CN116334041B - 一种鼠李糖苷酶突变体及其应用 - Google Patents
一种鼠李糖苷酶突变体及其应用 Download PDFInfo
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- 239000007787 solid Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
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- 239000012138 yeast extract Substances 0.000 description 1
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Abstract
本发明公开了一种鼠李糖苷酶突变体及其应用。所述突变体包括如SEQ ID No:1所示的氨基酸序列在第187位、第337位和第521位中的至少一个氨基酸处的突变。与现有技术中的鼠李糖苷酶相比,具有更优良的热稳定性以及更高的催化效率,突变体最适反应温度为80℃,相比野生酶提高了10度;在70℃保温6小时后活性仍没有检测到活性下降,且在70℃下测定的半衰期达10h,稳定性高。以120g/l朝藿定C为底物,在80℃、pH 8.0的条件下反应4h后,淫羊藿苷浓度达到118g/L,转化效率可达98.3%。该方法制备淫羊藿苷操作简单,成本低廉,可适用于大规模工业化生产,更具有市场竞争力。
Description
技术领域
本发明属于基因工程技术领域,具体涉及一种鼠李糖苷酶突变体及其应用。
背景技术
α-L-鼠李糖苷酶(α-L-rhamnosidase,EC 3.2.1.40)是一种能特异性地水解鼠李糖苷键的水解酶,广泛存在于动物、植物和微生物中。目前己报道大多数α-L-鼠李糖苷酶来源于微生物,其中,己表征的细菌源GH78家族α-L-鼠李糖苷酶有19个,大多数来源于革兰氏阳性细菌,包括芽孢杆菌属、乳酸杆菌属、梭菌属、链霉菌属和热微菌门属。
多数从植物中提取的糖苷类黄酮化合物都含有一个或者多个鼠李糖基,例如芦丁、橘皮苷、柚皮苷、新橙皮苷以及朝藿定C等。该系列黄酮化合物中,鼠李糖基的存在很大程度上影响了它们的溶解性以及生物活性。因此,利用α-L-鼠李糖苷酶,将其中的鼠李糖基水解不仅能够极大改善溶解性,提高人体对黄酮类化合物的吸收率,更能够提高黄酮化合物的生物利用率。α-L-鼠李糖苷酶已被广泛地应用在食品、医药等领域。食品工业中能应用于柑桔类果汁的去苦,饮料风味改善以及食品添加剂的生产等。在医药领域中,用于生产药物或者药物中间体以及对药物进行改性提高药效等。由于该酶具有良好的应用前景,因此吸引了国内外越来越多的研究者对其进行研究开发。
淫羊藿苷是一种具有8-异戊烯基黄酮母核的黄酮类物质,CAS号为489-32-7,已被证实在防治肝癌、乳腺癌、肺癌、结肠癌等方面的生物活性显著高于朝藿定C等其它多组分类黄酮类化合物。然而天然环境中,在淫羊藿草中淫羊藿苷的比例一般在2%以下,相对而言,朝藿定C的含量会比淫羊藿苷更丰富,通常是淫羊藿苷含量的5~6倍。与淫羊藿苷结构相比,朝藿定C在3位多一个α-1,2糖苷键连接鼠李糖,如果能将朝藿定C的3位上鼠李糖特异性切除,就可以将朝藿定C转化成淫羊藿苷。由此可见,筛选获得能特异性降解鼠李糖连接的α-1,2糖苷键的α-L-鼠李糖苷酶,研究淫羊藿苷的生物催化制备工艺,对其大规模工业生产具有重要意义。
酶法水解具有温和、高效、专一等特点,适合用于糖苷类黄酮化合物的水解,但现有的α-L-鼠李糖苷酶酶催化朝藿定C制备淫羊藿苷的转化浓度与效率离工业化生产还有一定的距离。如专利申请CN201810110773中,使用来源于多形拟杆菌(Bacteroidesthetaiotaomicron)的鼠李糖苷酶,4h内水解1g/L的朝藿定C,摩尔转化率达90.5%。专利申请CN201910435031公开了重组AnRhaE全细胞催化水解1g/L淫羊藿提取物样品,于37℃进行催化反应1.5h可将朝藿定C完全转化为淫羊藿苷。专利申请CN202010928885将来源于Aspergillus nidulans FGSC A4菌株的α-L-鼠李糖苷酶RhaE在毕赤酵母细胞中进行表达获得重组酶,50℃下反应2h,水解2g/L朝藿定C生成淫羊藿苷。专利申请CN 202111434208利用重组AmRhaE粗酶液催化水解淫羊藿提取物样品,催化水解4g/L朝藿定C,反应24h,转化率为97%。已报道的文献中转化浓度最高的是专利申请CN202111511848,公开了一种碱性嗜热鼠李糖苷酶,在80℃水浴转化100g/L朝藿定C,反应6h,转化率>98.5%。因此,挖掘更多能够高效催化朝藿定C生成淫羊藿苷的α-L-鼠李糖苷酶,以指导工业化的大规模生产淫羊藿苷具有重要价值。
发明内容
本发明的目的在于提供一种新来源的鼠李糖苷酶,并利用定点突变对其进行定向改造,提高其在催化生产淫羊藿苷中的热稳定性及催化效率。
本发明所采取的技术方案是:
本发明的第一方面,提供一种鼠李糖苷酶突变体,所述突变体包括如SEQ ID No.1所示的氨基酸序列在第187位、第337位和第521位中的至少一个氨基酸处的突变。
优选地,所述如SEQ ID No.1所示的氨基酸序列在第187位突变为异亮氨酸;所述如SEQ ID No.1所示的氨基酸序列在第337位突变为丙氨酸;所述如SEQ ID No.1所示的氨基酸序列在第521位突变为丝氨酸。
优选地,所述鼠李糖苷酶突变体的氨基酸序列如SEQ ID NO.2~SEQ ID NO.6任一项所示。
优选地,所述鼠李糖苷酶突变体的氨基酸序列如SEQ ID NO.6所示。
本发明的第二方面,提供一种核酸分子,所述核酸分子的核苷酸序列为:
(I)编码本发明第一方面所述的鼠李糖苷酶突变体;或
(II)与(I)所述的核苷酸序列互补的核苷酸序列。
优选地,所述核酸分子的核苷酸序列如SEQ ID NO.8~SEQ ID NO.12所示。
本发明的第三方面,提供一种重组载体,所述载体可表达本发明第一方面所述的鼠李糖苷酶或含有本发明第二方面所述的核酸分子。
优选地,所述载体包括所述载体包括pET、pCW、pUC或pPIC9k。
本发明的第四方面,提供一种重组细胞,所述重组细胞包含本发明第三方面所述的重组载体。所述重组细胞非动物或植物新品种。
优选地,所述重组细胞选自大肠杆菌、毕赤酵母、酿酒酵母、链霉菌、枯草芽孢杆菌等本领域熟知的能够用于表达目的蛋白的真核或原核细胞。
优选地,所述大肠杆菌包括E.coli BL21(DE3)或E.coli DH5α。
本发明的第五方面,提供本发明第一方面所述的鼠李糖苷酶突变体、本发明第二方面所述核酸分子、本发明第三方面所述重组载体、本发明第四方面所述重组细胞在以下至少一项中的应用:
(a)水解鼠李糖苷键中的应用;
(b)制备水解鼠李糖苷键的产品;
(c)生产淫羊藿苷;
(d)制备生产淫羊藿苷的产品。
优选地,所述鼠李糖苷键包括朝藿定C鼠李糖苷键、芦丁鼠李糖苷键、柚皮苷鼠李糖苷键、杨梅苷鼠李糖苷键、新橙皮苷鼠李糖苷键、柴胡皂苷C鼠李糖苷键、橙皮苷、芦鼠李糖苷键、槲皮苷鼠李糖苷键、薯蓣皂苷鼠李糖苷键中的至少一种。
本发明第六方面,提供一种鼠李糖苷酶突变体的制备方法,通过培养本发明第四方面所述的重组细胞获得鼠李糖苷酶突变体。
在本发明的一些实施方式中,在分离出鼠李糖苷酶突变体的粗酶液后,可以对其进行进一步的纯化以获得更高纯度的鼠李糖苷酶突变体。
在本发明的一些实施方式中,所述纯化方法包括亲和层析。
在本发明的一些优选实施方式中,所述亲和层析包括镍柱亲和层析。
本发明第七方面,提供一种淫羊藿苷的制备方法,使用本发明第一方面所述的鼠李糖苷酶突变体、本发明第二方面所述核酸分子、本发明第三方面所述重组载体、本发明第四方面所述重组细胞制备淫羊藿苷。
优选地,所述制备方法包括:将朝藿定C作为底物,使用本发明第一方面所述鼠李糖苷酶突变体催化朝藿定C制备淫羊藿苷。
优选地,所述催化的条件包括:70~90℃、pH 7~9。
优选地,所述催化的时间包括1~24h。
本发明的第八方面,提供一种产品,所述产品包括本发明第一方面所述的鼠李糖苷酶突变体、本发明第二方面所述核酸分子、本发明第三方面所述重组载体、本发明第四方面所述重组细胞。
优选地,所述产品包括食品、药品、催化剂等。
本发明的有益效果是:
