CN112280758A - 一种甾体5β还原酶变体及其用途 - Google Patents
一种甾体5β还原酶变体及其用途 Download PDFInfo
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- CN112280758A CN112280758A CN202011215626.6A CN202011215626A CN112280758A CN 112280758 A CN112280758 A CN 112280758A CN 202011215626 A CN202011215626 A CN 202011215626A CN 112280758 A CN112280758 A CN 112280758A
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Abstract
本发明涉及一种甾体5β还原酶变体及其用途。所述甾体5β还原酶变体具有甾体5β还原酶活性,其氨基酸序列包含R63K和R64H突变,能够利用NADH为辅酶,且具有选自以下的氨基酸序列:1)SEQ ID NO:1、3或5所示氨基酸序列;2)SEQ ID NO:1、3或5所示氨基酸序列经过取代、缺失或添加一个或多个氨基酸而得到的氨基酸序列;或3)与SEQ ID NO:1、3或5具有69%以上序列同一性的氨基酸序列。所述甾体5β还原酶变体的转化产物构象单一,只有β型还原产物出现,具有空间加氢特异性,产物中无α型结构,具有高效、稳定的特点。
Description
技术领域
本发明涉及基因工程和酶工程领域,更具体而言,本发明涉及一种甾体5β还原酶变体及其用途。
背景技术
甾体药物是广泛应用于临床的一大类药物,其大致分为类皮质激素和性激素两大类,对机体起着非常重要的调节作用,在医药工业中占有重要的地位。甾体药物的发现及成功合成是近半个世纪以来医药工业取得的最引人注目的两大进展之一。然而,利用化学法合成时,往往合成步骤多,效率低,价格昂贵。1950年Murray和Peterson利用黑根霉高效转化***为11α-羟基***,使从孕酮合成皮质酮只需要3步,这一研究成果引起了微生物学者的极大兴趣,此后开展了大量的微生物对甾体转化的研究工作。
有别于甾体化合物结构中的5α氢,胆酸类化合物5位上的氢为5β,是一个重要的化学结构,其化学合成难度大,急需高效专一的生物转化法。文献报道,所有甾体5β还原酶均以NADPH为辅酶(Herl V,Fischer G,Reva VA,Stiebritz M,Muller YA,Müller-Uri F,Kreis W:The VEP1 gene(At4g24220)encodes a short-chain dehydrogenase/reductasewith3-oxo-Delta4,5-steroid 5beta-reductase activity in Arabidopsis thalianaL.Biochimie 2009,91(4):517-525.),但是在实际工业生产中由于NADPH稳定性差,价格昂贵,不能工业化应用。
由于自然存在的酶在工业生产上会存在很多问题,例如对底物催化缓慢,稳定性差,或生产成本高等,不适用于工业应用。而对于甾体5β还原酶类,考虑最多的是酶对底物的催化速度和辅酶的选择性。因此,需要深入了解甾体5β还原酶的辅酶特异性问题。若甾体5β还原酶能够以NADH为辅酶,则将可以克服这些困难,因此甾体5β还原酶利用的辅酶由NADPH转变为NADH的研究具有重大应用价值。
发明内容
本发明的目的在于克服现有技术中存在的缺陷与不足,提供了一种甾体5β还原酶的变体,该变体的辅酶特异性由稳定性差的NADPH改变为价格低廉、性质稳定的NADH,该变体具有高效、稳定的特点,能够大大降低实际工业生产的成本。
不受具体理论约束,本发明提供的甾体5β还原酶变体将甾体转化为5β-甾体的方程式如下:
其中,R可为任何取代基,如羟丙基或乙酰基。
本发明通过对甾体5β还原酶进行改造,获得了一种甾体5β还原酶变体,所述甾体5β还原酶变体的氨基酸序列包括两个相邻位点的RR突变为相邻的KH。优选地,该变体的SEQID NO:1、3或5所示氨基酸序列具有R63K和R64H突变(R63K/R64H)。该突变使得甾体5β还原酶可利用NADPH和NADH为辅酶。
本发明提供了一种具有甾体5β还原酶活性的甾体5β还原酶变体,所述甾体5β还原酶变体的氨基酸序列包含R63K和R64H突变,能够利用NADH为辅酶,且具有选自以下的氨基酸序列:
1)SEQ ID NO:1、SEQ ID NO:3或SEQ ID NO:5所示氨基酸序列;
2)SEQ ID NO:1、SEQ ID NO:3或SEQ ID NO:5所示氨基酸序列经过取代、缺失或添加一个或多个氨基酸而得到的氨基酸序列;或
3)与SEQ ID NO:1、SEQ ID NO:3或SEQ ID NO:5具有69%以上序列同一性的氨基酸序列。
优选地,所述甾体5β还原酶变体能够利用NADPH和NADH为辅酶。
优选地,所述甾体5β还原酶变体是CmP5βR-LY,其氨基酸序列如SEQ ID NO:1所示,其编码多核苷酸序列如SEQ ID NO:2所示。具体序列如下:
SEQ ID NO:1
MSWWGAGAIGAAKKKLDDDEPTQSYESVALIIGVTGIVGNSLAEILPLSDTLGGPWKVYGVAKHPRPSWNADHPIDYIQCDVSNADDARSKLSPLTDVTHVFYVTWTNRESETENCEANGSMLRNVLRAVVPHAPNLRHVCLQTGTKHYLGPFTNVDGPHHDPPFTEDMPRLQIQNFYYTQEDVLFEEIKKKEGVTWSIHRPNMIFGFSPYSLMNIVGTLCVYAAICKHEGSPLMFPGSKKAWEGFMTASDADLIAEQQIWAAVDPYAKNEAFNCNNADIFKWKHLWKILAEQFGIEEYGFEEGKNLGLVEMMKGKERVWEEMVKENQLLEKKLDEVGVWWFADVILGVEGMIDSMNKSKEHGFLGFRNSNNSFISWIDKYKAFKIVP
