CN113943721A - 一种脂肪酶突变体及其应用 - Google Patents
一种脂肪酶突变体及其应用 Download PDFInfo
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- CN113943721A CN113943721A CN202111331999.4A CN202111331999A CN113943721A CN 113943721 A CN113943721 A CN 113943721A CN 202111331999 A CN202111331999 A CN 202111331999A CN 113943721 A CN113943721 A CN 113943721A
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Abstract
本发明属于酶工程技术领域，涉及一种脂肪酶突变体及其应用。所述的突变体在野生型脂肪酶SEQ ID NO.1所示氨基酸序列中突变多个氨基酸位点。突变体酶较野生型脂肪酶水解比活力提高了36.21倍，酯交换比活力提高了47.10倍。使用固定化酶进行生物柴油转化，转化率由野生型的85％提高至99％以上。利用本发明的突变体脂肪酶进行生物柴油的生产，显著降低了酶的发酵成本以及固定化酶制备成本，提高了生物柴油生产的转化率，更适合于工业应用。
Description
技术领域
本发明属于酶工程技术领域，涉及一种脂肪酶突变体及利用其合成生物柴油的应用。
背景技术
全球气候变暖是近年来各个国家都非常关注的问题。化石燃料的燃烧导致大气中二氧化碳大量积累是导致全球气温上升的重要原因之一。因此，迫切需要利用清洁、环保和可再生能源代替化石能源。而生物柴油因其优良的环保特性、较高的安全性和可再生性等成为化石燃料的良好代替品。得到了国家产业政策的大力支持。
目前生物柴油的制备主要有化学法，甘油酯化法和生物酶法。其中生物酶法主要是油料作物如大豆、油菜、棉、棕榈等，野生油料植物和工程微藻等水生植物油脂以及动物油脂、餐饮垃圾油等为原料，在脂肪酶的催化下与醇通过酯交换反应制备。与前两种方法相比具有反应条件温和、醇用量小、无污染等优点。但是在实际应用中发现，脂肪酶的表达量不高，活力较低或转化率较低。在实际生产中需要使用大量的酶，成而造成生产成本高居不下。因此，迫切需要开发一种转化率高、活力高的脂肪酶菌株，以降低生产成本。
本发明以Streptomyces sp.W007来源的脂肪酶为基础，通过基因工程及酶工程技术手段，对野生型脂肪酶(MAS)进行突变，所获得的脂肪酶突变体较MAS比活力显著提高，生物柴油转化率明显提高。显著降低了生物柴油生产的用酶成本，更适用于工业化生产应用。
发明内容
本发明的首要目的是提供一种高效合成生物柴油的脂肪酶突变体。该突变体较野生型的脂肪酶具有更高的比活力，更高的产物转化率。
为实现此目的，在基础的实施方案中，本发明提供一种脂肪酶突变体，是对SEQ IDNO.1所示氨基酸序列进行突变，突变的氨基酸位点包括D89G、V90A、A104V、E232D、V264I中的至少一个。
在一种优选的实施方案中，本发明突变体的突变方式包括以下14种中任一种：
D89G；V90A；A104V；E232D；V264I；A104V和D89G；A104V和V90A；A104V和E232D；A104V和V264I；A104V和V264I和D89G；A104V和V264I和V90A；A104V和V264I和E232D；A104V和V264I和E232D和D89G；A104V和V264I和E232D和V90A。
序列依次如SEQ ID NO.2-15所示。
进一步地，突变方式包括以下5种中的任一种：
A104V；A104V和V264I；A104V和V264I和E232D；A104V和V264I和E232D和D89G；A104V和V264I和E232D和V90A。
本发明的第二个目的是提供编码前述脂肪酶突变体的多核苷酸，以能够使编码的脂肪酶突变体较野生型脂肪酶具有更高的比活力，在合成生物柴油时具有更高的转化率。
为实现此目的，在基础的实施方案中，本发明提供编码前述脂肪酶突变体的多核苷酸。
本发明的第三个目的是提供上述的脂肪酶突变体的应用，以能够更好的制备生物柴油。
为实现此目的，在基础的实施方案中，本发明提供一种脂肪酶突变体的应用，所述的应用是在反应体系中，用植物油脂作为底物与醇反应合成生物柴油。
在一种优选的实施方案中，植物油脂包括：大豆油、葵花油、棕榈油、菜籽油中的至少一种，醇包括：甲醇、乙醇中的至少一种；优选底物为大豆油和甲醇。
进一步地，本发明所述的反应体系中含有植物油脂、脂肪酶突变体酶、水和醇，脂肪酶突变体酶活为3-8U/g底物植物油脂，8-12％水，反应初始醇浓度为5-7％，每间隔40-80min分别再依次递减添加体系的5％-2％的醇。
更进一步地，本发明所述的反应体系中，脂肪酶突变体酶活为3-8U/g底物大豆油，反应初始甲醇浓度为6％，每间隔1hr分别再添加体系的5％、4％、3％、2％的甲醇。
更进一步地，本发明的反应温度为35-45℃，反应时间为180-540min。反应搅拌转速为800-1200r/min。
在一种优选的实施方案中，本发明所述的脂肪酶突变体是固定化的脂肪酶突变体。
本发明的有益效果在于，利用本发明的脂肪酶突变体，能够使突变体较野生型脂肪酶比活力显著提高，固定化酶活力显著提高，发酵成本和固定化酶制备成本显著降低。同时，生物柴油转化率明显提高。
本发明选择Streptomyces sp.W007来源的脂肪酶MAS作为出发点，相比其它来源的脂肪酶具有更高的异源表达量，稳定性好，对底物亲和力高，转化率高。在此基础上，本发明通过基因工程及酶工程技术手段，对MAS进行突变，所获得的脂肪酶突变体较MAS比活力显著提高，生物柴油转化率明显提高。显著降低了发酵成本和固定化酶制备成本。因此更适合工业应用。
附图说明
图1为生物柴油的合成原理图。
具体实施方式
以下结合实施例对本发明作出进一步的说明，而非限制本发明。
其中MAS及其突变体的酶活力的测定方法如下。
水解活力测定：于装有预热至30℃的40ml底物乳剂(用0.1mol/L的氢氧化钠滴定液预调pH至6.5)的反应器中，加入0.1ml酶液或25mg固定化酶，开搅拌，水浴恒温30℃，用0.1mol/L的氢氧化钠滴定液调pH至7.00计时，保持pH7.00反应5分钟，记录氢氧化钠滴定液的消耗量。
酶活力单位：在一定反应条件下，每分钟消耗1μmol的NaOH为一个单位(1U)。
底物溶液的配制
乳化剂
分别称取阿拉伯胶10.0g，、氯化钠17.9g和磷酸二氢钾0.40g，量取甘油540ml。将大约100ml去离子水倒入200ml的烧杯中，开高速搅拌，将阿拉伯胶缓慢倒入水中，不断搅拌直至全部溶解。将称好的氯化钠和磷酸二氢钾用100ml去离子水完全溶解后转入1000ml容量瓶中，并将甘油全部加入。将阿拉伯胶溶液转入容量瓶中，充分混合均匀后用去离子水定容至刻度。
底物乳剂
称取三丁酸甘油酯12.50g，分别量取去离子水94ml和乳化剂20ml，混合，将混合液超声处理10min。超声处理后的溶液先用搅拌器搅拌至少20min，然后调节pH到4.75±0.05。
酯交换活力测定：
于50ml具塞锥形瓶中，加入大豆油20g和甲醇1ml，预热至37℃后加入1ml酶液或1g固定化酶与1ml水，于37℃的恒温振荡培养箱中恒温反应30min(振荡频率160rpm)，取样离心5min后取上层油样10μl于1.5ml规格的进样瓶中，进行气相分析，测定脂肪酸甲酯含量计算转化率。
脂肪酸甲酯含量测定条件如下：
柱子型号：HP-INNOWax(30m*0.320mm*0.25μm)
检测器：氢火焰离子化检测器(FID)
进样口温度：250℃
检测器温度：300℃
氢气流量：40ml/min
空气流量：400ml/min
柱温：210℃恒定7.5min，以20℃/min升至240℃，恒定13min
柱流量(氮气)：1.5ml/min
分流比：30：1
尾吹：30ml/min
进样量：1μl
酶活力单位：在一定反应条件下，每分钟生成1μmol的脂肪酸甲酯为一个单位(1U)。
实施例1：Streptomyces sp.W007来源的脂肪酶原核表达菌株的构建
下载GenBank中Streptomyces sp.W007来源的脂肪酶(MAS)的氨基酸序列(本文SEQ ID NO.1，对应GenBank登陆号：5H6G_A),提供给通用生物系统(安徽)有限公司进行编码核酸的全基因合成(采用大肠杆菌优选密码子)，构建至原核表达载体pET30a(+)中，原核表达载体酶切位点：5’端Nde I，3’端Xho I。将构建好的质粒pET30a(+)-MAS通过CaCl2热激转化法转化至大肠杆菌表达菌株BL21(DE3)中，涂布于含有50μg/ml Kanamycin的LB固体培养基平板，37℃过夜培养，平板上生长出的菌落即为脂肪酶原核表达重组菌株E.coli BL21(DE3)/pET30a(+)-MAS。