本发明提供了一种鼠李糖苷酶突变体、编码鼠李糖苷酶突变体的核酸分子以及包含上述核酸分子的表达载体和重组细胞。与现有技术中的鼠李糖苷酶相比,具有更优良的热稳定性以及更高的催化效率,突变体最适反应温度为80℃,相比野生酶提高了10度;;在70℃保温6小时后活性仍没有检测到活性下降,且在70℃下测定的半衰期达10h,稳定性高。利用大肠杆菌表达该CAbRha突变体V187I/N337A/L521S基因,以120g/l朝藿定C为底物,在80℃、pH 8.0的条件下反应4h后,淫羊藿苷浓度达到118g/L,转化效率可达98.3%。该方法制备淫羊藿苷操作简单,成本低廉、具有广阔的应用前景,可适用于大规模工业化生产,更具有市场竞争力。
附图说明
图1是显示在本发明实施方案条件下pH对突变体V187I/N337A/L521S的酶活性影响。
图2是显示在本发明实施方案条件下温度对野生型CAbRha及突变体V187I/N337A/L521S的酶活性影响。
图3是显示在本发明实施方案条件下突变体V187I/N337A/L521S的热稳定性。
图4是显示在本发明实施方案条件下野生型CAbRha及突变体V187I/N337 A/L521S转化朝藿定C生产淫羊藿苷的转化率。
图5是显示在本发明实施方案条件下突变体V187I/N337 A/L521S转化朝藿定C生产淫羊藿苷的高效液相色谱检测图。
具体实施方式
以下将结合实施例对本发明的构思及产生的技术效果进行清楚、完整地描述,以充分地理解本发明的目的、特征和效果。显然,所描述的实施例只是本发明的一部分实施例,而不是全部实施例,基于本发明的实施例,本领域的技术人员在不付出创造性劳动的前提下所获得的其他实施例,均属于本发明保护的范围。
朝藿定C及淫羊藿苷HPLC测定:色谱柱,C18;流动相为乙睛和水,比例为26:74,紫外检测器270nm,流速为l mL/min,柱温:30℃,进样量:10μL。
实施例1重组质粒pET-28a-CAbRha的转化
野生型CAbRha基因是由通用生物系统(安徽)股份有限公司合成来源于微生物Candidatus Aminicenantes bacterium(NCBI:PMP97063.1)的多肽的基因(经过密码子优化,氨基酸序列如SEQ ID NO.1)获得的。
SEQ ID NO.1:MIFSDKIFYHRRAKMILSLNLKNKKNNHLECYSQNRNSQIPKTKKISSGHGKNRKFSFFRFNRLASIAIFSFFLIFFMLTEISLPRPSTAGQAVSISGPGAPYDLRVEYLTNPLGVDVSKPRFFWKNAHPERGQVQSAYELIVSSSPDAQSADMWNSGKVNSDSSIQVVYEGKPLESNRTYYWKVRVWDAQGRVSPWSQVARFETGLFSASDWKGEWIGGENLLRKEFDLPAAPRRARVFISGLGYYELRINGRKVGDHVLDPGWTTFSKRVLYVTYDVTRALRPGRNAIGVMLGHGKYNDRALMFQLYVEGEDGNLVEIHSDGTWKTAPGPILEDNVYNGETYDARLEQPGWERPDFEEKNWKPAQKVKAPGGVLSAQLMPAIKVVDTIVPLTMTNPAPGVYVFDLGQNISGWAQLRVRGPRGTDVRLRFAELLYENGMINQENLRSSRAEDHYILKGEGEEVWEPRFTYHGFRYLEVTGFPGTPKIDSVRGRVVHTAVSPVGNFSCSKQILNDIQRIILWGQKTNLHSIPTDCDQRDERMGWMGDAQVTAEEAIMNYDMAAFYTNFLRDIRDVQGEDGSITDTVPHIWGSRPADPAWGTAYPLIAWYMFQYYGDKRVLEEHYDSLKKYVEFLRRKAENGLVKYSYYGDWVAIDKCPGSLVSAFYYYYDVKILAEAARVLGKQPEADLYLKLAAQIKDAFNREFLDPTTRNYAGGSQTANTLPLFIG。
编码该多肽的核苷酸序列如下所示:
SEQ ID NO.7:ATGATTTTCTCAGATAAAATATTTTATCACCGCCGTGCGAAAATGATCTTATCGCTGAACCTTAAGAACAAAAAGAACAACCACCTGGAGTGCTATAGCCAGAACAGGAACAGCCAGATCCCGAAAACCAAAAAGATCTCCAGCGGCCACGGCAAGAACCGCAAGTTTTCCTTTTTTCGTTTCAACCGACTGGCCAGTATTGCGATCTTTTCGTTCTTCCTGATTTTTTTCATGCTGACTGAAATTTCCCTGCCGCGTCCGAGCACCGCGGGTCAGGCAGTGTCTATCTCCGGCCCAGGTGCACCGTATGACCTGCGTGTGGAATATCTGACCAACCCGCTCGGCGTGGATGTTTCCAAACCTCGCTTCTTCTGGAAGAACGCCCATCCGGAACGTGGTCAGGTGCAGAGCGCGTATGAACTGATCGTTAGCTCTAGCCCCGACGCTCAAAGCGCAGACATGTGGAATAGCGGTAAGGTGAATAGCGATAGTTCTATCCAAGTGGTGTACGAGGGCAAGCCGTTGGAGAGCAATCGTACCTACTACTGGAAAGTGCGCGTTTGGGACGCGCAAGGTCGCGTCAGCCCGTGGAGCCAAGTTGCCCGTTTCGAAACGGGTCTGTTCAGCGCGAGCGATTGGAAAGGTGAGTGGATTGGTGGCGAAAACTTGCTGCGTAAAGAGTTTGATCTGCCAGCTGCGCCGCGTAGAGCGCGTGTGTTCATCTCTGGTCTTGGTTATTACGAGTTGCGCATCAACGGTCGCAAGGTCGGTGATCACGTTCTGGACCCGGGTTGGACCACCTTTAGCAAACGTGTACTCTACGTGACGTACGACGTTACCCGCGCGCTGAGACCGGGCCGTAATGCCATCGGTGTTATGCTGGGCCACGGCAAATACAACGACCGTGCGCTAATGTTTCAGCTGTACGTCGAAGGCGAGGACGGCAATCTCGTAGAGATCCACTCCGACGGTACTTGGAAGACGGCTCCGGGTCCGATTTTGGAGGATAATGTTTACAATGGTGAGACATACGACGCGCGTCTGGAGCAGCCGGGTTGGGAACGTCCGGATTTTGAGGAAAAAAACTGGAAACCGGCGCAAAAAGTGAAAGCTCCTGGCGGCGTCTTGTCTGCCCAACTGATGCCGGCGATTAAAGTTGTTGATACCATTGTGCCGTTGACCATGACCAACCCGGCGCCAGGTGTTTATGTGTTTGATCTGGGCCAGAATATTAGTGGCTGGGCACAATTGAGAGTTAGAGGCCCACGCGGTACGGACGTGCGGTTGCGTTTCGCCGAATTACTGTACGAGAACGGTATGATTAACCAGGAGAACCTCCGTAGCTCGCGGGCTGAAGACCACTATATTTTAAAGGGTGAAGGTGAGGAGGTTTGGGAACCGCGTTTCACTTACCATGGCTTCCGTTATCTGGAGGTGACCGGTTTTCCGGGCACTCCGAAGATCGATTCCGTTCGTGGCCGCGTGGTTCATACCGCCGTTTCCCCGGTTGGTAATTTTAGCTGCAGCAAGCAGATCCTAAACGATATTCAACGTATTATTCTGTGGGGCCAGAAAACCAACCTGCATTCGATCCCGACCGATTGTGATCAGCGTGATGAACGCATGGGTTGGATGGGCGACGCGCAAGTCACTGCGGAGGAAGCGATTATGAATTACGACATGGCAGCGTTTTATACCAACTTCCTGCGTGACATCCGTGACGTGCAGGGCGAGGATGGCTCCATTACCGACACCGTTCCGCATATTTGGGGTAGCCGACCGGCGGACCCTGCGTGGGGTACGGCATATCCGCTGATCGCGTGGTATATGTTCCAGTATTATGGAGACAAGCGCGTCTTGGAAGAGCACTACGATAGCCTGAAGAAATACGTCGAGTTCCTTAGACGTAAGGCTGAGAACGGGCTGGTGAAGTATAGCTATTACGGCGACTGGGTTGCAATTGATAAGTGCCCGGGCTCTCTGGTTTCAGCGTTCTATTACTACTACGACGTGAAAATCCTGGCTGAAGCGGCTCGCGTTTTGGGGAAACAACCGGAAGCCGACCTGTACCTGAAGCTGGCGGCACAGATCAAAGATGCCTTTAACCGTGAATTCCTGGATCCGACCACGCGTAATTACGCAGGTGGTTCACAAACCGCTAATACCCTGCCGTTGTTTATCGGC。