SEQ ID NO:2
ATGAGCTGGTGGGGCGCCGGTGCGATTGGTGCGGCGAAAAAGAAACTGGACGATGACGAGCCGACCCAGAGCTACGAGAGTGTTGCGCTGATCATCGGCGTTACGGGCATCGTTGGCAACAGTCTGGCGGAAATTCTGCCACTGAGCGATACGCTGGGTGGCCCGTGGAAAGTGTATGGTGTTGCGAAACATCCACGTCCAAGCTGGAATGCCGACCACCCGATCGACTACATCCAGTGCGACGTGAGTAACGCCGATGATGCGCGCAGCAAACTGAGCCCGCTGACCGATGTTACCCACGTGTTTTACGTGACGTGGACCAACCGCGAAAGCGAAACGGAAAACTGCGAAGCGAACGGCAGCATGCTGCGCAATGTGCTGCGCGCCGTTGTGCCACATGCCCCAAATCTGCGCCATGTGTGTCTGCAGACCGGCACGAAACACTATCTGGGCCCGTTTACGAATGTGGATGGCCCACACCACGACCCACCGTTCACGGAAGATATGCCGCGCCTCCAGATCCAGAACTTCTACTACACCCAAGAAGATGTGCTCTTTGAGGAGATCAAGAAAAAAGAAGGCGTGACGTGGAGCATCCACCGCCCAAATATGATCTTCGGCTTCAGCCCGTACAGTCTGATGAATATCGTGGGCACGCTGTGTGTGTACGCCGCCATCTGCAAACATGAGGGTAGTCCGCTGATGTTCCCGGGCAGTAAAAAAGCGTGGGAGGGCTTCATGACCGCCAGCGATGCCGACCTCATCGCCGAACAGCAGATTTGGGCGGCCGTGGACCCGTATGCCAAGAACGAGGCGTTCAACTGCAACAACGCCGACATCTTCAAGTGGAAACACCTCTGGAAAATTCTGGCCGAGCAGTTTGGCATTGAGGAGTACGGCTTCGAAGAAGGCAAGAACCTCGGTCTGGTGGAGATGATGAAAGGCAAGGAACGCGTGTGGGAGGAGATGGTTAAGGAAAACCAGCTGCTCGAGAAAAAGCTCGACGAGGTTGGCGTTTGGTGGTTTGCGGACGTTATTCTGGGTGTTGAGGGCATGATCGACAGTATGAATAAGAGCAAGGAACACGGCTTTCTGGGCTTCCGCAACAGCAACAACAGCTTCATTAGCTGGATCGATAAGTATAAAGCCTTCAAAATTGTGCCG
优选地,所述甾体5β还原酶变体是AtP5βR-LY,其氨基酸序列如SEQ ID NO:3所示,其编码多核苷酸序列如SEQ ID NO:4所示。
SEQ ID NO:3
MSWWWAGAIGAAKKKLDEDEPSQSFESVALIIGVTGIVGNSLAEILPLSDTPGGPWKVYGVAKHPRPTWNADHPIDYIQCDVSDAEDTRSKLSPLTDVTHVFYVTWTNRESESENCEANGSMLRNVLQAIIPYAPNLRHVCLQTGTKHYLGPFTNVDGPRHDPPFTEDMPRLQIQNFYYTQEDILFEEIKKIETVTWSIHRPNMIFGFSPYSLMNIVGTLCVYAAICKHEGSPLLFPGSKKAWEGFMTASDADLIAEQQIWAAVDPYAKNEAFNCNNADIFKWKHLWKILAEQFGIEEYGFEEGKNLGLVEMMKGKERVWEEMVKENQLQEKKLEEVGVWWFADVILGVEGMIDSMNKSKEYGFLGFRNSNNSFISWIDKYKAFKIVP
SEQ ID NO:4
ATGAGCTGGTGGTGGGCAGGTGCAATTGGTGCCGCCAAGAAGAAGCTGGATGAGGATGAACCGAGCCAGAGCTTTGAAAGCGTGGCCCTGATCATCGGTGTGACCGGCATCGTTGGCAATAGCCTGGCCGAAATCCTGCCGCTGAGCGATACCCCTGGTGGTCCGTGGAAAGTTTATGGTGTGGCAAAACATCCTCGTCCGACCTGGAACGCAGATCACCCGATTGACTACATCCAATGCGACGTGAGCGATGCAGAAGACACCCGTAGCAAACTGAGCCCGCTGACAGATGTGACCCACGTGTTCTACGTGACCTGGACCAACCGTGAAAGCGAGAGCGAAAATTGTGAGGCCAACGGCAGCATGCTGCGCAATGTGCTGCAGGCAATTATCCCGTACGCACCGAATCTGCGTCACGTGTGTCTGCAGACAGGCACCAAGCATTACCTGGGCCCGTTTACCAACGTTGATGGCCCTCGCCATGATCCTCCGTTTACCGAGGACATGCCGCGCCTGCAGATCCAGAATTTCTACTACACCCAAGAAGATATTCTGTTTGAAGAAATCAAAAAGATCGAAACCGTGACCTGGAGCATCCACCGCCCGAACATGATCTTTGGCTTCAGCCCGTATAGCCTGATGAACATCGTGGGCACACTGTGCGTGTACGCAGCCATCTGCAAGCACGAAGGTAGCCCGCTGCTGTTTCCGGGTAGCAAGAAAGCCTGGGAGGGCTTTATGACAGCAAGCGATGCCGACCTGATTGCCGAACAGCAGATTTGGGCCGCCGTGGATCCGTATGCCAAAAACGAGGCCTTCAACTGCAATAACGCCGATATTTTTAAATGGAAACATCTGTGGAAAATCCTGGCCGAGCAGTTTGGCATCGAAGAATACGGCTTCGAAGAAGGCAAGAACCTGGGCCTGGTTGAGATGATGAAAGGCAAGGAGCGCGTGTGGGAAGAAATGGTTAAGGAGAACCAGCTGCAGGAGAAAAAGCTGGAGGAAGTGGGTGTGTGGTGGTTCGCCGATGTGATCCTGGGCGTTGAAGGCATGATCGATAGTATGAATAAAAGCAAGGAATATGGCTTCCTGGGCTTTCGCAACAGCAACAACAGCTTTATTAGCTGGATTGATAAATATAAAGCATTTAAGATTGTGCCT