用灭菌枪头在上述LB固体培养基平板中小心挑取脂肪酶原核表达重组菌株单菌落，接种至含有20mL的LB液体培养基的三角瓶中，37℃，200r/min，振荡过夜培养。次日按照1％的接种量将摇瓶菌液接种至含有100mL TB液体培养基的三角瓶中，37℃，220r/min，振荡培养，并每隔1h测定培养液的OD值，待培养液OD值＝1.5时，补加终浓度为1％(m/v)的乳糖，25℃，220rpm继续培养4h-6h，停止培养。
实施例2：Streptomyces sp.W007来源的脂肪酶的纯化与固定化
利用MAS重组蛋白中所携带His标签，采用已活化的IDA树脂(购买于安诺伦(北京)生物科技有限公司，具体型号:His.Bind Resin，Ni-charged)对实施例1得到的发酵液进行蛋白纯化，具体方法如下：4℃，10000r/min，离心发酵液10min，弃上清，收集菌体，菌体用磷酸盐缓冲液(pH 7.5、0.1mol/L)反复洗涤两次，离心后将菌体浓缩5倍重悬于20ml磷酸盐缓冲液(pH 7.5、0.1mol/L)中。将上述处理后的菌液置于冰水中进行超声破碎直至澄清，超声破碎条件为：工作2s，间隔5s，超声功率500W。将上述破碎后的裂解液置于低温高速离心机中离心(12000rpm、4℃、20min)，收集上清，得到粗蛋白。将粗蛋白上样到已活化的IDA树脂上，用咪唑溶液(10mM-300mM)进行梯度洗脱，利用蛋白层析系统(Bio-Rad)进行实时监控，收集出现的稳定的蛋白峰，即为MAS重组蛋白纯化蛋白，用于固定化酶制备。
将纯化的MAS重组蛋白用于固定化酶的制备，具体方法为：
(1)固定化载体活化：准确量取60％(m/v)的戊二醛30ml，同磷酸氢二钾(K2HPO4·3H2O)4.76g加入600ml去离子水中，溶解后用去离子水定容至1000ml，并用磷酸溶液调节其pH为8.0。将氨基载体ECHA/S(意大利ResindionS.r.l公司)250g投入到上述溶液中，并于25℃低速搅拌活化2h，过滤收集载体，并用无菌去离子水冲洗5-10次后真空滤干备用。
(2)MAS重组蛋白的固定化：取一定量上述纯化后的MAS重组蛋白，用磷酸盐缓冲液(pH 6.0、0.5mol/L)稀释，然后加入50g经活化处理后的载体，于22℃、120rpm条件下固定化16h，所得固定化酶用磷酸盐缓冲液(pH 6.0、0.02mol/L)清洗3-5次，真空滤干后即得固定化酶终品。
实施例3：MAS原核表达菌株E.coli BL21(DE3)/pET30a(+)-MAS易错突变文库的构建
以pET30a(+)-MAS重组质粒作为PCR模板，常规的T7F/R作为通用引物(引物序列：T7F:5’-TAATACGACTCACTATAGGG-3’T7R:GCTAGTTATTGCTCAGCGG，分别见SEQ ID NO.16和17)对MAS基因进行易错PCR扩增，调整PCR扩增反应体系中Mg2+、Mn2+、dCTP和dTTP寡核苷酸浓度，使该突变体文库的碱基错配率仅为千分之二，即保证一个突变体仅有1到2个氨基酸发生突变。
易错PCR反应体系：
易错PCR反应条件：先95℃预变性5min；然后94℃变性30s，56℃退火30s，72℃延伸1min，共25个循环；最后72℃延伸10min。
将上述易错PCR产物取样2μL进行琼脂糖凝胶电泳检测，检测无误后用PCR产物纯化试剂盒进行纯化处理。在37℃条件下，用Nde I和Xho I限制性内切酶分别对PCR纯化产物和原核表达载体pET30a(+)进行双酶切，酶切产物切胶回收(其中回收PCR纯化产物片段大小约为820bp，回收载体pET30a(+)片段大小约为5300bp)后按照易错PCR产物：原核表达载体pET30a(+)为3：1的摩尔比进行混合，加入T4 DNA ligase于16℃过夜连接。第二天，通过电击转化的方法将连接产物转入大肠杆菌BL21(DE3)中构建工程菌，即可得到一个库容量大的随机突变体文库。
实施例4：MAS原核表达菌株E.coli BL21(DE3)/pET30a(+)-MAS易错突变文库的筛选
其筛选原理为：
氯化钙将水解反应产生的脂肪酸转化为盐酸，通过pH指示剂检测盐酸释放的H+。
具体方法如下：
用高温灭菌后的牙签，小心挑取突变体文库的单菌落(每根牙签挑取1个单菌落)，分别接种于96孔细胞培养板的不同孔中(每孔中已加入含50μg/ml卡那霉素的LB液体培养基)。将96孔细胞培养板置于恒温摇床中37℃，700rpm培养6小时，然后用8通道移液器在每孔加入终浓度为1％(m/v)的乳糖，25℃，250rpm诱导培养8小时。诱导培养完毕后，将96孔细胞培养板放入-86℃的超低温冰箱中冷冻2小时，取出放置于室温半小时后4000rpm，4℃离心20分钟后将每孔上清10μL转移至新的96孔板中，加入10ul双指示剂(溴百里酚蓝和酚红各0.5mg/ml溶于5mM pH 8.5的Tris/HCl中)，40ul 100mM氯化钙(溶于5mM pH 8.5的Tris/HCl中),50ul橄榄油溶液(按1:6v/v溶于DMF)。15min后观察颜色变化。
通过反复大量筛选验证(约170000个克隆子)、测序分析及酶活力测定，筛选到5株活力高于野生型的菌株。分别命名为MAS-1,MAS-2,MAS-3,MAS-4,MAS-5。其突变位点和活力情况如下表1。由表可知，突变株脂肪酶水解活力和酯交换活力均有不同程度提高。其中MAS-3活力提高最为显著，较野生型MAS-WT菌株水解活力和酯交换活力均提高4.43倍。因此，以MAS-3为出发菌株，进一步对表1中筛选到的有益位点进行叠加突变。以期进一步提高酶活力。
表1易错文库筛选的脂肪酶MAS突变菌株
| 酶 | 突变位点 | 水解活力(U/mg) | 酯交换活力(U/mg) |
| MAS-WT | 无 | 55.52 | 2.41 |
| MAS-1 | D89G | 89.66 | 3.49 |
| MAS-2 | V90A | 81.89 | 2.76 |
| MAS-3 | A104V | 246.02 | 10.67 |
| MAS-4 | E232D | 103.25 | 3.37 |
| MAS-5 | V264I | 96.23 | 3.80 |
实施例5：以MAS-3为出发菌株，不同叠加突变体菌株的构建与筛选
将表1中MAS-3表达菌株进行扩大培养，用质粒提取试剂盒(OMEGA)提取质粒，以质粒pET30a(+)-MAS-3为模板，将所获得的突变位点进行叠加突变，选择MAS-3氨基酸序列第89位、90位、232位和264位，分别设计定点突变引物，进行全质粒PCR反应，全质粒PCR产物经DpnI消化后转化至表达菌株BL21(DE3)中，经测序验证无误，即获得在MAS-3突变体基础上的各叠加突变体的表达菌株，分别命名为MAS-6,MAS-7,MAS-8,MAS-9。其突变位点和活力情况如下表2。由表可知，对其他4个有益位点进行叠加突变后，除V90A位点叠加活力未提高外，其它位点叠加后活力均有不同程度提高。其中MAS-9活力提高最为显著。较野生型MAS-WT菌株水解活力和酯交换活力分别提高13.12倍和15.90倍。因此，以MAS-9为出发菌株，进一步对筛选到的有益位点进行叠加突变。以期进一步提高酶活力。
表2以MAS-3为出发菌株的不同叠加突变体菌株
| 酶 | 突变位点 | 水解活力(U/mg) | 酯交换活力(U/mg) |
| MAS-WT | 无 | 55.52 | 2.41 |
| MAS-3 | A104V | 246.02 | 10.67 |
| MAS-6 | A104V，D89G | 313.84 | 13.51 |
| MAS-7 | A104V，V90A | 261.77 | 10.13 |
| MAS-8 | A104V，E232D | 562.59 | 28.10 |
| MAS-9 | A104V，V264I | 728.29 | 38.32 |
实施例6：以MAS-9为出发菌株，不同叠加突变体菌株的构建与筛选
将表2中MAS-9表达菌株进行扩大培养，用质粒提取试剂盒(OMEGA)提取质粒，以质粒pET30a(+)-MAS-9为模板，将所获得的突变位点进行叠加突变，选择MAS-9氨基酸序列第89位、90位和232位，分别设计定点突变引物，进行全质粒PCR反应，全质粒PCR产物经DpnI消化后转化至表达菌株BL21(DE3)中，经测序验证无误，即获得在MAS-9突变体基础上的各叠加突变体的表达菌株，分别命名为MAS-10,MAS-11,MAS-12。其突变位点和活力情况如下表3。由表可知，除V90A位点叠加活力未提高外，其它位点叠加后活力均有不同程度提高。其中MAS-12活力提高最为显著。较野生型MAS-WT菌株水解活力和酯交换活力分别提高32.38倍和39.26倍。因此，以MAS-12为出发菌株，进一步对筛选到的有益位点进行叠加突变。以期进一步提高酶活力。
表3以MAS-9为出发菌株的不同叠加突变体菌株
| 酶 | 突变位点 | 水解活力(U/mg) | 酯交换活力(U/mg) |