利用限制性内切酶Nde I和EcoR I,将获得的野生型CAbRha酶基因插入到表达质粒pET-28a(+)中,从而产生重组载体pET-28a-CAbRha。通过常规转化方法,将该重组载体转化到大肠杆菌E.coli BL21(DE3)中,转化得到含有野生型CAbRha基因的基因工程菌E.coliBL21(DE3)-pET-28a-CAbRha并保藏在-80℃的超低温冰箱中。
实施例2突变体的获得
采用定点突变技术获得单点突变或组合突变体,定点突变的引物采用诺唯赞公司提供的CE Design V1.04设计。突变引物见表1。
表1突变引物
突变 | 突变引物的核苷酸序列 | 对应序列号 |
V187I-F | AGTGCGCATTTTTGGGACGCGCAAGGTCGCGT | SEQ ID NO.13 |
V187I-R | TCCCAAAAATGCGCACTTTCCAGTAGTAGGTACG | SEQ ID NO.14 |
N337A-F | GGAGGATGCTTGTTTACAATGGTGAGACATACGACG | SEQ ID NO.15 |
N337A-R | GTAAACAAGCATCCTCCAAAATCGGACCCGGA | SEQ ID NO.16 |
L521S-F | TATTATTTCGGTGGGGCCAGAAAACCAACCTG | SEQ ID NO.17 |
C374T-R | GCCCCACCGAAATAATACGTTGAATATCGTTTAGGATCT | SEQ ID NO.18 |
定点突变以pET-28a-CAbRha重组质粒为模板,利用Vazyme 2xphanta master mix进行全质粒扩增,按照表2设置反应体系。
表2定点突变体系
名称 | 体积(μl) |
Vazyme 2xphanta master mix | 12.5μL |
pET-28a-CAbRha载体模板 | 1μL |
引物-F | 1μL |
引物-R | 1μL |
ddH2O | 加至25μL |
PCR程序为:94℃预变性3min,94℃变性30s,65℃退火30s,72℃延伸5min,反应30个循环后,再72℃延伸10min,最后4℃保温。PCR反应结束后,加入1μL DpnI消化酶,37℃反应2h消化模板,回收纯化后的PCR产物转化到E.coli BL21(DE3)感受态细胞中。
转化方法:取10μL PCR产物与100μL的诺唯赞商用E.coli BL21(DE3)感受态混匀冰浴20min后,42℃热激90s后迅速拿出,冰浴2min,加入400μL的LB液体培养基,37℃,200rpm,复苏60min,取100ul菌液涂布于含有50μg/mL卡那抗性的固体LB平板上,37℃恒温箱过夜培养。
次日从平板中挑选重组大肠杆菌BL21三株,将上述重组菌从平板分别接种到装有5mL含有相应抗性的液体LB培养基(LB(g/L):蛋白胨10,氯化钠10,酵母提取物5)的50mL的摇管中,加入相应抗性,在摇床上37℃恒温培养12h,转速200rpm。培养结束后抽提质粒,送通用公司进行测序。最后将测序结果与野生型酶蛋白核酸序列进行比对,确定突变是否成功。
按照上述方法先进行单个位点突变,再以进行过一次突变后的重组质粒为模板进行二次或三次突变,获得的突变体依据其突变位点分别命名为V187I、N337A、L521S、V187I/N337A、V187I/N337A/L521S,V187I的氨基酸序列如SEQ ID NO.2所示,核苷酸序列如SEQ IDNO.8所示;N337A的氨基酸序列如SEQ ID NO.3所示,核苷酸序列如SEQ ID NO.9所示、L521S的氨基酸序列如SEQ ID NO.4所示,核苷酸序列如SEQ ID NO.10所示;V187I/N337A的氨基酸序列如SEQ ID NO.5所示,核苷酸序列为SEQ ID NO.11所示;V187I/N337A/L521S的氨基酸序列如SEQ ID NO.6所示,核苷酸序列为SEQ ID NO.12所示。
其中,SEQ ID No.1所示的氨基酸序列第187位缬氨酸突变为异亮氨酸后的V187I的氨基酸序列如下所示:
MIFSDKIFYHRRAKMILSLNLKNKKNNHLECYSQNRNSQIPKTKKISSGHGKNRKFSFFRFNRLASIAIFSFFLIFFMLTEISLPRPSTAGQAVSISGPGAPYDLRVEYLTNPLGVDVSKPRFFWKNAHPERGQVQSAYELIVSSSPDAQSADMWNSGKVNSDSSIQVVYEGKPLESNRTYYWKVRIWDAQGRVSPWSQVARFETGLFSASDWKGEWIGGENLLRKEFDLPAAPRRARVFISGLGYYELRINGRKVGDHVLDPGWTTFSKRVLYVTYDVTRALRPGRNAIGVMLGHGKYNDRALMFQLYVEGEDGNLVEIHSDGTWKTAPGPILEDNVYNGETYDARLEQPGWERPDFEEKNWKPAQKVKAPGGVLSAQLMPAIKVVDTIVPLTMTNPAPGVYVFDLGQNISGWAQLRVRGPRGTDVRLRFAELLYENGMINQENLRSSRAEDHYILKGEGEEVWEPRFTYHGFRYLEVTGFPGTPKIDSVRGRVVHTAVSPVGNFSCSKQILNDIQRIILWGQKTNLHSIPTDCDQRDERMGWMGDAQVTAEEAIMNYDMAAFYTNFLRDIRDVQGEDGSITDTVPHIWGSRPADPAWGTAYPLIAWYMFQYYGDKRVLEEHYDSLKKYVEFLRRKAENGLVKYSYYGDWVAIDKCPGSLVSAFYYYYDVKILAEAARVLGKQPEADLYLKLAAQIKDAFNREFLDPTTRNYAGGSQTANTLPLFIG(SEQ ID NO.2);
核苷酸序列如下所示:
ATGATTTTCTCAGATAAAATATTTTATCACCGCCGTGCGAAAATGATCTTATCGCTGAACCTTAAGAACAAAAAGAACAACCACCTGGAGTGCTATAGCCAGAACAGGAACAGCCAGATCCCGAAAACCAAAAAGATCTCCAGCGGCCACGGCAAGAACCGCAAGTTTTCCTTTTTTCGTTTCAACCGACTGGCCAGTATTGCGATCTTTTCGTTCTTCCTGATTTTTTTCATGCTGACTGAAATTTCCCTGCCGCGTCCGAGCACCGCGGGTCAGGCAGTGTCTATCTCCGGCCCAGGTGCACCGTATGACCTGCGTGTGGAATATCTGACCAACCCGCTCGGCGTGGATGTTTCCAAACCTCGCTTCTTCTGGAAGAACGCCCATCCGGAACGTGGTCAGGTGCAGAGCGCGTATGAACTGATCGTTAGCTCTAGCCCCGACGCTCAAAGCGCAGACATGTGGAATAGCGGTAAGGTGAATAGCGATAGTTCTATCCAAGTGGTGTACGAGGGCAAGCCGTTGGAGAGCAATCGTACCTACTACTGGAAAGTGCGCATTTGGGACGCGCAAGGTCGCGTCAGCCCGTGGAGCCAAGTTGCCCGTTTCGAAACGGGTCTGTTCAGCGCGAGCGATTGGAAAGGTGAGTGGATTGGTGGCGAAAACTTGCTGCGTAAAGAGTTTGATCTGCCAGCTGCGCCGCGTAGAGCGCGTGTGTTCATCTCTGGTCTTGGTTATTACGAGTTGCGCATCAACGGTCGCAAGGTCGGTGATCACGTTCTGGACCCGGGTTGGACCACCTTTAGCAAACGTGTACTCTACGTGACGTACGACGTTACCCGCGCGCTGAGACCGGGCCGTAATGCCATCGGTGTTATGCTGGGCCACGGCAAATACAACGACCGTGCGCTAATGTTTCAGCTGTACGTCGAAGGCGAGGACGGCAATCTCGTAGAGATCCACTCCGACGGTACTTGGAAGACGGCTCCGGGTCCGATTTTGGAGGATAATGTTTACAATGGTGAGACATACGACGCGCGTCTGGAGCAGCCGGGTTGGGAACGTCCGGATTTTGAGGAAAAAAACTGGAAACCGGCGCAAAAAGTGAAAGCTCCTGGCGGCGTCTTGTCTGCCCAACTGATGCCGGCGATTAAAGTTGTTGATACCATTGTGCCGTTGACCATGACCAACCCGGCGCCAGGTGTTTATGTGTTTGATCTGGGCCAGAATATTAGTGGCTGGGCACAATTGAGAGTTAGAGGCCCACGCGGTACGGACGTGCGGTTGCGTTTCGCCGAATTACTGTACGAGAACGGTATGATTAACCAGGAGAACCTCCGTAGCTCGCGGGCTGAAGACCACTATATTTTAAAGGGTGAAGGTGAGGAGGTTTGGGAACCGCGTTTCACTTACCATGGCTTCCGTTATCTGGAGGTGACCGGTTTTCCGGGCACTCCGAAGATCGATTCCGTTCGTGGCCGCGTGGTTCATACCGCCGTTTCCCCGGTTGGTAATTTTAGCTGCAGCAAGCAGATCCTAAACGATATTCAACGTATTATTCTGTGGGGCCAGAAAACCAACCTGCATTCGATCCCGACCGATTGTGATCAGCGTGATGAACGCATGGGTTGGATGGGCGACGCGCAAGTCACTGCGGAGGAAGCGATTATGAATTACGACATGGCAGCGTTTTATACCAACTTCCTGCGTGACATCCGTGACGTGCAGGGCGAGGATGGCTCCATTACCGACACCGTTCCGCATATTTGGGGTAGCCGACCGGCGGACCCTGCGTGGGGTACGGCATATCCGCTGATCGCGTGGTATATGTTCCAGTATTATGGAGACAAGCGCGTCTTGGAAGAGCACTACGATAGCCTGAAGAAATACGTCGAGTTCCTTAGACGTAAGGCTGAGAACGGGCTGGTGAAGTATAGCTATTACGGCGACTGGGTTGCAATTGATAAGTGCCCGGGCTCTCTGGTTTCAGCGTTCTATTACTACTACGACGTGAAAATCCTGGCTGAAGCGGCTCGCGTTTTGGGGAAACAACCGGAAGCCGACCTGTACCTGAAGCTGGCGGCACAGATCAAAGATGCCTTTAACCGTGAATTCCTGGATCCGACCACGCGTAATTACGCAGGTGGTTCACAAACCGCTAATACCCTGCCGTTGTTTATCGGC(SEQ IDNO.8);
SEQ ID No.1所示的氨基酸序列第337位天冬酰胺突变为丙氨酸后的N337A的氨基酸序列如下所示:
MIFSDKIFYHRRAKMILSLNLKNKKNNHLECYSQNRNSQIPKTKKISSGHGKNRKFSFFRFNRLASIAIFSFFLIFFMLTEISLPRPSTAGQAVSISGPGAPYDLRVEYLTNPLGVDVSKPRFFWKNAHPERGQVQSAYELIVSSSPDAQSADMWNSGKVNSDSSIQVVYEGKPLESNRTYYWKVRVWDAQGRVSPWSQVARFETGLFSASDWKGEWIGGENLLRKEFDLPAAPRRARVFISGLGYYELRINGRKVGDHVLDPGWTTFSKRVLYVTYDVTRALRPGRNAIGVMLGHGKYNDRALMFQLYVEGEDGNLVEIHSDGTWKTAPGPILEDAVYNGETYDARLEQPGWERPDFEEKNWKPAQKVKAPGGVLSAQLMPAIKVVDTIVPLTMTNPAPGVYVFDLGQNISGWAQLRVRGPRGTDVRLRFAELLYENGMINQENLRSSRAEDHYILKGEGEEVWEPRFTYHGFRYLEVTGFPGTPKIDSVRGRVVHTAVSPVGNFSCSKQILNDIQRIILWGQKTNLHSIPTDCDQRDERMGWMGDAQVTAEEAIMNYDMAAFYTNFLRDIRDVQGEDGSITDTVPHIWGSRPADPAWGTAYPLIAWYMFQYYGDKRVLEEHYDSLKKYVEFLRRKAENGLVKYSYYGDWVAIDKCPGSLVSAFYYYYDVKILAEAARVLGKQPEADLYLKLAAQIKDAFNREFLDPTTRNYAGGSQTANTLPLFIG(SEQ ID NO.3);
核苷酸序列如下:
ATGATTTTCTCAGATAAAATATTTTATCACCGCCGTGCGAAAATGATCTTATCGC
TGAACCTTAAGAACAAAAAGAACAACCACCTGGAGTGCTATAGCCAGAACAGGA
ACAGCCAGATCCCGAAAACCAAAAAGATCTCCAGCGGCCACGGCAAGAACCGCA
AGTTTTCCTTTTTTCGTTTCAACCGACTGGCCAGTATTGCGATCTTTTCGTTCTTCCT
GATTTTTTTCATGCTGACTGAAATTTCCCTGCCGCGTCCGAGCACCGCGGGTCAGG
CAGTGTCTATCTCCGGCCCAGGTGCACCGTATGACCTGCGTGTGGAATATCTGACC
AACCCGCTCGGCGTGGATGTTTCCAAACCTCGCTTCTTCTGGAAGAACGCCCATCC
GGAACGTGGTCAGGTGCAGAGCGCGTATGAACTGATCGTTAGCTCTAGCCCCGAC
GCTCAAAGCGCAGACATGTGGAATAGCGGTAAGGTGAATAGCGATAGTTCTATCCA
AGTGGTGTACGAGGGCAAGCCGTTGGAGAGCAATCGTACCTACTACTGGAAAGTG
CGCGTTTGGGACGCGCAAGGTCGCGTCAGCCCGTGGAGCCAAGTTGCCCGTTTCG
AAACGGGTCTGTTCAGCGCGAGCGATTGGAAAGGTGAGTGGATTGGTGGCGAAA
ACTTGCTGCGTAAAGAGTTTGATCTGCCAGCTGCGCCGCGTAGAGCGCGTGTGTTC
ATCTCTGGTCTTGGTTATTACGAGTTGCGCATCAACGGTCGCAAGGTCGGTGATCA
CGTTCTGGACCCGGGTTGGACCACCTTTAGCAAACGTGTACTCTACGTGACGTACG
ACGTTACCCGCGCGCTGAGACCGGGCCGTAATGCCATCGGTGTTATGCTGGGCCAC
GGCAAATACAACGACCGTGCGCTAATGTTTCAGCTGTACGTCGAAGGCGAGGACG
GCAATCTCGTAGAGATCCACTCCGACGGTACTTGGAAGACGGCTCCGGGTCCGATT
TTGGAGGATGCTGTTTACAATGGTGAGACATACGACGCGCGTCTGGAGCAGCCGG
GTTGGGAACGTCCGGATTTTGAGGAAAAAAACTGGAAACCGGCGCAAAAAGTGA
AAGCTCCTGGCGGCGTCTTGTCTGCCCAACTGATGCCGGCGATTAAAGTTGTTGAT
ACCATTGTGCCGTTGACCATGACCAACCCGGCGCCAGGTGTTTATGTGTTTGATCT
GGGCCAGAATATTAGTGGCTGGGCACAATTGAGAGTTAGAGGCCCACGCGGTACG
GACGTGCGGTTGCGTTTCGCCGAATTACTGTACGAGAACGGTATGATTAACCAGGA
GAACCTCCGTAGCTCGCGGGCTGAAGACCACTATATTTTAAAGGGTGAAGGTGAG
