tgaagatggt gagggaaagt tcttcggtgt ttcctccttg 900
actcctctgt tcaaggctct tggtaagggt aagctgaaca ccaagtacga cccaagagac 960
tgtactgaca accacgctag accaaagaag tgtgctaact tgtccgactg caacggttac 1020
ggttactgtc caaaggactc cagagacaag aaggctactt gcgactgttt tccaggtttg 1080
accggtaaga cctgcaacaa gattacctgt ccatccaact gcacttcccc atctcatggt 1140
acttgtaccg gtccaaacat ctgcacttgt atcccaccat tcactggtga gaactgtgct 1200
actttggctg ttccagctag atacgagact gaagaatctg gtggtgctga tggtgatgac 1260
ccagctattt ggattcaccc aactgacaag accaagtcca gaatcatcac caccgttaag 1320
tccgaagttg gttccggttt gggtgtctac gatctgaagg gtaagagaac cggtggtgtt 1380
tcaggtggtg aacctaacaa cgttgacgtc ttgtacggtg ttgagttcgc cggtagaaag 1440
gttgacttgg ctgttgctgc ttgtagagct gacgacacca tctgtatcta cgagatcact 1500
ccaaccggtg acttggttac tattccaggt ggtgttcaac cattgccacc agctgtcaaa 1560
gagctggaaa agaagttcaa ggtctacggt tcctgcgtct accactctcc aaagactggt 1620
gcttaccaca tcttcgtcaa ctccaagtcc tccttgtact tgcagttcca gttgtccgct 1680
actactgacg gaaagttgaa cactaccctg gtcagacact tctacgctgg taacagaggt 1740
caagttgagg gttgtgttgt tgacgacgaa aactcttccc tgttcttggg tgaagaacca 1800
tacggtattt ggtcctacga tgctgaacca gaccaacctg ctgttggtac tttggttgac 1860
aacactgtcg ttgacggtgg taagttgcac gctgatgttg agggtgttac cttggtttac 1920
ggaaagacca agaaagaggg ttacatcatc gtttcttgcc agggttactc cgagtacaac 1980
atctaccaaa gatacccacc acacgagttc gtcatgtcct tctctatccc tgacaacaaa 2040
gagaagggcg ttgacagagt taccaacact gacggtatta ctgccgttgg tgccaacttg 2100
ggtaaagaat ggccatacgg aatggttgtt gtccacgacg atgttaacga agctgctggt 2160
ggtggcgtta gagctgatgc tacttttaag attgtcggtc tgggtgacat cttgggtaac 2220
aaggctgtga aagagttggg tttgctgaag ggtgttgatg agaactggga cccaagaaag 2280
gtcgac 2286
<210> 4
<211> 21
<212> DNA
<213> 人工序列（Artificial sequence)
<400> 4
gactggttcc aattgacaag c 21
<210> 5
<211> 21
<212> DNA
<213> 人工序列（Artificial sequence)
<400> 5
gcaaatggca ttctgacatc c 21
Claims (4)
1.一种制备β螺旋桨型重组植酸酶r-AoPhytase的方法，其特征在于包括如下步骤：
步骤1：将重组表达菌株以1:1000v/v接种至10mL-1L BMGY培养基中，220rpm摇床30℃培养48h，测得OD600＝2-8，获得培养物；
步骤2：将所述培养物以1500rpm离心10min，弃去上清，利用100mL-5L BMMY培养基，重悬沉淀，测得OD600＝0.4-4.0，220rpm摇床30℃诱导72h，获得培养液；
步骤3：将所述培养液于4℃，8000rpm离心10min，收集上清，制备得到粗酶液；
步骤4：在所述粗酶液中加入5mL Ni NTA Beads 6FF，4℃温孵2h，将温孵产物加入空柱管，收集流穿液；
步骤5：利用20mM咪唑缓冲液清洗填料，洗脱杂质；然后用40-500mmol/L咪唑缓冲液分级洗脱目的蛋白，获得纯化的r-AoPhytase蛋白；
所述重组表达菌株的保藏编号为CCTCC NO：M 2019775。
2.根据权利要求1所述的一种制备β螺旋桨型重组植酸酶r-AoPhytase的方法，其特征在于，所述重组表达菌株的制备方法为：
首先通过化学合成r-AoPhytase基因，利用EcoRⅠ和SalⅠ双酶切并与表达质粒载体pPICZαA连接，将连接产物转化感受态DH5α细胞，获得表达载体pPICZαA-AoPhytase；然后通过SacⅠ酶使表达载体线性化，并将线性化的质粒电转化表达宿主细胞毕赤酵母GS115，获得可重组表达r-AoPhytase的重组工程菌株，所述r-AoPhytase基因的核苷酸序列如SEQ IDNO:3所示。
3.根据权利要求1或2所述的一种制备β螺旋桨型重组植酸酶r-AoPhytase的方法，其特征在于，所述植酸酶r-AoPhytase的氨基酸序列如SEQ ID NO:1所示。
4.一种β螺旋桨型重组植酸酶r-AoPhytase在制备用于饲料加工和生产的植酸酶制剂中的应用，所述重组植酸酶r-AoPhytase的氨基酸序列如SEQ ID NO:1所示。
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1616659A (zh) * | 2004-10-13 | 2005-05-18 | 江南大学 | 一种耐高温、高比活植酸酶基因及其克隆和表达 |
| CN101063113A (zh) * | 2006-04-30 | 2007-10-31 | 中国农业科学院饲料研究所 | 一种新的植酸酶的克隆和表达 |
| CN101426907A (zh) * | 2006-04-30 | 2009-05-06 | 中国农业科学院饲料研究所 | 一种新的植酸酶的克隆和表达 |
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| CN101063113A (zh) * | 2006-04-30 | 2007-10-31 | 中国农业科学院饲料研究所 | 一种新的植酸酶的克隆和表达 |
| CN101426907A (zh) * | 2006-04-30 | 2009-05-06 | 中国农业科学院饲料研究所 | 一种新的植酸酶的克隆和表达 |
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