| MAS-WT | 无 | 55.52 | 2.41 |
| MAS-3 | A104V | 246.02 | 10.67 |
| MAS-9 | A104V，V264I | 728.29 | 38.32 |
| MAS-10 | A104V，V264I，D89G | 1468.26 | 73.92 |
| MAS-11 | A104V，V264I，V90A | 645.54 | 28.95 |
| MAS-12 | A104V，V264I，E232D | 1797.77 | 94.61 |
实施例7：以MAS-12为出发菌株，不同叠加突变体菌株的构建与筛选
将表3中MAS-12表达菌株进行扩大培养，用质粒提取试剂盒(OMEGA)提取质粒，以质粒pET30a(+)-MAS-12为模板，将所获得的突变位点进行叠加突变，选择MAS-12氨基酸序列第89位、90位，分别设计定点突变引物，进行全质粒PCR反应，全质粒PCR产物经DpnI消化后转化至表达菌株BL21(DE3)中，经测序验证无误，即获得在MAS-12突变体基础上的各叠加突变体的表达菌株，分别命名为MAS-13和MAS-14。其突变位点和活力情况如下表4。由表可知，V90A位点叠加突变活力未提高。而D89G位点叠加后较野生型MAS-WT菌株水解活力和酯交换活力分别提高36.21倍和47.10倍。为所有突变株中最高酶活力。
表4以MAS-12为出发菌株的不同叠加突变体菌株
| 酶 | 突变位点 | 水解活力(U/mg) | 酯交换活力(U/mg) |
| MAS-WT | 无 | 55.52 | 2.41 |
| MAS-3 | A104V | 246.02 | 10.67 |
| MAS-9 | A104V，V264I | 728.29 | 38.32 |
| MAS-12 | A104V，V264I，E232D | 1797.77 | 94.61 |
| MAS-13 | A104V，V264I，E232D，D89G | 2010.35 | 113.51 |
| MAS-14 | A104V，V264I，E232D，V90A | 1684.26 | 88.58 |
实施例8：不同MAS突变体的发酵、纯化
采用与MAS-WT基本相同的方法进行MAS-3、MAS-9、MAS-12和MAS-13的发酵与纯化，所得蛋白纯化样品连同实施例2得到的MAS-WT纯化样一起进行SDS-PAGE电泳纯度检测(电泳条件：分离胶浓度10％，电压100V，电流50mA，电泳时间120min)，结果纯度均在95％以上。
实施例9：不同突变株与MAS-WT野生株固定化酶催化的生物柴油合成反应比较
将MAS-3、MAS-9、MAS-12、MAS-13和MAS-WT分别制备固定化酶后(方法同实施例2)催化生物柴油合成反应。
反应条件：100g大豆油+10ml水+6ml甲醇置于500ml圆底三口瓶中，温度维持在40℃，搅拌转速在1000rpm，混匀后加入500U酯交换活力的脂肪酶固定化酶，开始反应。反应1hr后加入5ml甲醇，反应到2hr后再加入4ml甲醇，反应3hr后再加入3ml甲醇，反应4hr后再加入2ml甲醇，8hr反应结束。反应过程中分别取2hr、4hr、6hr和8hr样各1ml，离心5min后取上层油样10μl于1.5ml规格的进样瓶中，进行气相分析，测定脂肪酸甲酯含量计算转化率。固定化酶活力和反应结果如下表5。由表可知，各检测时间点MAS-WT转化率均低于各突变体酶转化率。且MAS-WT转化率最低为85.32％，而MAS-9、MAS-12和MAS-13转化率均超过了99％，较MAS-WT提升明显。
表5野生型和不同突变体脂肪酶固定化酶合成生物柴油比较
显然，本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样，倘若对本发明的这些修改和变型属于本发明权利要求及其同等技术的范围之内，则本发明也意图包含这些改动和变型在内。上述实施例或实施方式只是对本发明的举例说明，本发明也可以以其它的特定方式或其它的特定形式实施，而不偏离本发明的要旨或本质特征。因此，描述的实施方式从任何方面来看均应视为说明性而非限定性的。本发明的范围应由附加的权利要求说明，任何与权利要求的意图和范围等效的变化也应包含在本发明的范围内。
序列表
<110> 湖南福来格生物技术有限公司
<120> 一种脂肪酶突变体及其应用
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Val Val Leu Val His Gly Thr Phe Gly Asn Ser Ile Asp Asn Trp Leu
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Val Leu Ala Pro Tyr Leu Val Asn Arg Gly Tyr Cys Val Phe Ser Leu
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Ile Asp Lys Ser Ala Glu Gln Leu Asp Val Phe Val Asp Lys Val Leu
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Val Asn Ala Leu Val Gly Ile Ala Pro Asp Asn His Gly Thr Thr Leu
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Met Ala Thr Ala Thr Ala Ala Thr Pro Ala Ala Glu Ala Thr Ser Arg
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Gly Trp Asn Asp Tyr Ser Cys Lys Pro Ser Ala Ala His Pro Arg Pro
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Val Val Leu Val His Gly Thr Phe Gly Asn Ser Ile Asp Asn Trp Leu
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Val Leu Ala Pro Tyr Leu Val Asn Arg Gly Tyr Cys Val Phe Ser Leu
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Asp Tyr Gly Gln Leu Pro Gly Val Pro Phe Phe His Gly Leu Gly Pro
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Ile Asp Lys Ser Ala Glu Gln Leu Gly Val Phe Val Asp Lys Val Leu
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Asp Ala Thr Gly Ala Pro Lys Ala Asp Leu Val Gly His Ser Gln Gly
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Gly Met Met Pro Asn Tyr Tyr Leu Lys Phe Leu Gly Gly Ala Asp Lys
115 120 125
Val Asn Ala Leu Val Gly Ile Ala Pro Asp Asn His Gly Thr Thr Leu
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Ile Ser Asp Asn Thr Pro Gly Leu Ala Asp Gln Val Ala Gly Ser Pro
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Leu Cys Pro Val Asp Leu Ser Glu His Val Ala Ile Gly Thr Ile Asp
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Thr Pro Thr Thr Cys Ala Ser Val Ile Gly
260 265
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Met Ala Thr Ala Thr Ala Ala Thr Pro Ala Ala Glu Ala Thr Ser Arg
1 5 10 15
Gly Trp Asn Asp Tyr Ser Cys Lys Pro Ser Ala Ala His Pro Arg Pro