GAGGTTTGGGAACCGCGTTTCACTTACCATGGCTTCCGTTATCTGGAGGTGACCGG
TTTTCCGGGCACTCCGAAGATCGATTCCGTTCGTGGCCGCGTGGTTCATACCGCCG
TTTCCCCGGTTGGTAATTTTAGCTGCAGCAAGCAGATCCTAAACGATATTCAACGTA
TTATTCTGTGGGGCCAGAAAACCAACCTGCATTCGATCCCGACCGATTGTGATCAG
CGTGATGAACGCATGGGTTGGATGGGCGACGCGCAAGTCACTGCGGAGGAAGCGA
TTATGAATTACGACATGGCAGCGTTTTATACCAACTTCCTGCGTGACATCCGTGACG
TGCAGGGCGAGGATGGCTCCATTACCGACACCGTTCCGCATATTTGGGGTAGCCGA
CCGGCGGACCCTGCGTGGGGTACGGCATATCCGCTGATCGCGTGGTATATGTTCCA
GTATTATGGAGACAAGCGCGTCTTGGAAGAGCACTACGATAGCCTGAAGAAATAC
GTCGAGTTCCTTAGACGTAAGGCTGAGAACGGGCTGGTGAAGTATAGCTATTACGG
CGACTGGGTTGCAATTGATAAGTGCCCGGGCTCTCTGGTTTCAGCGTTCTATTACTA
CTACGACGTGAAAATCCTGGCTGAAGCGGCTCGCGTTTTGGGGAAACAACCGGAA
GCCGACCTGTACCTGAAGCTGGCGGCACAGATCAAAGATGCCTTTAACCGTGAATT
CCTGGATCCGACCACGCGTAATTACGCAGGTGGTTCACAAACCGCTAATACCCTGC
CGTTGTTTATCGGC(SEQ ID NO.9);
SEQ ID No.1所示的氨基酸序列第521位亮氨酸突变为丝氨酸后的L521S的氨基酸序列如下所示:
MIFSDKIFYHRRAKMILSLNLKNKKNNHLECYSQNRNSQIPKTKKISSGHGKNRKFSFFRFNRLASIAIFSFFLIFFMLTEISLPRPSTAGQAVSISGPGAPYDLRVEYLTNPLGVDVSKPRFFWKNAHPERGQVQSAYELIVSSSPDAQSADMWNSGKVNSDSSIQVVYEGKPLESNRTYYWKVRVWDAQGRVSPWSQVARFETGLFSASDWKGEWIGGENLLRKEFDLPAAPRRARVFISGLGYYELRINGRKVGDHVLDPGWTTFSKRVLYVTYDVTRALRPGRNAIGVMLGHGKYNDRALMFQLYVEGEDGNLVEIHSDGTWKTAPGPILEDNVYNGETYDARLEQPGWERPDFEEKNWKPAQKVKAPGGVLSAQLMPAIKVVDTIVPLTMTNPAPGVYVFDLGQNISGWAQLRVRGPRGTDVRLRFAELLYENGMINQENLRSSRAEDHYILKGEGEEVWEPRFTYHGFRYLEVTGFPGTPKIDSVRGRVVHTAVSPVGNFSCSKQILNDIQRIISWGQKTNLHSIPTDCDQRDERMGWMGDAQVTAEEAIMNYDMAAFYTNFLRDIRDVQGEDGSITDTVPHIWGSRPADPAWGTAYPLIAWYMFQYYGDKRVLEEHYDSLKKYVEFLRRKAENGLVKYSYYGDWVAIDKCPGSLVSAFYYYYDVKILAEAARVLGKQPEADLYLKLAAQIKDAFNREFLDPTTRNYAGGSQTANTLPLFIG(SEQ ID NO.4);
核苷酸序列如下:
ATGATTTTCTCAGATAAAATATTTTATCACCGCCGTGCGAAAATGATCTTATCGC
TGAACCTTAAGAACAAAAAGAACAACCACCTGGAGTGCTATAGCCAGAACAGGA
ACAGCCAGATCCCGAAAACCAAAAAGATCTCCAGCGGCCACGGCAAGAACCGCA
AGTTTTCCTTTTTTCGTTTCAACCGACTGGCCAGTATTGCGATCTTTTCGTTCTTCCT
GATTTTTTTCATGCTGACTGAAATTTCCCTGCCGCGTCCGAGCACCGCGGGTCAGG
CAGTGTCTATCTCCGGCCCAGGTGCACCGTATGACCTGCGTGTGGAATATCTGACC
AACCCGCTCGGCGTGGATGTTTCCAAACCTCGCTTCTTCTGGAAGAACGCCCATCC
GGAACGTGGTCAGGTGCAGAGCGCGTATGAACTGATCGTTAGCTCTAGCCCCGAC
GCTCAAAGCGCAGACATGTGGAATAGCGGTAAGGTGAATAGCGATAGTTCTATCCA
AGTGGTGTACGAGGGCAAGCCGTTGGAGAGCAATCGTACCTACTACTGGAAAGTG
CGCGTTTGGGACGCGCAAGGTCGCGTCAGCCCGTGGAGCCAAGTTGCCCGTTTCG
AAACGGGTCTGTTCAGCGCGAGCGATTGGAAAGGTGAGTGGATTGGTGGCGAAA
ACTTGCTGCGTAAAGAGTTTGATCTGCCAGCTGCGCCGCGTAGAGCGCGTGTGTTC
ATCTCTGGTCTTGGTTATTACGAGTTGCGCATCAACGGTCGCAAGGTCGGTGATCA
CGTTCTGGACCCGGGTTGGACCACCTTTAGCAAACGTGTACTCTACGTGACGTACG
ACGTTACCCGCGCGCTGAGACCGGGCCGTAATGCCATCGGTGTTATGCTGGGCCAC
GGCAAATACAACGACCGTGCGCTAATGTTTCAGCTGTACGTCGAAGGCGAGGACG
GCAATCTCGTAGAGATCCACTCCGACGGTACTTGGAAGACGGCTCCGGGTCCGATT
TTGGAGGATAATGTTTACAATGGTGAGACATACGACGCGCGTCTGGAGCAGCCGG
GTTGGGAACGTCCGGATTTTGAGGAAAAAAACTGGAAACCGGCGCAAAAAGTGA
AAGCTCCTGGCGGCGTCTTGTCTGCCCAACTGATGCCGGCGATTAAAGTTGTTGAT
ACCATTGTGCCGTTGACCATGACCAACCCGGCGCCAGGTGTTTATGTGTTTGATCT
GGGCCAGAATATTAGTGGCTGGGCACAATTGAGAGTTAGAGGCCCACGCGGTACG
GACGTGCGGTTGCGTTTCGCCGAATTACTGTACGAGAACGGTATGATTAACCAGGA
GAACCTCCGTAGCTCGCGGGCTGAAGACCACTATATTTTAAAGGGTGAAGGTGAG
GAGGTTTGGGAACCGCGTTTCACTTACCATGGCTTCCGTTATCTGGAGGTGACCGG
TTTTCCGGGCACTCCGAAGATCGATTCCGTTCGTGGCCGCGTGGTTCATACCGCCG
TTTCCCCGGTTGGTAATTTTAGCTGCAGCAAGCAGATCCTAAACGATATTCAACGTA
TTATTTCGTGGGGCCAGAAAACCAACCTGCATTCGATCCCGACCGATTGTGATCAG
CGTGATGAACGCATGGGTTGGATGGGCGACGCGCAAGTCACTGCGGAGGAAGCGA
TTATGAATTACGACATGGCAGCGTTTTATACCAACTTCCTGCGTGACATCCGTGACG
TGCAGGGCGAGGATGGCTCCATTACCGACACCGTTCCGCATATTTGGGGTAGCCGA
CCGGCGGACCCTGCGTGGGGTACGGCATATCCGCTGATCGCGTGGTATATGTTCCA
GTATTATGGAGACAAGCGCGTCTTGGAAGAGCACTACGATAGCCTGAAGAAATAC
GTCGAGTTCCTTAGACGTAAGGCTGAGAACGGGCTGGTGAAGTATAGCTATTACGG