优选地,所述甾体5β还原酶变体是DlP5βR-LY,其氨基酸序列如SEQ ID NO:5所示,其编码多核苷酸序列如SEQ ID NO:6所示。
SEQ ID NO:5
MSWWWAGAIGAAKKRLEEDDAQPKHSSVALIVGVTGIIGNSLAEILPLADTPGGPWKVYGVAKHTRPAWHEDNPINYVQCDISDPDDSQAKLSPLTDVTHVFYVTWANRSTEQENCEANSKMFRNVLDAVIPNCPNLKHISLQTGRKHYMGPFESYGKIESHDPPYTEDLPRLKYMNFYYDLEDIMLEEVEKKEGLTWSVHRPGNIFGFSPYSMMNLVGTLCVYAAICKHEGKVLRFTGCKAAWDGYSDCSDADLIAEHHIWAAVDPYAKNEAFNVSNGDVFKWKHFWKVLAEQFGVGCGEYEEGVDLKLQDLMKGKEPVWEEIVRENGLTPTKLKDVGIWWFGDVILGNECFLDSMNKSKEHGFLGFRNSKNAFISWIDKAKAYKIVP
SEQ ID NO:6
ATGAGCTGGTGGTGGGCCGGGGCGATTGGTGCCGCAAAAAAACGTCTGGAAGAAGATGATGCACAGCCGAAACATAGCAGCGTTGCACTGATTGTTGGTGTTACCGGTATTATTGGTAATAGCCTGGCAGAAATTCTGCCGCTGGCAGATACCCCGGGTGGTCCGTGGAAAGTTTATGGTGTTGCAAAACATACCCGTCCGGCATGGCATGAAGATAATCCGATTAATTATGTTCAGTGTGATATTAGCGATCCGGATGATAGCCAGGCAAAACTGAGCCCGCTGACCGATGTTACCCATGTTTTTTATGTTACCTGGGCAAATCGTAGCACCGAACAGGAAAATTGTGAAGCAAATAGCAAAATGTTTCGTAATGTTCTGGATGCAGTTATTCCGAATTGTCCGAATCTGAAACATATTAGCCTGCAGACCGGTCGTAAACATTATATGGGTCCGTTTGAAAGCTATGGTAAAATTGAAAGCCATGATCCGCCGTATACCGAAGATCTGCCGCGTCTGAAATATATGAATTTTTATTATGATCTGGAAGATATTATGCTGGAAGAAGTTGAAAAAAAAGAAGGTCTGACCTGGAGCGTTCATCGTCCGGGTAATATTTTTGGTTTTAGCCCGTATAGCATGATGAATCTGGTTGGTACCCTGTGTGTTTATGCAGCAATTTGTAAACATGAAGGTAAAGTTCTGCGTTTTACCGGTTGTAAAGCAGCATGGGATGGTTATAGCGATTGTAGCGATGCAGATCTGATTGCAGAACATCATATTTGGGCAGCAGTTGATCCGTATGCAAAAAATGAAGCATTTAATGTTAGCAATGGTGATGTTTTTAAATGGAAACATTTTTGGAAAGTTCTGGCAGAACAGTTTGGTGTTGGTTGTGGTGAATATGAAGAAGGTGTTGATCTGAAACTGCAGGATCTGATGAAAGGTAAAGAACCGGTTTGGGAAGAAATTGTTCGTGAAAATGGTCTGACCCCGACCAAACTGAAAGATGTTGGTATTTGGTGGTTTGGTGATGTTATTCTGGGTAATGAATGTTTTCTGGATAGCATGAATAAAAGCAAAGAACATGGTTTTCTGGGTTTTCGTAATAGCAAAAATGCATTTATTAGCTGGATTGATAAAGCAAAAGCATATAAAATTGTTCCG
优选地,所述甾体5β还原酶变体催化底物所获得的产物5位的氢构象为β型。此还原酶具有空间加氢特异性,产物中无5α型氢结构。
本发明还提供了编码所述甾体5β还原酶变体的分离的多核苷酸、包含所述多核苷酸的表达载体以及包含所述载体的宿主细胞。优选地,所述多核苷酸序列如SEQ ID NO:2、SEQ ID NO:4或SEQ ID NO:6所示。
优选地,所述表达载体为真核或原核生物的染色体。优选地,所述表达载体选自真核表达载体或原核表达载体。优选地,所述表达载体为质粒,优选为pcDNA3.1或pET26b(+)。
所述宿主细胞选自真核细胞或原核细胞。
更优选地,所述真核细胞是真菌细胞,更进一步优选为酵母菌。
更优选地,所述原核细胞选自大肠杆菌、分枝杆菌、假单胞菌、红球菌、节杆菌、枯草杆菌或放线菌细胞;更进一步优选为大肠杆菌T7 express或BL21(DE3)细胞。
本发明还提供了一种甾体5β还原酶组合物,其包含所述甾体5β还原酶变体。
本发明还提供了一种制备所述甾体5β还原酶变体的方法,所述方法包括以下步骤:
(1)在有助于生产所述甾体5β还原酶变体的条件下培养含有表达载体的宿主细胞,以及
(2)从得到的培养液中获得所述的甾体5β还原酶变体。
本发明还提供了所述甾体5β还原酶变体、所述多核苷酸、所述表达载体、所述宿主细胞或所述甾体5β还原酶组合物在制备5β-氢的甾体中的用途。优选地,所述5β-氢的甾体为5β-孕甾烷-3,20-二酮、20-羟甲基-5β-孕甾-3-酮(5β-PHM)或胆酸类化合物。更优选地,所述胆酸类化合物为具有脱氧胆酸骨架结构的化合物,例如脱氧胆酸。