20 25 30
Val Val Leu Val His Gly Thr Phe Gly Asn Ser Ile Asp Asn Trp Leu
35 40 45
Val Leu Ala Pro Tyr Leu Val Asn Arg Gly Tyr Cys Val Phe Ser Leu
50 55 60
Asp Tyr Gly Gln Leu Pro Gly Val Pro Phe Phe His Gly Leu Gly Pro
65 70 75 80
Ile Asp Lys Ser Ala Glu Gln Leu Asp Ala Phe Val Asp Lys Val Leu
85 90 95
Asp Ala Thr Gly Ala Pro Lys Ala Asp Leu Val Gly His Ser Gln Gly
100 105 110
Gly Met Met Pro Asn Tyr Tyr Leu Lys Phe Leu Gly Gly Ala Asp Lys
115 120 125
Val Asn Ala Leu Val Gly Ile Ala Pro Asp Asn His Gly Thr Thr Leu
130 135 140
Leu Gly Leu Thr Lys Leu Leu Pro Phe Phe Pro Gly Val Glu Lys Phe
145 150 155 160
Ile Ser Asp Asn Thr Pro Gly Leu Ala Asp Gln Val Ala Gly Ser Pro
165 170 175
Phe Ile Thr Lys Leu Thr Ala Gly Gly Asp Thr Val Pro Gly Val Arg
180 185 190
Tyr Thr Val Ile Ala Thr Lys Tyr Asp Gln Val Val Thr Pro Tyr Arg
195 200 205
Thr Gln Tyr Leu Asp Gly Pro Asn Val Arg Asn Val Leu Leu Gln Asp
210 215 220
Leu Cys Pro Val Asp Leu Ser Glu His Val Ala Ile Gly Thr Ile Asp
225 230 235 240
Arg Ile Ala Phe His Glu Val Ala Asn Ala Leu Asp Pro Ala Arg Ala
245 250 255
Thr Pro Thr Thr Cys Ala Ser Val Ile Gly
260 265
<210> 4
<211> 266
<212> PRT
<213> 人工序列(Artificial Sequence)
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Met Ala Thr Ala Thr Ala Ala Thr Pro Ala Ala Glu Ala Thr Ser Arg
1 5 10 15
Gly Trp Asn Asp Tyr Ser Cys Lys Pro Ser Ala Ala His Pro Arg Pro
20 25 30
Val Val Leu Val His Gly Thr Phe Gly Asn Ser Ile Asp Asn Trp Leu
35 40 45
Val Leu Ala Pro Tyr Leu Val Asn Arg Gly Tyr Cys Val Phe Ser Leu
50 55 60
Asp Tyr Gly Gln Leu Pro Gly Val Pro Phe Phe His Gly Leu Gly Pro
65 70 75 80
Ile Asp Lys Ser Ala Glu Gln Leu Asp Val Phe Val Asp Lys Val Leu
85 90 95
Asp Ala Thr Gly Ala Pro Lys Val Asp Leu Val Gly His Ser Gln Gly
100 105 110
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115 120 125
Val Asn Ala Leu Val Gly Ile Ala Pro Asp Asn His Gly Thr Thr Leu
130 135 140
Leu Gly Leu Thr Lys Leu Leu Pro Phe Phe Pro Gly Val Glu Lys Phe
145 150 155 160
Ile Ser Asp Asn Thr Pro Gly Leu Ala Asp Gln Val Ala Gly Ser Pro
165 170 175
Phe Ile Thr Lys Leu Thr Ala Gly Gly Asp Thr Val Pro Gly Val Arg
180 185 190
Tyr Thr Val Ile Ala Thr Lys Tyr Asp Gln Val Val Thr Pro Tyr Arg
195 200 205
Thr Gln Tyr Leu Asp Gly Pro Asn Val Arg Asn Val Leu Leu Gln Asp
210 215 220
Leu Cys Pro Val Asp Leu Ser Glu His Val Ala Ile Gly Thr Ile Asp
225 230 235 240
Arg Ile Ala Phe His Glu Val Ala Asn Ala Leu Asp Pro Ala Arg Ala
245 250 255
Thr Pro Thr Thr Cys Ala Ser Val Ile Gly
260 265
<210> 5
<211> 266
<212> PRT
<213> 人工序列(Artificial Sequence)
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Met Ala Thr Ala Thr Ala Ala Thr Pro Ala Ala Glu Ala Thr Ser Arg
1 5 10 15
Gly Trp Asn Asp Tyr Ser Cys Lys Pro Ser Ala Ala His Pro Arg Pro
20 25 30
Val Val Leu Val His Gly Thr Phe Gly Asn Ser Ile Asp Asn Trp Leu
35 40 45
Val Leu Ala Pro Tyr Leu Val Asn Arg Gly Tyr Cys Val Phe Ser Leu
50 55 60
Asp Tyr Gly Gln Leu Pro Gly Val Pro Phe Phe His Gly Leu Gly Pro
65 70 75 80
Ile Asp Lys Ser Ala Glu Gln Leu Asp Val Phe Val Asp Lys Val Leu
85 90 95
Asp Ala Thr Gly Ala Pro Lys Ala Asp Leu Val Gly His Ser Gln Gly
100 105 110
Gly Met Met Pro Asn Tyr Tyr Leu Lys Phe Leu Gly Gly Ala Asp Lys
115 120 125
Val Asn Ala Leu Val Gly Ile Ala Pro Asp Asn His Gly Thr Thr Leu
130 135 140
Leu Gly Leu Thr Lys Leu Leu Pro Phe Phe Pro Gly Val Glu Lys Phe
145 150 155 160
Ile Ser Asp Asn Thr Pro Gly Leu Ala Asp Gln Val Ala Gly Ser Pro
165 170 175
Phe Ile Thr Lys Leu Thr Ala Gly Gly Asp Thr Val Pro Gly Val Arg
180 185 190
Tyr Thr Val Ile Ala Thr Lys Tyr Asp Gln Val Val Thr Pro Tyr Arg
195 200 205
Thr Gln Tyr Leu Asp Gly Pro Asn Val Arg Asn Val Leu Leu Gln Asp
210 215 220
Leu Cys Pro Val Asp Leu Ser Asp His Val Ala Ile Gly Thr Ile Asp
225 230 235 240
Arg Ile Ala Phe His Glu Val Ala Asn Ala Leu Asp Pro Ala Arg Ala
245 250 255
Thr Pro Thr Thr Cys Ala Ser Val Ile Gly