CGACTGGGTTGCAATTGATAAGTGCCCGGGCTCTCTGGTTTCAGCGTTCTATTACTA
CTACGACGTGAAAATCCTGGCTGAAGCGGCTCGCGTTTTGGGGAAACAACCGGAA
GCCGACCTGTACCTGAAGCTGGCGGCACAGATCAAAGATGCCTTTAACCGTGAATT
CCTGGATCCGACCACGCGTAATTACGCAGGTGGTTCACAAACCGCTAATACCCTGC
CGTTGTTTATCGGC(SEQ ID NO.10);
SEQ ID No.1所示的氨基酸序列第187位缬氨酸突变为异亮氨酸、第337位天冬酰胺突变为丙氨酸后的V187I/N337A的氨基酸序列如下所示:
MIFSDKIFYHRRAKMILSLNLKNKKNNHLECYSQNRNSQIPKTKKISSGHGKNRKFSFFRFNRLASIAIFSFFLIFFMLTEISLPRPSTAGQAVSISGPGAPYDLRVEYLTNPLGVDVSKPRFFWKNAHPERGQVQSAYELIVSSSPDAQSADMWNSGKVNSDSSIQVVYEGKPLESNRTYYWKVRIWDAQGRVSPWSQVARFETGLFSASDWKGEWIGGENLLRKEFDLPAAPRRARVFISGLGYYELRINGRKVGDHVLDPGWTTFSKRVLYVTYDVTRALRPGRNAIGVMLGHGKYNDRALMFQLYVEGEDGNLVEIHSDGTWKTAPGPILEDAVYNGETYDARLEQPGWERPDFEEKNWKPAQKVKAPGGVLSAQLMPAIKVVDTIVPLTMTNPAPGVYVFDLGQNISGWAQLRVRGPRGTDVRLRFAELLYENGMINQENLRSSRAEDHYILKGEGEEVWEPRFTYHGFRYLEVTGFPGTPKIDSVRGRVVHTAVSPVGNFSCSKQILNDIQRIILWGQKTNLHSIPTDCDQRDERMGWMGDAQVTAEEAIMNYDMAAFYTNFLRDIRDVQGEDGSITDTVPHIWGSRPADPAWGTAYPLIAWYMFQYYGDKRVLEEHYDSLKKYVEFLRRKAENGLVKYSYYGDWVAIDKCPGSLVSAFYYYYDVKILAEAARVLGKQPEADLYLKLAAQIKDAFNREFLDPTTRNYAGGSQTANTLPLFIG(SEQ ID NO.5);
核苷酸序列如下所示:
ATGATTTTCTCAGATAAAATATTTTATCACCGCCGTGCGAAAATGATCTTATCGCTGAACCTTAAGAACAAAAAGAACAACCACCTGGAGTGCTATAGCCAGAACAGGAACAGCCAGATCCCGAAAACCAAAAAGATCTCCAGCGGCCACGGCAAGAACCGCAAGTTTTCCTTTTTTCGTTTCAACCGACTGGCCAGTATTGCGATCTTTTCGTTCTTCCTGATTTTTTTCATGCTGACTGAAATTTCCCTGCCGCGTCCGAGCACCGCGGGTCAGGCAGTGTCTATCTCCGGCCCAGGTGCACCGTATGACCTGCGTGTGGAATATCTGACCAACCCGCTCGGCGTGGATGTTTCCAAACCTCGCTTCTTCTGGAAGAACGCCCATCCGGAACGTGGTCAGGTGCAGAGCGCGTATGAACTGATCGTTAGCTCTAGCCCCGACGCTCAAAGCGCAGACATGTGGAATAGCGGTAAGGTGAATAGCGATAGTTCTATCCAAGTGGTGTACGAGGGCAAGCCGTTGGAGAGCAATCGTACCTACTACTGGAAAGTGCGCATTTGGGACGCGCAAGGTCGCGTCAGCCCGTGGAGCCAAGTTGCCCGTTTCGAAACGGGTCTGTTCAGCGCGAGCGATTGGAAAGGTGAGTGGATTGGTGGCGAAAACTTGCTGCGTAAAGAGTTTGATCTGCCAGCTGCGCCGCGTAGAGCGCGTGTGTTCATCTCTGGTCTTGGTTATTACGAGTTGCGCATCAACGGTCGCAAGGTCGGTGATCACGTTCTGGACCCGGGTTGGACCACCTTTAGCAAACGTGTACTCTACGTGACGTACGACGTTACCCGCGCGCTGAGACCGGGCCGTAATGCCATCGGTGTTATGCTGGGCCACGGCAAATACAACGACCGTGCGCTAATGTTTCAGCTGTACGTCGAAGGCGAGGACGGCAATCTCGTAGAGATCCACTCCGACGGTACTTGGAAGACGGCTCCGGGTCCGATTTTGGAGGATGCTGTTTACAATGGTGAGACATACGACGCGCGTCTGGAGCAGCCGGGTTGGGAACGTCCGGATTTTGAGGAAAAAAACTGGAAACCGGCGCAAAAAGTGAAAGCTCCTGGCGGCGTCTTGTCTGCCCAACTGATGCCGGCGATTAAAGTTGTTGATACCATTGTGCCGTTGACCATGACCAACCCGGCGCCAGGTGTTTATGTGTTTGATCTGGGCCAGAATATTAGTGGCTGGGCACAATTGAGAGTTAGAGGCCCACGCGGTACGGACGTGCGGTTGCGTTTCGCCGAATTACTGTACGAGAACGGTATGATTAACCAGGAGAACCTCCGTAGCTCGCGGGCTGAAGACCACTATATTTTAAAGGGTGAAGGTGAGGAGGTTTGGGAACCGCGTTTCACTTACCATGGCTTCCGTTATCTGGAGGTGACCGGTTTTCCGGGCACTCCGAAGATCGATTCCGTTCGTGGCCGCGTGGTTCATACCGCCGTTTCCCCGGTTGGTAATTTTAGCTGCAGCAAGCAGATCCTAAACGATATTCAACGTATTATTCTGTGGGGCCAGAAAACCAACCTGCATTCGATCCCGACCGATTGTGATCAGCGTGATGAACGCATGGGTTGGATGGGCGACGCGCAAGTCACTGCGGAGGAAGCGATTATGAATTACGACATGGCAGCGTTTTATACCAACTTCCTGCGTGACATCCGTGACGTGCAGGGCGAGGATGGCTCCATTACCGACACCGTTCCGCATATTTGGGGTAGCCGACCGGCGGACCCTGCGTGGGGTACGGCATATCCGCTGATCGCGTGGTATATGTTCCAGTATTATGGAGACAAGCGCGTCTTGGAAGAGCACTACGATAGCCTGAAGAAATACGTCGAGTTCCTTAGACGTAAGGCTGAGAACGGGCTGGTGAAGTATAGCTATTACGGCGACTGGGTTGCAATTGATAAGTGCCCGGGCTCTCTGGTTTCAGCGTTCTATTACTACTACGACGTGAAAATCCTGGCTGAAGCGGCTCGCGTTTTGGGGAAACAACCGGAAGCCGACCTGTACCTGAAGCTGGCGGCACAGATCAAAGATGCCTTTAACCGTGAATTCCTGGATCCGACCACGCGTAATTACGCAGGTGGTTCACAAACCGCTAATACCCTGCCGTTGTTTATCGGC(SEQ IDNO.11);
SEQ ID No.1所示的氨基酸序列第187位缬氨酸突变为异亮氨酸、第337位天冬酰胺突变为丙氨酸、第521位亮氨酸突变为丝氨酸后的V187I/N337A/L521S的氨基酸序列如下所示:
