CN116676359A - 一种利用酶法合成阿霉素的方法 - Google Patents
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Abstract
本发明提供一种利用酶法合成阿霉素的方法，即一种通过细胞色素P450酶DoxA，利用过氧化氢催化柔红霉素转化为阿霉素的方法。本发明实现了一步酶法替代目前阿霉素合成中复杂和污染严重的多步化学拆分，并通过优化条件使其产量达到23.5％，为工业合成阿霉素提供了一条新的简便途径；该方法反应条件温和，易控制；酶催化专一性强，无副产物产生；催化剂易于从反应体系中除去，显著提升了过程绿色指数，具有较好的经济性。
Description
技术领域
本发明属于生物制剂制备技术领域，具体涉及一种利用酶法合成阿霉素的方法，即一种利用H2O2酶法合成阿霉素(DXR)的方法。
背景技术
阿霉素(Doxorubicin，DXR，化学结构见图1)是从波赛链霉菌青灰变种(Streptomyces peucetius var.caesius)的发酵液中提取的一种糖苷类抗生素，是目前临床上使用的一种广谱抗肿瘤药物，对多种肿瘤均有治疗作用，其作用机制是通过抑制RNA和DNA复制，从而抑制肿瘤细胞的增生。尽管DXR有心脏毒性的副作用，但凭借其突出的抗癌优势，仍然是全世界治疗癌症的优先选择之一。
目前阿霉素的主要工业生产方式为半化学合成，即以微生物发酵获得的中间产物柔红霉素为原料，随后需要经过七步化学反应才能实现C-14位羟基的引入，收率仅为33％，整个过程难度大，操作复杂、收率低且环境污染严重，造成阿霉素生产成本偏高。
发明内容
本发明提供一种利用酶法合成阿霉素的方法，即一种通过细胞色素P450酶DoxA，利用过氧化氢(H2O2)催化柔红霉素(DNR)转化为阿霉素(DXR)的方法(图2)。
本发明提供一种天蓝淡红链霉菌(S.coeruleorubidus)来源的的DoxA酶，其氨基酸序列如下(SEQ ID NO:1)：
VAVDPFACPMMTMQRKPEVHDAFREAGPVVEVNAPAGGPAWVITDDALAREVLADPRFVKDPDLAPAAWRGVDDGLDIPVPELRPFTLIAVDGEAHRRLRRIHAPAFNPRRLAERTDRIAAIAGRLLTELADTSGRSGKPAELIGGFAYHFPLLVICELLGVPVTDPAMAREAVSVLKALGLGGPQSGGGDGTDPAGGVPDTSALESLLLEAVHSARRNDTPTMTRVLYERAQAEFGSVSDDQLVYMITGLIFAGHDTTGSFLGFLLAEVLAGRLAADADEDAVSRFVEEALRYHPPVPYTLWRFAATEVTIGGVRLPRGAPVLVDIEGTNTDGRHHDDPHAFHPDRPSWRRLTFGDGPHYCIGEQLAQLESRTMIGVLRSRFPEARLAVPYDELRWSRKGAQTARLTELPVWLR；
其编码基因的核苷酸序列如下(SEQ ID NO:2)：
gtggccgtcgacccgttcgcgtgtcccatgatgaccatgcagcgcaagcccgaggtgcacgacgccttccgggaggcgggcccggtcgtcgaggtgaacgcccccgcgggcggacccgcctgggtcatcaccgatgacgccctcgcccgcgaggtgctggccgatccccggttcgtgaaggaccccgacctcgcccccgccgcctggcggggggtggacgacggtctcgacatccccgttccggagctgcgtccgttcacgctcatcgccgtggacggcgaggcccaccggcgcctgcgccgcatccacgcacccgcgttcaacccgcgccggctggccgagcggacggatcgcatcgccgccatcgccggccggctgctcaccgaactcgccgacacttccggccggtcgggcaaaccggccgagctgatcggcggtttcgcgtaccacttcccgctgttggtcatctgcgagctgctcggcgtgccggtcaccgatccggcgatggcccgcgaggccgtcagcgttctcaaggcactcggcctcggcggcccgcagagcggcgggggtgacggcacggaccctgccgggggcgtgccggacacctcggccctggagagcctgctcctcgaagccgtgcactcggcccggcggaacgacaccccgaccatgacccgcgtgctgtacgaacgcgcgcaggccgagttcggctcggtctccgacgaccagctcgtctacatgatcaccgggctcatcttcgccggccacgacaccaccggctccttcctgggcttcctgctcgcggaggtgctggcgggccgcctcgcggcggacgccgacgaggacgccgtctcccggttcgtggaggaggcgctgcgctaccacccgccggtgccctacacgttgtggaggttcgctgccacggaggtgaccatcggcggcgtccggctgccccgcggagcgccggtgctggtggacatcgagggcaccaacaccgacggccgccatcacgacgacccgcacgccttccacccggaccgtccctcgtggcggcggctcaccttcggcgacgggccgcactactgcatcggggagcagctcgcccagctggagtcgcgcacgatgatcggcgtactgcgcagcaggttccccgaggcccgactggccgtgccgtacgacgagttgcggtggtcccggaagggggcccagacggcgcggctcaccgaactgcccgtctggctgcgctga；