260 265
<210> 6
<211> 266
<212> PRT
<213> 人工序列(Artificial Sequence)
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Met Ala Thr Ala Thr Ala Ala Thr Pro Ala Ala Glu Ala Thr Ser Arg
1 5 10 15
Gly Trp Asn Asp Tyr Ser Cys Lys Pro Ser Ala Ala His Pro Arg Pro
20 25 30
Val Val Leu Val His Gly Thr Phe Gly Asn Ser Ile Asp Asn Trp Leu
35 40 45
Val Leu Ala Pro Tyr Leu Val Asn Arg Gly Tyr Cys Val Phe Ser Leu
50 55 60
Asp Tyr Gly Gln Leu Pro Gly Val Pro Phe Phe His Gly Leu Gly Pro
65 70 75 80
Ile Asp Lys Ser Ala Glu Gln Leu Asp Val Phe Val Asp Lys Val Leu
85 90 95
Asp Ala Thr Gly Ala Pro Lys Ala Asp Leu Val Gly His Ser Gln Gly
100 105 110
Gly Met Met Pro Asn Tyr Tyr Leu Lys Phe Leu Gly Gly Ala Asp Lys
115 120 125
Val Asn Ala Leu Val Gly Ile Ala Pro Asp Asn His Gly Thr Thr Leu
130 135 140
Leu Gly Leu Thr Lys Leu Leu Pro Phe Phe Pro Gly Val Glu Lys Phe
145 150 155 160
Ile Ser Asp Asn Thr Pro Gly Leu Ala Asp Gln Val Ala Gly Ser Pro
165 170 175
Phe Ile Thr Lys Leu Thr Ala Gly Gly Asp Thr Val Pro Gly Val Arg
180 185 190
Tyr Thr Val Ile Ala Thr Lys Tyr Asp Gln Val Val Thr Pro Tyr Arg
195 200 205
Thr Gln Tyr Leu Asp Gly Pro Asn Val Arg Asn Val Leu Leu Gln Asp
210 215 220
Leu Cys Pro Val Asp Leu Ser Glu His Val Ala Ile Gly Thr Ile Asp
225 230 235 240
Arg Ile Ala Phe His Glu Val Ala Asn Ala Leu Asp Pro Ala Arg Ala
245 250 255
Thr Pro Thr Thr Cys Ala Ser Ile Ile Gly
260 265
<210> 7
<211> 266
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Met Ala Thr Ala Thr Ala Ala Thr Pro Ala Ala Glu Ala Thr Ser Arg
1 5 10 15
Gly Trp Asn Asp Tyr Ser Cys Lys Pro Ser Ala Ala His Pro Arg Pro
20 25 30
Val Val Leu Val His Gly Thr Phe Gly Asn Ser Ile Asp Asn Trp Leu
35 40 45
Val Leu Ala Pro Tyr Leu Val Asn Arg Gly Tyr Cys Val Phe Ser Leu
50 55 60
Asp Tyr Gly Gln Leu Pro Gly Val Pro Phe Phe His Gly Leu Gly Pro
65 70 75 80
Ile Asp Lys Ser Ala Glu Gln Leu Gly Val Phe Val Asp Lys Val Leu
85 90 95
Asp Ala Thr Gly Ala Pro Lys Val Asp Leu Val Gly His Ser Gln Gly
100 105 110
Gly Met Met Pro Asn Tyr Tyr Leu Lys Phe Leu Gly Gly Ala Asp Lys
115 120 125
Val Asn Ala Leu Val Gly Ile Ala Pro Asp Asn His Gly Thr Thr Leu
130 135 140
Leu Gly Leu Thr Lys Leu Leu Pro Phe Phe Pro Gly Val Glu Lys Phe
145 150 155 160
Ile Ser Asp Asn Thr Pro Gly Leu Ala Asp Gln Val Ala Gly Ser Pro
165 170 175
Phe Ile Thr Lys Leu Thr Ala Gly Gly Asp Thr Val Pro Gly Val Arg
180 185 190
Tyr Thr Val Ile Ala Thr Lys Tyr Asp Gln Val Val Thr Pro Tyr Arg
195 200 205
Thr Gln Tyr Leu Asp Gly Pro Asn Val Arg Asn Val Leu Leu Gln Asp
210 215 220
Leu Cys Pro Val Asp Leu Ser Glu His Val Ala Ile Gly Thr Ile Asp
225 230 235 240
Arg Ile Ala Phe His Glu Val Ala Asn Ala Leu Asp Pro Ala Arg Ala
245 250 255
Thr Pro Thr Thr Cys Ala Ser Val Ile Gly
260 265
<210> 8
<211> 266
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Met Ala Thr Ala Thr Ala Ala Thr Pro Ala Ala Glu Ala Thr Ser Arg
1 5 10 15
Gly Trp Asn Asp Tyr Ser Cys Lys Pro Ser Ala Ala His Pro Arg Pro
20 25 30
Val Val Leu Val His Gly Thr Phe Gly Asn Ser Ile Asp Asn Trp Leu
35 40 45
Val Leu Ala Pro Tyr Leu Val Asn Arg Gly Tyr Cys Val Phe Ser Leu
50 55 60
Asp Tyr Gly Gln Leu Pro Gly Val Pro Phe Phe His Gly Leu Gly Pro
65 70 75 80
Ile Asp Lys Ser Ala Glu Gln Leu Asp Ala Phe Val Asp Lys Val Leu
85 90 95
Asp Ala Thr Gly Ala Pro Lys Val Asp Leu Val Gly His Ser Gln Gly
100 105 110
Gly Met Met Pro Asn Tyr Tyr Leu Lys Phe Leu Gly Gly Ala Asp Lys
115 120 125
Val Asn Ala Leu Val Gly Ile Ala Pro Asp Asn His Gly Thr Thr Leu
130 135 140
Leu Gly Leu Thr Lys Leu Leu Pro Phe Phe Pro Gly Val Glu Lys Phe
145 150 155 160
Ile Ser Asp Asn Thr Pro Gly Leu Ala Asp Gln Val Ala Gly Ser Pro
165 170 175
Phe Ile Thr Lys Leu Thr Ala Gly Gly Asp Thr Val Pro Gly Val Arg
180 185 190
Tyr Thr Val Ile Ala Thr Lys Tyr Asp Gln Val Val Thr Pro Tyr Arg
195 200 205
Thr Gln Tyr Leu Asp Gly Pro Asn Val Arg Asn Val Leu Leu Gln Asp
210 215 220