本发明还提供了一种制备5β-氢的甾体的方法,所述方法包括使用所述甾体5β还原酶变体、所述多核苷酸、所述表达载体、所述宿主细胞或所述甾体5β还原酶组合物制备5β-氢的甾体。优选地,所述5β-氢的甾体为5β-孕甾烷-3,20-二酮、20-羟甲基-5β-孕甾-3-酮或胆酸类化合物。更优选地,所述胆酸类化合物为具有脱氧胆酸骨架结构的化合物,例如脱氧胆酸。
本发明的有益效果包括:
(1)本发明获得的以NADH为辅酶的甾体5β还原酶变体相较于利用NADPH为辅酶的甾体5β还原酶具有高效、稳定的特点,能够大大降低实际工业生产的成本;
(2)本发明的方法为微生物转化法,反应条件温和,对环境没有污染;
(3)应用本发明的甾体5β还原酶变体进行转化,生产成本低,工艺简单,经济效益可观;
(4)本发明的甾体5β还原酶变体可以以NADH为辅酶转化***生成5β-孕甾烷-3,20-二酮,或将20-羟甲基孕甾-3-酮(PHM)转化为20-羟甲基-5β-孕甾-3-酮(5β-PHM),且转化产物构象单一,只有β型还原产物出现,此还原酶具有空间加氢特异性,产物中无α型结构。
附图说明
图1显示了TLC法检测CmP5βR及CmP5βR-LY利用NADH转化***的产物点图,其中,“–”:CmP5βR以NADH为辅酶反应4h的产物情况;“+”:CmP5βR-LY以NADH为辅酶反应4h的产物情况;“5β”:5β-孕甾烷-3,20-二酮标准品(Rf=0.5);“Pro”:***标准品(Rf=0.4)。
图2显示了GC分析5α-孕甾烷-3,20-二酮标准品、5β-孕甾烷-3,20-二酮标准品和***标准品混合峰图,其中,1.5β-孕甾烷-3,20-二酮标准品,tR=25.106min;2.5α-孕甾烷-3,20-二酮标准品,tR=25.741min;3.***标准品,tR=26.776min。
图3显示了GC检测CmP5βR-LY利用NADH为辅酶,特异性转化***为5β-孕甾烷-3,20-二酮所获得的样品中各物质的色谱图,其中,1.产物峰5β-孕甾烷-3,20-二酮,tR=25.144min;3.底物峰***,tR=26.780min。
图4-1显示了CmP5βR-LY以NADH为辅酶转化***所得产物的质谱图。
图4-2显示了CmP5βR-LY利用NADH转化***的产物的1H谱图。
图4-3显示了CmP5βR-LY利用NADH转化***的产物的13C谱图。
图5显示了TLC法检测CmP5βR及CmP5βR-LY利用NADH转化20-羟甲基孕甾-3-酮(PHM)的产物点图,其中,“1”:20-羟甲基孕甾-3-酮(PHM)底物标准品(Rf=0.5);“2”:CmP5βR以NADH为辅酶反应4h的产物情况;“3”:CmP5βR-LY以NADH为辅酶反应4h的产物情况。
图6-1显示了CmP5βR-LY以NADH为辅酶转化PHM所得产物的质谱图。
图6-2显示了CmP5βR-LY利用NADH转化PHM的产物的1H谱图。
图6-3显示了CmP5βR-LY利用NADH转化PHM的产物的13C谱图。
具体实施方式
以下参照具体的实施例来说明本发明。本领域技术人员能够理解,这些实施例仅用于说明本发明,其不以任何方式限制本发明的范围。
下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的菌株、质粒、试剂盒等,如无特殊说明,均为市售购买产品。
实施例1甾体5β还原酶变体CmP5βR-LY基因的获取
通过对甾体5β还原酶CmP5βR的生物信息学分析发现相应的第63/64位氨基酸参与辅酶的结合,与辅酶的专一性直接相关。替代该位点的氨基酸可使得甾体5β还原酶CmP5βR利用NADH为辅酶。通过基因的突变和表达,筛选获得可利用NADH为辅酶的突变体,可催化***转化为5β-孕甾烷-3,20-二酮。
采用以下步骤获取编码甾体5β还原酶变体CmP5βR-LY的基因:
(1)甾体5β还原酶CmP5βR基因全序列DNA的获得
以CmP5βR氨基酸序列SEQ ID NO:7(GenBank:ALB78110),通过反向翻译法转换成基因的核苷酸序列SEQ ID NO:8,全合成CmP5βR基因DNA。设计CmP5βR基因引物序列SEQ IDNO:9和SEQ ID NO:10,以CmP5βR基因DNA为模板,通过PCR扩增出具有酶切位点NdeI/HindIII的CmP5βR基因的全序列DNA片段。
SEQ ID NO:7
MSWWGAGAIGAAKKKLDDDEPTQSYESVALIIGVTGIVGNSLAEILPLSDTLGGPWKVYGVARRPRPSWNADHPIDYIQCDVSNADDARSKLSPLTDVTHVFYVTWTNRESETENCEANGSMLRNVLRAVVPHAPNLRHVCLQTGTKHYLGPFTNVDGPHHDPPFTEDMPRLQIQNFYYTQEDVLFEEIKKKEGVTWSIHRPNMIFGFSPYSLMNIVGTLCVYAAICKHEGSPLMFPGSKKAWEGFMTASDADLIAEQQIWAAVDPYAKNEAFNCNNADIFKWKHLWKILAEQFGIEEYGFEEGKNLGLVEMMKGKERVWEEMVKENQLLEKKLDEVGVWWFADVILGVEGMIDSMNKSKEHGFLGFRNSNNSFISWIDKYKAFKIVP
SEQ ID NO:8