MIFSDKIFYHRRAKMILSLNLKNKKNNHLECYSQNRNSQIPKTKKISSGHGKNRKFSFFRFNRLASIAIFSFFLIFFMLTEISLPRPSTAGQAVSISGPGAPYDLRVEYLTNPLGVDVSKPRFFWKNAHPERGQVQSAYELIVSSSPDAQSADMWNSGKVNSDSSIQVVYEGKPLESNRTYYWKVRIWDAQGRVSPWSQVARFETGLFSASDWKGEWIGGENLLRKEFDLPAAPRRARVFISGLGYYELRINGRKVGDHVLDPGWTTFSKRVLYVTYDVTRALRPGRNAIGVMLGHGKYNDRALMFQLYVEGEDGNLVEIHSDGTWKTAPGPILEDAVYNGETYDARLEQPGWERPDFEEKNWKPAQKVKAPGGVLSAQLMPAIKVVDTIVPLTMTNPAPGVYVFDLGQNISGWAQLRVRGPRGTDVRLRFAELLYENGMINQENLRSSRAEDHYILKGEGEEVWEPRFTYHGFRYLEVTGFPGTPKIDSVRGRVVHTAVSPVGNFSCSKQILNDIQRIISWGQKTNLHSIPTDCDQRDERMGWMGDAQVTAEEAIMNYDMAAFYTNFLRDIRDVQGEDGSITDTVPHIWGSRPADPAWGTAYPLIAWYMFQYYGDKRVLEEHYDSLKKYVEFLRRKAENGLVKYSYYGDWVAIDKCPGSLVSAFYYYYDVKILAEAARVLGKQPEADLYLKLAAQIKDAFNREFLDPTTRNYAGGSQTANTLPLFIG(SEQ ID NO.6);
核苷酸序列如下所示:
ATGATTTTCTCAGATAAAATATTTTATCACCGCCGTGCGAAAATGATCTTATCGCTGAACCTTAAGAACAAAAAGAACAACCACCTGGAGTGCTATAGCCAGAACAGGAACAGCCAGATCCCGAAAACCAAAAAGATCTCCAGCGGCCACGGCAAGAACCGCAAGTTTTCCTTTTTTCGTTTCAACCGACTGGCCAGTATTGCGATCTTTTCGTTCTTCCTGATTTTTTTCATGCTGACTGAAATTTCCCTGCCGCGTCCGAGCACCGCGGGTCAGGCAGTGTCTATCTCCGGCCCAGGTGCACCGTATGACCTGCGTGTGGAATATCTGACCAACCCGCTCGGCGTGGATGTTTCCAAACCTCGCTTCTTCTGGAAGAACGCCCATCCGGAACGTGGTCAGGTGCAGAGCGCGTATGAACTGATCGTTAGCTCTAGCCCCGACGCTCAAAGCGCAGACATGTGGAATAGCGGTAAGGTGAATAGCGATAGTTCTATCCAAGTGGTGTACGAGGGCAAGCCGTTGGAGAGCAATCGTACCTACTACTGGAAAGTGCGCATTTGGGACGCGCAAGGTCGCGTCAGCCCGTGGAGCCAAGTTGCCCGTTTCGAAACGGGTCTGTTCAGCGCGAGCGATTGGAAAGGTGAGTGGATTGGTGGCGAAAACTTGCTGCGTAAAGAGTTTGATCTGCCAGCTGCGCCGCGTAGAGCGCGTGTGTTCATCTCTGGTCTTGGTTATTACGAGTTGCGCATCAACGGTCGCAAGGTCGGTGATCACGTTCTGGACCCGGGTTGGACCACCTTTAGCAAACGTGTACTCTACGTGACGTACGACGTTACCCGCGCGCTGAGACCGGGCCGTAATGCCATCGGTGTTATGCTGGGCCACGGCAAATACAACGACCGTGCGCTAATGTTTCAGCTGTACGTCGAAGGCGAGGACGGCAATCTCGTAGAGATCCACTCCGACGGTACTTGGAAGACGGCTCCGGGTCCGATTTTGGAGGATGCTGTTTACAATGGTGAGACATACGACGCGCGTCTGGAGCAGCCGGGTTGGGAACGTCCGGATTTTGAGGAAAAAAACTGGAAACCGGCGCAAAAAGTGAAAGCTCCTGGCGGCGTCTTGTCTGCCCAACTGATGCCGGCGATTAAAGTTGTTGATACCATTGTGCCGTTGACCATGACCAACCCGGCGCCAGGTGTTTATGTGTTTGATCTGGGCCAGAATATTAGTGGCTGGGCACAATTGAGAGTTAGAGGCCCACGCGGTACGGACGTGCGGTTGCGTTTCGCCGAATTACTGTACGAGAACGGTATGATTAACCAGGAGAACCTCCGTAGCTCGCGGGCTGAAGACCACTATATTTTAAAGGGTGAAGGTGAGGAGGTTTGGGAACCGCGTTTCACTTACCATGGCTTCCGTTATCTGGAGGTGACCGGTTTTCCGGGCACTCCGAAGATCGATTCCGTTCGTGGCCGCGTGGTTCATACCGCCGTTTCCCCGGTTGGTAATTTTAGCTGCAGCAAGCAGATCCTAAACGATATTCAACGTATTATTTCGTGGGGCCAGAAAACCAACCTGCATTCGATCCCGACCGATTGTGATCAGCGTGATGAACGCATGGGTTGGATGGGCGACGCGCAAGTCACTGCGGAGGAAGCGATTATGAATTACGACATGGCAGCGTTTTATACCAACTTCCTGCGTGACATCCGTGACGTGCAGGGCGAGGATGGCTCCATTACCGACACCGTTCCGCATATTTGGGGTAGCCGACCGGCGGACCCTGCGTGGGGTACGGCATATCCGCTGATCGCGTGGTATATGTTCCAGTATTATGGAGACAAGCGCGTCTTGGAAGAGCACTACGATAGCCTGAAGAAATACGTCGAGTTCCTTAGACGTAAGGCTGAGAACGGGCTGGTGAAGTATAGCTATTACGGCGACTGGGTTGCAATTGATAAGTGCCCGGGCTCTCTGGTTTCAGCGTTCTATTACTACTACGACGTGAAAATCCTGGCTGAAGCGGCTCGCGTTTTGGGGAAACAACCGGAAGCCGACCTGTACCTGAAGCTGGCGGCACAGATCAAAGATGCCTTTAACCGTGAATTCCTGGATCCGACCACGCGTAATTACGCAGGTGGTTCACAAACCGCTAATACCCTGCCGTTGTTTATCGGC(SEQ IDNO.12)。
先进行单个位点突变依次得到重组表达菌E.coli BL21(DE3)-pET-28a-V187I、E.coli BL21(DE3)-pET-28a-N337A、E.coli BL21(DE3)-pET-28a-L521S,再以进行过一次突变后的重组质粒为模板进行二次或三次突变;最后得到了测序正确的包含CAbRha突变体的双位点以及三位点突变重组表达菌E.coli BL21(DE3)-pET-28a-V187I/N337A、E.coliBL21(DE3)-pET-28a-V187I/N337A/L 521S。
实施例3重组菌大肠杆菌发酵培养
将实施例1、2中得到的6株重组菌大肠杆菌菌株分别接种于5ml含卡那霉素(50μg/mL)抗性的LB培养基中,37℃下培养8h后以2%接种量转接至50mL TB发酵培养基中,先于37℃、200rpm恒温培养至菌体OD600达0.6时转至30℃、200rpm进行摇瓶诱导发酵16h。发酵结束后,8000rpm 6min离心收集菌体,使用50mM pH8.0 Tris-盐酸缓冲液将菌泥重悬后超声破碎(超1秒,停2秒,10min),破碎液经12000rmp离心20min后所得上清即为重组酶CAbRha以及突变体酶。
实施例4利用CAbRha及其突变体酶生产淫羊藿苷
筛选可用于生产高浓度淫羊藿苷的CAbRha酶突变体,反应条件相同:取2mL实施例3中的得到的野生型CAbRha酶及突变体CAbRha胞外酶液,分别加入含有朝藿定C的50mMpH8.0 Tris-盐酸缓冲液,朝藿定C终浓度为50g/L,在60℃水浴条件下反应2h,再100℃灭活30min以终止该反应。12000rpm,离心1min,取上清液,稀释一定倍数后通过高效液相色谱法测量朝藿定C和淫羊藿苷的浓度,朝藿定C最终转化率显示在下表3中。
表3朝藿定C转化率
催化酶 | 朝藿定C转化率 |
野生型CAbRha | 71% |
V187I | 91% |
N337A | 88% |
L521S | 85% |
V187I/N337A | 95% |