Leu Cys Pro Val Asp Leu Ser Glu His Val Ala Ile Gly Thr Ile Asp
225 230 235 240
Arg Ile Ala Phe His Glu Val Ala Asn Ala Leu Asp Pro Ala Arg Ala
245 250 255
Thr Pro Thr Thr Cys Ala Ser Val Ile Gly
260 265
<210> 9
<211> 266
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Met Ala Thr Ala Thr Ala Ala Thr Pro Ala Ala Glu Ala Thr Ser Arg
1 5 10 15
Gly Trp Asn Asp Tyr Ser Cys Lys Pro Ser Ala Ala His Pro Arg Pro
20 25 30
Val Val Leu Val His Gly Thr Phe Gly Asn Ser Ile Asp Asn Trp Leu
35 40 45
Val Leu Ala Pro Tyr Leu Val Asn Arg Gly Tyr Cys Val Phe Ser Leu
50 55 60
Asp Tyr Gly Gln Leu Pro Gly Val Pro Phe Phe His Gly Leu Gly Pro
65 70 75 80
Ile Asp Lys Ser Ala Glu Gln Leu Asp Val Phe Val Asp Lys Val Leu
85 90 95
Asp Ala Thr Gly Ala Pro Lys Val Asp Leu Val Gly His Ser Gln Gly
100 105 110
Gly Met Met Pro Asn Tyr Tyr Leu Lys Phe Leu Gly Gly Ala Asp Lys
115 120 125
Val Asn Ala Leu Val Gly Ile Ala Pro Asp Asn His Gly Thr Thr Leu
130 135 140
Leu Gly Leu Thr Lys Leu Leu Pro Phe Phe Pro Gly Val Glu Lys Phe
145 150 155 160
Ile Ser Asp Asn Thr Pro Gly Leu Ala Asp Gln Val Ala Gly Ser Pro
165 170 175
Phe Ile Thr Lys Leu Thr Ala Gly Gly Asp Thr Val Pro Gly Val Arg
180 185 190
Tyr Thr Val Ile Ala Thr Lys Tyr Asp Gln Val Val Thr Pro Tyr Arg
195 200 205
Thr Gln Tyr Leu Asp Gly Pro Asn Val Arg Asn Val Leu Leu Gln Asp
210 215 220
Leu Cys Pro Val Asp Leu Ser Asp His Val Ala Ile Gly Thr Ile Asp
225 230 235 240
Arg Ile Ala Phe His Glu Val Ala Asn Ala Leu Asp Pro Ala Arg Ala
245 250 255
Thr Pro Thr Thr Cys Ala Ser Val Ile Gly
260 265
<210> 10
<211> 266
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Met Ala Thr Ala Thr Ala Ala Thr Pro Ala Ala Glu Ala Thr Ser Arg
1 5 10 15
Gly Trp Asn Asp Tyr Ser Cys Lys Pro Ser Ala Ala His Pro Arg Pro
20 25 30
Val Val Leu Val His Gly Thr Phe Gly Asn Ser Ile Asp Asn Trp Leu
35 40 45
Val Leu Ala Pro Tyr Leu Val Asn Arg Gly Tyr Cys Val Phe Ser Leu
50 55 60
Asp Tyr Gly Gln Leu Pro Gly Val Pro Phe Phe His Gly Leu Gly Pro
65 70 75 80
Ile Asp Lys Ser Ala Glu Gln Leu Asp Val Phe Val Asp Lys Val Leu
85 90 95
Asp Ala Thr Gly Ala Pro Lys Val Asp Leu Val Gly His Ser Gln Gly
100 105 110
Gly Met Met Pro Asn Tyr Tyr Leu Lys Phe Leu Gly Gly Ala Asp Lys
115 120 125
Val Asn Ala Leu Val Gly Ile Ala Pro Asp Asn His Gly Thr Thr Leu
130 135 140
Leu Gly Leu Thr Lys Leu Leu Pro Phe Phe Pro Gly Val Glu Lys Phe
145 150 155 160
Ile Ser Asp Asn Thr Pro Gly Leu Ala Asp Gln Val Ala Gly Ser Pro
165 170 175
Phe Ile Thr Lys Leu Thr Ala Gly Gly Asp Thr Val Pro Gly Val Arg
180 185 190
Tyr Thr Val Ile Ala Thr Lys Tyr Asp Gln Val Val Thr Pro Tyr Arg
195 200 205
Thr Gln Tyr Leu Asp Gly Pro Asn Val Arg Asn Val Leu Leu Gln Asp
210 215 220
Leu Cys Pro Val Asp Leu Ser Glu His Val Ala Ile Gly Thr Ile Asp
225 230 235 240
Arg Ile Ala Phe His Glu Val Ala Asn Ala Leu Asp Pro Ala Arg Ala
245 250 255
Thr Pro Thr Thr Cys Ala Ser Ile Ile Gly
260 265
<210> 11
<211> 266
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Met Ala Thr Ala Thr Ala Ala Thr Pro Ala Ala Glu Ala Thr Ser Arg
1 5 10 15
Gly Trp Asn Asp Tyr Ser Cys Lys Pro Ser Ala Ala His Pro Arg Pro
20 25 30
Val Val Leu Val His Gly Thr Phe Gly Asn Ser Ile Asp Asn Trp Leu
35 40 45
Val Leu Ala Pro Tyr Leu Val Asn Arg Gly Tyr Cys Val Phe Ser Leu
50 55 60
Asp Tyr Gly Gln Leu Pro Gly Val Pro Phe Phe His Gly Leu Gly Pro
65 70 75 80
Ile Asp Lys Ser Ala Glu Gln Leu Gly Val Phe Val Asp Lys Val Leu
85 90 95
Asp Ala Thr Gly Ala Pro Lys Val Asp Leu Val Gly His Ser Gln Gly
100 105 110
Gly Met Met Pro Asn Tyr Tyr Leu Lys Phe Leu Gly Gly Ala Asp Lys
115 120 125
Val Asn Ala Leu Val Gly Ile Ala Pro Asp Asn His Gly Thr Thr Leu
130 135 140
Leu Gly Leu Thr Lys Leu Leu Pro Phe Phe Pro Gly Val Glu Lys Phe
145 150 155 160
Ile Ser Asp Asn Thr Pro Gly Leu Ala Asp Gln Val Ala Gly Ser Pro
165 170 175
Phe Ile Thr Lys Leu Thr Ala Gly Gly Asp Thr Val Pro Gly Val Arg
180 185 190