ATGAGCTGGTGGGGCGCCGGTGCGATTGGTGCGGCGAAAAAGAAACTGGACGATGACGAGCCGACCCAGAGCTACGAGAGTGTTGCGCTGATCATCGGCGTTACGGGCATCGTTGGCAACAGTCTGGCGGAAATTCTGCCACTGAGCGATACGCTGGGTGGCCCGTGGAAAGTGTATGGTGTTGCGCGTCGCCCACGTCCAAGCTGGAATGCCGACCACCCGATCGACTACATCCAGTGCGACGTGAGTAACGCCGATGATGCGCGCAGCAAACTGAGCCCGCTGACCGATGTTACCCACGTGTTTTACGTGACGTGGACCAACCGCGAAAGCGAAACGGAAAACTGCGAAGCGAACGGCAGCATGCTGCGCAATGTGCTGCGCGCCGTTGTGCCACATGCCCCAAATCTGCGCCATGTGTGTCTGCAGACCGGCACGAAACACTATCTGGGCCCGTTTACGAATGTGGATGGCCCACACCACGACCCACCGTTCACGGAAGATATGCCGCGCCTCCAGATCCAGAACTTCTACTACACCCAAGAAGATGTGCTCTTTGAGGAGATCAAGAAAAAAGAAGGCGTGACGTGGAGCATCCACCGCCCAAATATGATCTTCGGCTTCAGCCCGTACAGTCTGATGAATATCGTGGGCACGCTGTGTGTGTACGCCGCCATCTGCAAACATGAGGGTAGTCCGCTGATGTTCCCGGGCAGTAAAAAAGCGTGGGAGGGCTTCATGACCGCCAGCGATGCCGACCTCATCGCCGAACAGCAGATTTGGGCGGCCGTGGACCCGTATGCCAAGAACGAGGCGTTCAACTGCAACAACGCCGACATCTTCAAGTGGAAACACCTCTGGAAAATTCTGGCCGAGCAGTTTGGCATTGAGGAGTACGGCTTCGAAGAAGGCAAGAACCTCGGTCTGGTGGAGATGATGAAAGGCAAGGAACGCGTGTGGGAGGAGATGGTTAAGGAAAACCAGCTGCTCGAGAAAAAGCTCGACGAGGTTGGCGTTTGGTGGTTTGCGGACGTTATTCTGGGTGTTGAGGGCATGATCGACAGTATGAATAAGAGCAAGGAACACGGCTTTCTGGGCTTCCGCAACAGCAACAACAGCTTCATTAGCTGGATCGATAAGTATAAAGCCTTCAAAATTGTGCCG
SEQ ID NO:9:
CmP5βR-F AAAAACATATGAGCTGGTGGGGCGCCGGTG
SEQ ID NO:10:
CmP5βR-R AAAAAAAGCTTCGGCACAATTTTGAAGGCTT
(2)表达载体pET26b-CmP5βR的构建
将甾体5β还原酶DNA片段和pET26b(+)质粒分别用NdeI/HindIII酶切并回收,连接,化学法转化E.coli DH5α感受态细胞,并筛选重组菌,提取质粒验证成功后命名为pET26b-CmP5βR。
(3)CmP5βR基因63/64位定点饱和突变
设计CmP5βR63/64位定点饱和突变引物SEQ ID NO:11和SEQ ID NO:12,以pET26b-CmP5βR质粒DNA为模板,通过重叠延伸PCR法将甾体5β还原酶CmP5βR进行定点饱和突变。
SEQ ID NO:11:
R(63X)m CCAGCTTGGACGTGGNNNNNNCGCAACACCATACAC
SEQ ID NO:12:
F(63X)m GGTGTATGGTGTTGCGNNNNNNCCACGTCCAAGCTGG
重叠延伸PCR法的具体方法如下:
A.以pET26b-CmP5βR质粒DNA为模板,以CmP5βR-F(SEQ ID NO:9)和R(63X)m(SEQID NO:11)为引物,通过PCR扩增获得突变基因片段PCR1;
B.以pET26b-CmP5βR质粒DNA为模板,以F(63X)m(SEQ ID NO:12)和CmP5βR-R(SEQID NO:10)为引物,通过PCR扩增获得突变基因片段PCR2;
C.以PCR1和PCR2的混合物DNA为模板,以CmP5βR-F(SEQ ID NO:9)和CmP5βR-R(SEQID NO:10)为引物,通过PCR扩增获得具有酶切位点NdeI/HindIII且含有定点饱和突变基因的DNA片段PCR3;
(4)CmP5βR基因定点饱和突变株的转化子筛选及检测分析
将定点饱和突变获得的DNA片段PCR3使用限制性内切酶NdeI/HindIII酶切后,酶连于已使用限制性内切酶NdeI/HindIII酶切后的pET26b(+)质粒,电转化于E.coli T7感受态细胞,获得大量转化子。将每个转化子进行基因表达,并进行底物的转化(实施例2),产物萃取后进行TLC和GC检测和定性定量分析,筛选成功可利用NADH为辅酶的突变质粒命名为pET26b-CmP5βR-LY,获得编码甾体5β还原酶变体CmP5βR-LY的基因(SEQ ID NO:2),该变体CmP5βR-LY具有R63K/R64H突变。
其中,提取产物5β-孕甾烷-3,20-二酮的方法如下:
将产物用2倍体积乙酸乙酯萃取一次,收集有机相,真空干燥后得5β-孕甾烷-3,20-二酮样品,将该样品进行TLC检测(实施例3)及GC检测(实施例4)。
实施例2CmP5βR-LY酶以NADH为辅酶制备5辅酶孕甾烷-3,20-二酮
采用以下步骤制备5β-孕甾烷-3,20-二酮:
(1)将含有CmP5βR-LY编码基因的pET26b(+)载体重组表达质粒转化进高效表达菌株E.coli T7感受态细胞中,获得重组菌。
(2)接重组菌单菌落于LBK(LB培养基含50μg/mL硫酸卡那霉素)培养基中,37℃过夜培养。其中,LB培养基由以下组分组成:
胰蛋白胨 10g/L
酵母提取物 5g/L
氯化钠 10g/L
LB培养基配制方法如下:定量称取上述培养基成分溶于800mL去离子水中,用5mol/L NaOH调pH至7.4,用去离子水定容至1L。在121℃高压下蒸汽灭菌20min,即得。
(3)取50μL菌液转接至5mL新鲜LBK培养基中,37℃培养2h至OD(600nm)值约等于0.6。随后加入IPTG至终浓度为1mM,于22℃培养过夜20h。
(4)离心收集诱导后菌体,以Tris-HCl(pH 5.5)缓冲液洗涤两次。将细胞低温超声波裂解,得裂解液。
(5)取上述裂解液300μL,再加入辅酶NADH至终浓度为3mg/mL,加入底物***至终浓度0.2mg/mL。整个反应体系在40℃条件下反应4h后,加入2倍体积乙酸乙酯终止反应过程。