本发明再一个方面提供所述的DoxA酶的一种用途，是在催化柔红霉素生成阿霉素中的应用；
本发明还提供一种合成阿霉素的方法，是使用所述的DoxA酶，利用H2O2作为唯一的氧和电子供体，来催化柔红霉素生成阿霉素；
更进一步的，所述的反应中H2O2浓度为100mM、DoxA浓度为6μM、柔红霉素浓度为100μM，30℃反应30min，获得最高的阿霉素产率。
本发明实现了一步酶法替代目前阿霉素合成中复杂和污染严重的多步化学拆分，并通过优化条件使其产量达到23.5％，为工业合成阿霉素提供了一条新的简便途径；该方法反应条件温和，易控制；酶催化专一性强，无副产物产生；催化剂易于从反应体系中除去，显著提升了过程绿色指数，具有较好的经济性。
附图说明
图1：阿霉素的化学结构图，
图2：DoxA酶利用H2O2催化柔红霉素氧化为阿霉素的合成路径图，
图3：DoxA酶的SDS-PAGE电泳检测图，
图4：DoxA酶利用不同浓度H2O2催化阿霉素合成的HPLC检测图谱，
图5：阿霉素DXR产量优化柱状图，
具体实施方式
本发明以柔红霉素为原料，P450 DoxA酶作为催化酶，利用H2O2作为唯一的氧和电子供体，合成了具有更高应用价值的阿霉素。
实施例1：DoxA蛋白利用H2O2催化柔红霉素DNR转化为阿霉素DXR
1)DoxA蛋白的诱导表达
将高效表达DoxA蛋白的大肠杆菌菌株BL21(DE3)划线接种至LB(含50μg/mL Kan)平板，37℃培养24h。挑取单克隆于50mL LB(含50μg/mL Kan)培养基，37℃、220rpm摇床培养过夜。按1％接种量接种至含500mL TB或LB(含50μg/mL Kan)培养基的2L三角瓶中，37℃、200rpm培养至OD600介于0.8～1，加入终浓度200μM的IPTG、500μM的5-氨基乙酰丙酸(5-ALA)和500μM的维生素B1(VB1)，随后，在18℃、150rpm条件下培养18-20h诱导蛋白表达。
(2)DoxA蛋白的纯化
离心收集菌体，加入30-40mL Lysis buffer(50mM NaH2PO4，300mM NaCl，10mM咪唑，10％甘油，pH 8.0)涡旋振荡重悬菌体，超声破碎细胞后10000rpm，4℃离心60min，上清液与Ni-NTA 4℃孵育60min。然后，使用Wash buffer(50mM NaH2PO4，300mM NaCl，20mM咪唑，10％甘油，pH 8.0)对杂蛋白进行洗脱；用Elution buffer(50mM NaH2PO4，300mM NaCl，250mM咪唑，10％甘油，pH 8.0)对目标蛋白进行洗脱，使用50KD超滤管浓缩蛋白溶液；最后使用PD-10脱盐柱去除咪唑，缓冲溶液使用Desalting buffer(50mM NaH2PO4，300mM NaCl，10％甘油，pH 8.0)；SDS-PAGE检测到明显的可溶性DoxA蛋白(图3)。
(3)DoxA利用H2O2催化柔红霉素生成阿霉素
以100μM柔红霉素为底物，加入2μM DoxA和不同浓度H2O2(500μM、1mM、5mM、10mM、20mM、40mM)，30℃反应2h，向反应体系中加入两倍体积的甲醇混匀终止反应，高速离心后，取上清进行高效液相色谱(HPLC)检测。检测结果显示，当H2O2浓度为10mM时，检测到DXR产生，产率仅为5％；随着提高H2O2浓度，阿霉素产率明显提高(图4)。
实施例2：优化反应体系提高DXR产率
H2O2作为强氧化剂，一定程度上会影响P450 DoxA酶活性，为了最大限度提高DoxA蛋白酶活，进而提高阿霉素产率，利用在线软件SPSSAU设计了正交试验表(表1)。
表1：SPSSAU设计正交试验表
筛选出了最优的反应体系和反应时间即：H2O2 100mM、DoxA6μM、柔红霉素100μM，30℃反应30min，阿霉素产率由5％提高至23.5％(图5)。
Claims (7)
1.一种DoxA酶，其特征在于，所述的DoxA酶的氨基酸序列为SEQ ID NO:1。
2.如权利要求1所述的DoxA酶，其特征在于，所述的DoxA酶是在序列为SEQ ID NO:1的氨基酸上取代、缺失、添加一个或数个氨基或获得的衍生酶。
3.一种基因，其特征在于，所述基因编码权利要求1或2所述的DoxA酶。
4.如权利要求3所述的基因，其特征在于，所述的基因的核苷酸序列为SEQ ID NO:2。
5.权利要求1或2所述的DoxA酶在催化柔红霉素生成阿霉素中的应用。
6.一种合成阿霉素的方法，其特征在于，所述的方法是利用H2O2作为唯一的氧和电子供体，使用权利要求1或2所述的DoxA酶来催化柔红霉素生成阿霉素。
7.如权利要求6所述的方法，其特征在于，所述的方法，在反应体系中H2O2浓度为100mM、DoxA浓度为6μM、柔红霉素浓度为100μM，30℃反应30min。
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