Tyr Thr Val Ile Ala Thr Lys Tyr Asp Gln Val Val Thr Pro Tyr Arg
195 200 205
Thr Gln Tyr Leu Asp Gly Pro Asn Val Arg Asn Val Leu Leu Gln Asp
210 215 220
Leu Cys Pro Val Asp Leu Ser Glu His Val Ala Ile Gly Thr Ile Asp
225 230 235 240
Arg Ile Ala Phe His Glu Val Ala Asn Ala Leu Asp Pro Ala Arg Ala
245 250 255
Thr Pro Thr Thr Cys Ala Ser Ile Ile Gly
260 265
<210> 12
<211> 266
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Met Ala Thr Ala Thr Ala Ala Thr Pro Ala Ala Glu Ala Thr Ser Arg
1 5 10 15
Gly Trp Asn Asp Tyr Ser Cys Lys Pro Ser Ala Ala His Pro Arg Pro
20 25 30
Val Val Leu Val His Gly Thr Phe Gly Asn Ser Ile Asp Asn Trp Leu
35 40 45
Val Leu Ala Pro Tyr Leu Val Asn Arg Gly Tyr Cys Val Phe Ser Leu
50 55 60
Asp Tyr Gly Gln Leu Pro Gly Val Pro Phe Phe His Gly Leu Gly Pro
65 70 75 80
Ile Asp Lys Ser Ala Glu Gln Leu Asp Ala Phe Val Asp Lys Val Leu
85 90 95
Asp Ala Thr Gly Ala Pro Lys Val Asp Leu Val Gly His Ser Gln Gly
100 105 110
Gly Met Met Pro Asn Tyr Tyr Leu Lys Phe Leu Gly Gly Ala Asp Lys
115 120 125
Val Asn Ala Leu Val Gly Ile Ala Pro Asp Asn His Gly Thr Thr Leu
130 135 140
Leu Gly Leu Thr Lys Leu Leu Pro Phe Phe Pro Gly Val Glu Lys Phe
145 150 155 160
Ile Ser Asp Asn Thr Pro Gly Leu Ala Asp Gln Val Ala Gly Ser Pro
165 170 175
Phe Ile Thr Lys Leu Thr Ala Gly Gly Asp Thr Val Pro Gly Val Arg
180 185 190
Tyr Thr Val Ile Ala Thr Lys Tyr Asp Gln Val Val Thr Pro Tyr Arg
195 200 205
Thr Gln Tyr Leu Asp Gly Pro Asn Val Arg Asn Val Leu Leu Gln Asp
210 215 220
Leu Cys Pro Val Asp Leu Ser Glu His Val Ala Ile Gly Thr Ile Asp
225 230 235 240
Arg Ile Ala Phe His Glu Val Ala Asn Ala Leu Asp Pro Ala Arg Ala
245 250 255
Thr Pro Thr Thr Cys Ala Ser Ile Ile Gly
260 265
<210> 13
<211> 266
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Met Ala Thr Ala Thr Ala Ala Thr Pro Ala Ala Glu Ala Thr Ser Arg
1 5 10 15
Gly Trp Asn Asp Tyr Ser Cys Lys Pro Ser Ala Ala His Pro Arg Pro
20 25 30
Val Val Leu Val His Gly Thr Phe Gly Asn Ser Ile Asp Asn Trp Leu
35 40 45
Val Leu Ala Pro Tyr Leu Val Asn Arg Gly Tyr Cys Val Phe Ser Leu
50 55 60
Asp Tyr Gly Gln Leu Pro Gly Val Pro Phe Phe His Gly Leu Gly Pro
65 70 75 80
Ile Asp Lys Ser Ala Glu Gln Leu Asp Val Phe Val Asp Lys Val Leu
85 90 95
Asp Ala Thr Gly Ala Pro Lys Val Asp Leu Val Gly His Ser Gln Gly
100 105 110
Gly Met Met Pro Asn Tyr Tyr Leu Lys Phe Leu Gly Gly Ala Asp Lys
115 120 125
Val Asn Ala Leu Val Gly Ile Ala Pro Asp Asn His Gly Thr Thr Leu
130 135 140
Leu Gly Leu Thr Lys Leu Leu Pro Phe Phe Pro Gly Val Glu Lys Phe
145 150 155 160
Ile Ser Asp Asn Thr Pro Gly Leu Ala Asp Gln Val Ala Gly Ser Pro
165 170 175
Phe Ile Thr Lys Leu Thr Ala Gly Gly Asp Thr Val Pro Gly Val Arg
180 185 190
Tyr Thr Val Ile Ala Thr Lys Tyr Asp Gln Val Val Thr Pro Tyr Arg
195 200 205
Thr Gln Tyr Leu Asp Gly Pro Asn Val Arg Asn Val Leu Leu Gln Asp
210 215 220
Leu Cys Pro Val Asp Leu Ser Asp His Val Ala Ile Gly Thr Ile Asp
225 230 235 240
Arg Ile Ala Phe His Glu Val Ala Asn Ala Leu Asp Pro Ala Arg Ala
245 250 255
Thr Pro Thr Thr Cys Ala Ser Ile Ile Gly
260 265
<210> 14
<211> 266
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Met Ala Thr Ala Thr Ala Ala Thr Pro Ala Ala Glu Ala Thr Ser Arg
1 5 10 15
Gly Trp Asn Asp Tyr Ser Cys Lys Pro Ser Ala Ala His Pro Arg Pro
20 25 30
Val Val Leu Val His Gly Thr Phe Gly Asn Ser Ile Asp Asn Trp Leu
35 40 45
Val Leu Ala Pro Tyr Leu Val Asn Arg Gly Tyr Cys Val Phe Ser Leu
50 55 60
Asp Tyr Gly Gln Leu Pro Gly Val Pro Phe Phe His Gly Leu Gly Pro
65 70 75 80
Ile Asp Lys Ser Ala Glu Gln Leu Gly Val Phe Val Asp Lys Val Leu
85 90 95
Asp Ala Thr Gly Ala Pro Lys Val Asp Leu Val Gly His Ser Gln Gly
100 105 110
Gly Met Met Pro Asn Tyr Tyr Leu Lys Phe Leu Gly Gly Ala Asp Lys
115 120 125
Val Asn Ala Leu Val Gly Ile Ala Pro Asp Asn His Gly Thr Thr Leu
130 135 140
Leu Gly Leu Thr Lys Leu Leu Pro Phe Phe Pro Gly Val Glu Lys Phe
145 150 155 160
Ile Ser Asp Asn Thr Pro Gly Leu Ala Asp Gln Val Ala Gly Ser Pro