(6)涡旋萃取上述反应产物,取上清挥干溶剂,复溶即可进行TLC检测、GC检测、MS检测及NMR检测,对产物定性和定量分析。
结果显示,CmP5βR-LY基因表达的蛋白酶以***为底物,NADH为辅酶,反应生成5β-孕甾烷-3,20-二酮,底物转化率为70%。
图4显示了CmP5βR-LY利用NADH转化***的产物的MS、NMR-1H、NMR-13C谱图。
由此可证,甾体5β还原酶变体CmP5βR-LY可以以NADH为辅酶转化***生成5β-孕甾烷-3,20-二酮,且转化产物构象单一,只有β型还原产物出现,此还原酶具有空间加氢特异性,产物中无α型结构。
实施例3TLC法检测生物转化产物
将对照品***和5β-孕甾烷-3,20-二酮标准品及生物转化获得的5β-孕甾烷-3,20-二酮样品点样于薄层层析板上,于石油醚:乙酸乙酯(3:1,V/V)的展开剂中展开30min。
将展开后的薄层层析板喷淋茴香醛试剂,于200℃高温喷烤显色。
如图1所示,“–”为CmP5βR基因以NADH为辅酶反应4h的产物情况;“+”为CmP5βR-LY以NADH为辅酶反应4h的产物情况;“5β”为5β-孕甾烷-3,20-二酮标准品(Rf=0.5);“Pro”为***标准品(Rf=0.4)。
由图1可知,CmP5βR基因不能以NADH为辅酶进行生物转化,CmP5βR-LY基因能以NADH为辅酶进行生物转化。
实施例4GC法检测生物转化产物
固体样品的制备方法:将生物转化获得的5β-孕甾烷-3,20-二酮样品用乙酸乙酯溶解,配制1mg/mL的溶液,并通过0.22μm的有机膜过滤除杂,滤液利用气相色谱法分析产物的含量。
GC分析条件包括:
仪器:Agilent Technologies 7890D
色谱柱为Agilent HP-5;检测器为FID检测器;进样量1μl,进样口温度为220℃;检测器温度为300℃;
采用程序升温:初温150℃,4min,升温速率5L/min,升到280℃,5min。
取样品1μl,注入气相色谱仪,记录色谱图;另取***、5α-孕甾烷-3,20-二酮和5β-孕甾烷-3,20-二酮标准品25mg,精密称定,置25ml容量瓶中,加乙酸乙酯溶解并稀释至刻度,摇匀,作为对照品溶液,同法测定,按外标法以峰面积计算出供试品中***、5α-孕甾烷-3,20-二酮标准品、5β-孕甾烷-3,20-二酮的浓度。
计算公式如下:
Ax为供试品中***(5α-孕甾烷-3,20-二酮标准品,5β-孕甾烷-3,20-二酮)峰面积,
Ar为对照品中***(5α-孕甾烷-3,20-二酮标准品,5β-孕甾烷-3,20-二酮)峰面积,
Cr为对照品中***(5α-孕甾烷-3,20-二酮标准品,5β-孕甾烷-3,20-二酮)的浓度(mg/mL),
进样要求:含量测定的样品,单样双针,平行测定。
利用各物质的出峰时间不同,对比***标准品、5α-孕甾烷-3,20-二酮标准品及5β-孕甾烷-3,20-二酮标准品来检测产物的生成。
图2显示了GC分析5α-孕甾烷-3,20-二酮标准品、5β-孕甾烷-3,20-二酮标准品和***标准品混合峰图,其中,1.5β-孕甾烷-3,20-二酮标准品,tR=25.106min;2.5α-孕甾烷-3,20-二酮标准品,tR=25.741min;3.***标准品,tR=26.776min。
图3显示了实施例1中CmP5βR-LY突变体利用NADH转化***生成5β-孕甾烷-3,20-二酮所获得的样品中各物质的出峰时间。其中,1:产物5β-孕甾烷-3,20-二酮,保留时间T=25.144min;3:底物***,保留时间T=26.780min。
由图3显示的各物质出峰时间可见,CmP5βR-LY突变体以NADH为辅酶,将***转化成5β-孕甾烷-3,20-二酮,而不转化为5α-孕甾烷-3,20-二酮,该CmP5βR-LY突变体具有空间加氢特异性。
实施例5_AtP5β5基因及DlP5及R基因产物的辅酶特异性转换
两个与CmP5βR同源的蛋白序列,AtP5βR和DlP5βR,与CmP5βR的同源性分别为94%和69%。通过与CmP5βR的序列比对,在AtP5βR和DlP5βR序列里找到了与CmP5βR的R63/R64相应氨基酸。
表2甾体5β还原酶的结合口袋和催化位点的序列分析
其中,AtP5βR(EF579963)蛋白的氨基酸序列如SEQ ID NO:13所示。DlP5βR(AY585867)蛋白的氨基酸序列如SEQ ID NO:14所示。AtP5βR与本发明的甾体5β还原酶CmP5βR具有94%的氨基酸同一性,是高度同源的蛋白。DlP5βR与本发明的甾体5β还原酶CmP5βR具有69%的氨基酸同一性,是低度同源的蛋白。
合成基因,将AtP5βR的反转录序列SEQ ID NO:19和DlP5βR的反转录序列SEQ IDNO:20进行全基因合成。设计AtP5βR基因引物SEQ ID NO:15和SEQ ID NO:16及DlP5βR基因引物SEQ ID NO:17和SEQ ID NO:18,以AtP5βR基因、DlP5βR基因DNA为模板,通过PCR扩增出具有酶切位点NdeI/HindIII的AtP5βR基因的全序列DNA片段和DlP5βR基因的全序列DNA片段,并克隆到表达质粒的NdeI/HindIII位点。
上述序列如下所示。
SEQ ID NO:13
MSWWWAGAIGAAKKKLDEDEPSQSFESVALIIGVTGIVGNSLAEILPLSDTPGGPWKVYGVARRPRPTWNADHPIDYIQCDVSDAEDTRSKLSPLTDVTHVFYVTWTNRESESENCEANGSMLRNVLQAIIPYAPNLRHVCLQTGTKHYLGPFTNVDGPRHDPPFTEDMPRLQIQNFYYTQEDILFEEIKKIETVTWSIHRPNMIFGFSPYSLMNIVGTLCVYAAICKHEGSPLLFPGSKKAWEGFMTASDADLIAEQQIWAAVDPYAKNEAFNCNNADIFKWKHLWKILAEQFGIEEYGFEEGKNLGLVEMMKGKERVWEEMVKENQLQEKKLEEVGVWWFADVILGVEGMIDSMNKSKEYGFLGFRNSNNSFISWIDKYKAFKIVP