V187I/N337A/L521S | 99% |
结果显示,反应2h后,野生型CAbRha酶体系催化朝藿定C生成淫羊藿苷的转化率约为71%,而V187I/N337A/L521S突变体体系中转化率最高达到了99%,相比野生型CAbRha转化率提高了28%。由此表明,突变体V187I/N337A/L521S的催化活性要显著高于野生型CAbRha酶,这在工业生产中可大大降低酶的用量以及缩短反应时间,因而降低生产成本。
实施例5野生型MsDPE及突变体V187I/N337A/L521S酶的纯化
为了纯化野生型MsDPE及筛选出的催化活性最高的V187I/N337A/L521S突变体酶。采用GE公司的AKTA prime plus层析系统,使用5mL的HisTrap HP镍柱亲和层析。层析柱用pH 8.0,0.5M NaCl,20mM磷酸钠缓冲(缓冲液A)预平衡,洗脱缓冲液为pH 8.0,0.5M NaCl,0.5M咪唑,20mM磷酸缓冲(缓冲液B),采用0%-100%缓冲B梯度洗脱,总洗脱时间为50min,将收集的活性蛋白装入透析袋(27mm,MW:7000),4℃下透析16h,每隔8h更换一次透析缓冲。透析结束后进行冷冻干燥,得到冻干粉用于测定蛋白浓度和活性。
实施例6野生型CAbRha及突变体V187I/N337A/L521S酶活测定
取1.5ml EP离心管,加入460μl 50mM pH8.0的Tris-HCl缓冲液,20μl 10mM硝基苯α-L-基鼠李糖苷(pNPR),先在80℃水浴5min,再向其中加入20μL适当蛋白浓度酶液,充分混合均匀。按照每孔100μL吸取反应液至96孔酶标板,置于合适的温度进行酶促反应,以加入100μL 1M的Na2C03终止反应,并于405nm处测定吸光值。每组三个平行,每10min终止一组反应并测定吸光度。
酶活定义:在pH8.0和80℃的反应条件下,每分钟生成1μmo1的对硝基苯酚所需酶量为1个酶活单位(U)。
实施例7pH和温度对酶活性的影响
为了研究不同pH和温度对野生型CAbRha及突变体V187I/N337A/L521S酶活性的影响,按照实施例6所述的酶活测定方法,在不同的温度和pH条件下将pNPR作为底物测定对其活性的影响,且比较了在不同温度和pH下的酶活性。
为了研究pH的作用,分别使用50mM柠檬酸钠pH 5~6、50mM磷酸钠pH7~8、50mMTris-HCl pH7.5~9、50mM甘氨酸-NaOH pH9~11的缓冲溶液,观察表现出最大活性的pH。分别加入突变体V187I/N337A/L521S酶冻干粉溶解液(稀释到合适的倍数),各自在80℃的条件下反应10min,再以Na2C03终止反应,并测量其酶活性。
为了研究温度的作用,使用50mM pH8.0的Tris-HCl缓冲液,分别加入野生型CAbRha或突变体V187I/N337A/L521S酶冻干粉溶解液(稀释到合适的倍数),各自在温度范围50-90℃的条件下反应10min,再以Na2C03终止反应,并测量其酶活性。
图1的结果显示该突变体V187I/N337A/L521S酶的最适pH为8,图2显示突变体的最适反应温度为80℃,相比野生型提高了10度。
实施例8酶的热稳定性
为了研究酶的热稳定性,突变体V187I/N337A/L521S酶分别置于50℃、60℃、70℃、80℃和90℃水浴条件下保温后,每隔2小时取样,以pNPR作为底物按照实施例6的方法测定其残余活性。
结果显示(如图3)突变体V187I/N337A/L521S具有优良的热稳定性,在60℃保温12小时后活性仍没有检测到活性下降,在70℃保温6小时后活性仍没有检测到活性下降,在80℃保温6小时后,活性仍然保持在80%以上。
酶半衰期测定:
测定突变体V187I/N337A/L521S酶的半衰期,分别将纯酶溶液稀释到0.5mg/mL,置于70℃水浴保温,每隔一定时间取样,按上述实施例6酶活测定方法,在70℃下,使用50mMpH8.0的Tris-HCl缓冲液,测定残余活力。将相对活力数值取对数后与时间拟合。按照如下公式计算酶在不同温度下的失活速率常数(kD)以及半衰期(t1/2):
一级失活Arrihenius方程:
Ar=A0exp(-kD·t);
其中,A0为初始酶活力,U/mg of protein;Ar为残余酶活力,U/mg of protein;t为时间,h。
失活半衰期方程:
t1/2=ln 0.5/(-kD)=0.693/kD;
实验结果突变体V187I/N337A/L521S酶在70℃下测定的半衰期达10h。
实施例9利用野生型CAbRha及突变体V187I/N337A/L521S酶生产淫羊藿苷
该野生型CAbRha及突变体V187I/N337A/L521S酶的反应分别在pH8.0 50mM的Tris-HCl缓冲溶液中,反应体系中含有120g/l的朝藿定C,20%(v/v)实施例3中得到的野生型CAbRha或突变酶胞外酶液,温度80℃条件下进行6h,从而容许该反应充分地进行。然后,取样500μl,100℃灭活20min以终止该反应,12000rpm,离心1min,取上清液,稀释一定倍数后通过高效液相色谱法测量朝藿定C和淫羊藿苷的浓度。
结果显示在图4中,野生型CAbRha酶转化朝藿定C生产淫羊藿苷的转化率在4h时为70%,而该突变体V187I/N337A/L521S酶反应4h时的转化率已经达到平衡的98.3%(液相检测结果如图5所示),明显高于野生型。
上述具体实施方式对本发明作了详细说明,但是本发明不限于上述实施例,在所属技术领域普通技术人员所具备的知识范围内,还可以在不脱离本发明宗旨的前提下作出各种变化。此外,在不冲突的情况下,本发明的实施例及实施例中的特征可以相互组合。
Claims (13)
1.一种鼠李糖苷酶突变体,其特征在于:所述突变体包括如SEQ ID No.1所示的氨基酸序列在第187位、第337位和第521位中的至少一个氨基酸处的突变;
所述如SEQ ID No.1所示的氨基酸序列在第187位突变为异亮氨酸;
所述如SEQ ID No.1所示的氨基酸序列在第337位突变为丙氨酸;
所述如SEQ ID No.1所示的氨基酸序列在第521位突变为丝氨酸。
2.根据权利要求1所述的鼠李糖苷酶突变体,其特征在于,所述鼠李糖苷酶突变体的氨基酸序列如SEQ ID NO.2~SEQ ID NO.6任一项所示。
3.根据权利要求2所述的鼠李糖苷酶突变体,其特征在于,所述鼠李糖苷酶突变体的氨基酸序列如SEQ ID NO.6所示。
4.一种核酸分子,其特征在于,所述核酸分子包含:
(I)编码权利要求1~3任一项所述的鼠李糖苷酶突变体的核苷酸序列;或
(II)与(I)所述的核苷酸序列互补的核苷酸序列。
5.根据权利要求4所述的核酸分子,其特征在于,所述核酸分子的核苷酸序列如SEQ IDNO.8~SEQ ID NO.12任一项所示。
6.一种重组载体,其特征在于,所述载体可表达权利要求1~3任一项所述的鼠李糖苷酶突变体或含有权利要求4~5任一项所述的核酸分子。
7.一种重组细胞,其特征在于,所述重组细胞包含权利要求6所述的重组载体。
8.权利要求1~3任一项所述的鼠李糖苷酶突变体和/或权利要求7所述重组细胞在以下至少一项中的应用:
(a)水解鼠李糖苷键中的应用;
(b)制备水解鼠李糖苷键的产品;
(c)生产淫羊藿苷;
(d)制备生产淫羊藿苷的产品。
9.权利要求4~5任一项所述核酸分子和/或权利要求6所述重组载体在制备水解鼠李糖苷键和/或生产淫羊藿苷的产品中的应用。
10.根据权利要求8或9所述的应用,其特征在于,所述鼠李糖苷键包括朝藿定C鼠李糖苷键、芦丁鼠李糖苷键、柚皮苷鼠李糖苷键、杨梅苷鼠李糖苷键、新橙皮苷鼠李糖苷键、柴胡皂苷C鼠李糖苷键、橙皮苷鼠李糖苷键、槲皮苷鼠李糖苷键、薯蓣皂苷鼠李糖苷键中的至少一种。
11.一种用于水解鼠李糖苷键和/或生产淫羊藿苷的产品,其特征在于,所述产品包含权利要求1~3任一项所述的鼠李糖苷酶突变体、权利要求4~5任一项所述核酸分子、权利要求6所述重组载体和/或权利要求7所述重组细胞。
12.一种鼠李糖苷酶突变体的制备方法,通过培养权利要求7所述的重组细胞获得鼠李糖苷酶突变体。
13.淫羊藿苷的制备方法,其特征在于,以朝藿定C作为底物,使用权利要求1~3任一项所述的鼠李糖苷酶突变体、权利要求4~5任一项所述核酸分子、权利要求6所述重组载体和/或权利要求7所述重组细胞制备淫羊藿苷。
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