165 170 175
Phe Ile Thr Lys Leu Thr Ala Gly Gly Asp Thr Val Pro Gly Val Arg
180 185 190
Tyr Thr Val Ile Ala Thr Lys Tyr Asp Gln Val Val Thr Pro Tyr Arg
195 200 205
Thr Gln Tyr Leu Asp Gly Pro Asn Val Arg Asn Val Leu Leu Gln Asp
210 215 220
Leu Cys Pro Val Asp Leu Ser Asp His Val Ala Ile Gly Thr Ile Asp
225 230 235 240
Arg Ile Ala Phe His Glu Val Ala Asn Ala Leu Asp Pro Ala Arg Ala
245 250 255
Thr Pro Thr Thr Cys Ala Ser Ile Ile Gly
260 265
<210> 15
<211> 266
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Met Ala Thr Ala Thr Ala Ala Thr Pro Ala Ala Glu Ala Thr Ser Arg
1 5 10 15
Gly Trp Asn Asp Tyr Ser Cys Lys Pro Ser Ala Ala His Pro Arg Pro
20 25 30
Val Val Leu Val His Gly Thr Phe Gly Asn Ser Ile Asp Asn Trp Leu
35 40 45
Val Leu Ala Pro Tyr Leu Val Asn Arg Gly Tyr Cys Val Phe Ser Leu
50 55 60
Asp Tyr Gly Gln Leu Pro Gly Val Pro Phe Phe His Gly Leu Gly Pro
65 70 75 80
Ile Asp Lys Ser Ala Glu Gln Leu Asp Ala Phe Val Asp Lys Val Leu
85 90 95
Asp Ala Thr Gly Ala Pro Lys Val Asp Leu Val Gly His Ser Gln Gly
100 105 110
Gly Met Met Pro Asn Tyr Tyr Leu Lys Phe Leu Gly Gly Ala Asp Lys
115 120 125
Val Asn Ala Leu Val Gly Ile Ala Pro Asp Asn His Gly Thr Thr Leu
130 135 140
Leu Gly Leu Thr Lys Leu Leu Pro Phe Phe Pro Gly Val Glu Lys Phe
145 150 155 160
Ile Ser Asp Asn Thr Pro Gly Leu Ala Asp Gln Val Ala Gly Ser Pro
165 170 175
Phe Ile Thr Lys Leu Thr Ala Gly Gly Asp Thr Val Pro Gly Val Arg
180 185 190
Tyr Thr Val Ile Ala Thr Lys Tyr Asp Gln Val Val Thr Pro Tyr Arg
195 200 205
Thr Gln Tyr Leu Asp Gly Pro Asn Val Arg Asn Val Leu Leu Gln Asp
210 215 220
Leu Cys Pro Val Asp Leu Ser Asp His Val Ala Ile Gly Thr Ile Asp
225 230 235 240
Arg Ile Ala Phe His Glu Val Ala Asn Ala Leu Asp Pro Ala Arg Ala
245 250 255
Thr Pro Thr Thr Cys Ala Ser Ile Ile Gly
260 265
<210> 16
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
taatacgact cactataggg 20
<210> 17
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
gctagttatt gctcagcgg 19
Claims (10)
1.一种脂肪酶突变体，其特征在于：是对SEQ ID NO.1所示氨基酸序列进行突变，突变的氨基酸位点包括D89G、V90A、A104V、E232D、V264I中的至少一个。
2.根据权利要求1所述的突变体，其特征在于：突变方式包括以下14种中的任一种：D89G；V90A；A104V；E232D；V264I；A104V和D89G；A104V和V90A；A104V和E232D；A104V和V264I；A104V和V264I和D89G；A104V和V264I和V90A；A104V和V264I和E232D；A104V和V264I和E232D和D89G；A104V和V264I和E232D和V90A。
3.根据权利要求2所述的突变体，其特征在于：突变方式包括以下5种中的任一种：A104V；A104V和V264I；A104V和V264I和E232D；A104V和V264I和E232D和D89G；A104V和V264I和E232D和V90A。
4.根据权利要求2所述的突变体，其特征在于：序列依次如SEQ ID NO.2-15所示。
5.一种编码权利要求1-4所述的任一脂肪酶突变体的核苷酸。
6.权利要求1-4所述的脂肪酶突变体的应用，其特征在于：用植物油脂作为底物与醇反应合成生物柴油。
7.根据权利要求6所述的应用，其特征在于，植物油脂包括：大豆油、葵花油、棕榈油、菜籽油中的至少一种，醇包括：甲醇、乙醇中的至少一种；优选底物为大豆油和甲醇。
8.根据权利要求6所述的应用，其特征在于：反应体系中含有植物油脂、脂肪酶突变体酶、水和醇，脂肪酶突变体酶活为3-8U/g底物植物油脂，8-12％水，反应初始醇浓度为5-7％，每间隔40-80min分别再依次递减添加体系的5％-2％的醇。
9.根据权利要求6所述的应用，其特征在于：反应温度为35-45℃，反应时间为180-540min；反应搅拌转速为800-1200r/min。
10.根据权利要求6所述的应用，其特征在于：所述的脂肪酶突变体是固定化的脂肪酶突变体。
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107916257A (zh) * | 2018-01-09 | 2018-04-17 | 华南理工大学 | T1脂肪酶突变体和应用 |
| CN107960104A (zh) * | 2015-07-06 | 2018-04-24 | 诺维信公司 | 减少气味的方法 |
| AU2017285439A1 (en) * | 2016-06-17 | 2018-12-20 | Arcadia Biosciences, Inc. | Plants with reduced lipase 1 activity |
| CN110540980A (zh) * | 2019-09-07 | 2019-12-06 | 华南理工大学 | 一种海洋链霉菌脂肪酶突变体及其应用 |
-
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107960104A (zh) * | 2015-07-06 | 2018-04-24 | 诺维信公司 | 减少气味的方法 |
| AU2017285439A1 (en) * | 2016-06-17 | 2018-12-20 | Arcadia Biosciences, Inc. | Plants with reduced lipase 1 activity |
| CN107916257A (zh) * | 2018-01-09 | 2018-04-17 | 华南理工大学 | T1脂肪酶突变体和应用 |
| CN110540980A (zh) * | 2019-09-07 | 2019-12-06 | 华南理工大学 | 一种海洋链霉菌脂肪酶突变体及其应用 |
Non-Patent Citations (2)
| Title |
|---|
| ZHAO,Z.等: ""Chain A, Putative secreted lipase，Accession no:5H6B_A"", 《GENBANK》 * |
| 姜占宝 等: ""定点突变引入二硫键提高华根霉脂肪酶热稳定性的研究"", 《工业微生物》 * |
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