SEQ ID NO:14
MSWWWAGAIGAAKKRLEEDDAQPKHSSVALIVGVTGIIGNSLAEILPLADTPGGPWKVYGVARRTRPAWHEDNPINYVQCDISDPDDSQAKLSPLTDVTHVFYVTWANRSTEQENCEANSKMFRNVLDAVIPNCPNLKHISLQTGRKHYMGPFESYGKIESHDPPYTEDLPRLKYMNFYYDLEDIMLEEVEKKEGLTWSVHRPGNIFGFSPYSMMNLVGTLCVYAAICKHEGKVLRFTGCKAAWDGYSDCSDADLIAEHHIWAAVDPYAKNEAFNVSNGDVFKWKHFWKVLAEQFGVGCGEYEEGVDLKLQDLMKGKEPVWEEIVRENGLTPTKLKDVGIWWFGDVILGNECFLDSMNKSKEHGFLGFRNSKNAFISWIDKAKAYKIVP
SEQ ID NO:15
AtP5βR-F AAAAACATATGAGCTGGTGGTGGGCAGGTG
SEQ ID NO:16
AtP5βR-R AAAAAAAGCTTAGGCACAATCTTAAATG
SEQ ID NO:17
DlP5βR-F AAAAACATATGAGCTGGTGGTGGGCCGGG
SEQ ID NO:18
DlP5βR-R AAAAAAAGCTTCGGAACAATTTTATATGC
SEQ ID NO:19
ATGAGCTGGTGGTGGGCAGGTGCAATTGGTGCCGCCAAGAAGAAGCTGGATGAGGATGAACCGAGCCAGAGCTTTGAAAGCGTGGCCCTGATCATCGGTGTGACCGGCATCGTTGGCAATAGCCTGGCCGAAATCCTGCCGCTGAGCGATACCCCTGGTGGTCCGTGGAAAGTTTATGGTGTGGCACGTCGCCCTCGTCCGACCTGGAACGCAGATCACCCGATTGACTACATCCAATGCGACGTGAGCGATGCAGAAGACACCCGTAGCAAACTGAGCCCGCTGACAGATGTGACCCACGTGTTCTACGTGACCTGGACCAACCGTGAAAGCGAGAGCGAAAATTGTGAGGCCAACGGCAGCATGCTGCGCAATGTGCTGCAGGCAATTATCCCGTACGCACCGAATCTGCGTCACGTGTGTCTGCAGACAGGCACCAAGCATTACCTGGGCCCGTTTACCAACGTTGATGGCCCTCGCCATGATCCTCCGTTTACCGAGGACATGCCGCGCCTGCAGATCCAGAATTTCTACTACACCCAAGAAGATATTCTGTTTGAAGAAATCAAAAAGATCGAAACCGTGACCTGGAGCATCCACCGCCCGAACATGATCTTTGGCTTCAGCCCGTATAGCCTGATGAACATCGTGGGCACACTGTGCGTGTACGCAGCCATCTGCAAGCACGAAGGTAGCCCGCTGCTGTTTCCGGGTAGCAAGAAAGCCTGGGAGGGCTTTATGACAGCAAGCGATGCCGACCTGATTGCCGAACAGCAGATTTGGGCCGCCGTGGATCCGTATGCCAAAAACGAGGCCTTCAACTGCAATAACGCCGATATTTTTAAATGGAAACATCTGTGGAAAATCCTGGCCGAGCAGTTTGGCATCGAAGAATACGGCTTCGAAGAAGGCAAGAACCTGGGCCTGGTTGAGATGATGAAAGGCAAGGAGCGCGTGTGGGAAGAAATGGTTAAGGAGAACCAGCTGCAGGAGAAAAAGCTGGAGGAAGTGGGTGTGTGGTGGTTCGCCGATGTGATCCTGGGCGTTGAAGGCATGATCGATAGTATGAATAAAAGCAAGGAATATGGCTTCCTGGGCTTTCGCAACAGCAACAACAGCTTTATTAGCTGGATTGATAAATATAAAGCATTTAAGATTGTGCCT
SEQ ID NO:20
ATGAGCTGGTGGTGGGCCGGGGCGATTGGTGCCGCAAAAAAACGTCTGGAAGAAGATGATGCACAGCCGAAACATAGCAGCGTTGCACTGATTGTTGGTGTTACCGGTATTATTGGTAATAGCCTGGCAGAAATTCTGCCGCTGGCAGATACCCCGGGTGGTCCGTGGAAAGTTTATGGTGTTGCACGTCGTACCCGTCCGGCATGGCATGAAGATAATCCGATTAATTATGTTCAGTGTGATATTAGCGATCCGGATGATAGCCAGGCAAAACTGAGCCCGCTGACCGATGTTACCCATGTTTTTTATGTTACCTGGGCAAATCGTAGCACCGAACAGGAAAATTGTGAAGCAAATAGCAAAATGTTTCGTAATGTTCTGGATGCAGTTATTCCGAATTGTCCGAATCTGAAACATATTAGCCTGCAGACCGGTCGTAAACATTATATGGGTCCGTTTGAAAGCTATGGTAAAATTGAAAGCCATGATCCGCCGTATACCGAAGATCTGCCGCGTCTGAAATATATGAATTTTTATTATGATCTGGAAGATATTATGCTGGAAGAAGTTGAAAAAAAAGAAGGTCTGACCTGGAGCGTTCATCGTCCGGGTAATATTTTTGGTTTTAGCCCGTATAGCATGATGAATCTGGTTGGTACCCTGTGTGTTTATGCAGCAATTTGTAAACATGAAGGTAAAGTTCTGCGTTTTACCGGTTGTAAAGCAGCATGGGATGGTTATAGCGATTGTAGCGATGCAGATCTGATTGCAGAACATCATATTTGGGCAGCAGTTGATCCGTATGCAAAAAATGAAGCATTTAATGTTAGCAATGGTGATGTTTTTAAATGGAAACATTTTTGGAAAGTTCTGGCAGAACAGTTTGGTGTTGGTTGTGGTGAATATGAAGAAGGTGTTGATCTGAAACTGCAGGATCTGATGAAAGGTAAAGAACCGGTTTGGGAAGAAATTGTTCGTGAAAATGGTCTGACCCCGACCAAACTGAAAGATGTTGGTATTTGGTGGTTTGGTGATGTTATTCTGGGTAATGAATGTTTTCTGGATAGCATGAATAAAAGCAAAGAACATGGTTTTCTGGGTTTTCGTAATAGCAAAAATGCATTTATTAGCTGGATTGATAAAGCAAAAGCATATAAAATTGTTCCG
按照与实施例1相似的方法将AtP5βR基因的全序列DNA片段和DlP5βR基因的全序列DNA片段进行定点突变,基因分别命名为AtP5βR-LY基因(其编码的甾体5β还原酶变体AtP5βR-LY的氨基酸序列如SEQ ID NO:3所示,其多核苷酸序列如SEQ ID NO:4所示)和DlP5βR-LY基因(其编码的甾体5β还原酶变体DlP5βR-LY的氨基酸序列如SEQ ID NO:5所示,其多核苷酸序列如SEQ ID NO:6所示)。突变体AtP5βR-LY和DlP5βR-LY序列均具有R63K/R64H氨基酸转换。
将AtP5βR基因、AtP5βR-LY基因、DlP5βR基因及DlP5βR-LY基因表达的蛋白,利用NADH为辅酶催化底物***进行反应,产物送至TLC和GC检测,结果见下表:
表3表达蛋白利用NADH转化产物积累统计表
结果显示,AtP5βR-LY基因、DlP5βR-LY基因表达的蛋白酶以***为底物,NADH为辅酶,反应生成5β-孕甾烷-3,20-二酮,且转化产物构象单一,只有β型还原产物出现,无α型结构,此还原酶具有空间加氢特异性;AtP5βR基因、DlP5βR基因表达的蛋白酶以***为底物,NADH为辅酶,无产物生成,其不可利用NADH为辅酶催化底物生成产物。这表明无论是具有与本发明的甾体5β还原酶CmP5βR高度同源性的甾体5β还原酶AtP5βR,还是具有与本发明的甾体5β还原酶CmP5βR低度同源性的甾体5β还原酶DlP5βR,在进行定点突变R63K/R64H后,均可利用NADH为辅酶,可见本发明R63K/R64H突变可成功改变甾体5β还原酶的辅酶专一性。
实施例6CmP5β5及CmP5例R-LY酶以NADH为辅酶转化20-羟甲基孕甾-3-酮(PHM)
采用以下步骤转化PHM:
(1)以实施例2的方法,获得CmP5βR及CmP5βR-LY基因表达蛋白的裂解液。
(2)取上述裂解液300μL,再加入辅酶NADH至终浓度为3mg/mL,加入底物PHM至终浓度0.2mg/mL。整个反应体系在40℃条件下反应4h后,加入2倍体积乙酸乙酯终止反应过程。
(3)涡旋萃取上述反应产物,取上清挥干溶剂,复溶即可进行TLC检测、MS检测及NMR检测,对产物定性和定量分析。
图5显示了TLC法检测CmP5βR及CmP5βR-LY利用NADH转化20-羟甲基孕甾-3-酮(PHM)的产物点图,其中,“1”:PHM底物标准品(Rf=0.5);“2”:CmP5βR以NADH为辅酶反应4h的产物情况;“3”:CmP5βR-LY以NADH为辅酶反应4h的产物情况。
图6显示了CmP5βR-LY利用NADH转化PHM的产物的MS、NMR-1H、NMR-13C谱图。
由此可证明,甾体5β还原酶变体CmP5βR-LY可以以NADH为辅酶转化PHM生成5β-PHM,且转化产物构象单一,只有β型还原产物出现,此还原酶具有空间加氢特异性,产物中无α型结构。
以上对本发明具体实施方式的描述并不限制本发明,本领域技术人员可以根据本发明作出各种改变或变形,只要不脱离本发明的精神,均应属于本发明所附权利要求的范围。
序列表
<110> 沈阳博泰生物制药有限公司
<120> 一种甾体5β还原酶变体及其用途
<130> DIC18110079R
<150> 2019111284283
<151> 2019-11-18
<160> 20
<170> SIPOSequenceListing 1.0
<210> 1
<211> 388
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met Ser Trp Trp Gly Ala Gly Ala Ile Gly Ala Ala Lys Lys Lys Leu
1 5 10 15
Asp Asp Asp Glu Pro Thr Gln Ser Tyr Glu Ser Val Ala Leu Ile Ile
20 25 30
Gly Val Thr Gly Ile Val Gly Asn Ser Leu Ala Glu Ile Leu Pro Leu
35 40 45
Ser Asp Thr Leu Gly Gly Pro Trp Lys Val Tyr Gly Val Ala Lys His
50 55 60
Pro Arg Pro Ser Trp Asn Ala Asp His Pro Ile Asp Tyr Ile Gln Cys
65 70 75 80
Asp Val Ser Asn Ala Asp Asp Ala Arg Ser Lys Leu Ser Pro Leu Thr
85 90 95
Asp Val Thr His Val Phe Tyr Val Thr Trp Thr Asn Arg Glu Ser Glu
100 105 110
Thr Glu Asn Cys Glu Ala Asn Gly Ser Met Leu Arg Asn Val Leu Arg
115 120 125
Ala Val Val Pro His Ala Pro Asn Leu Arg His Val Cys Leu Gln Thr
130 135 140
Gly Thr Lys His Tyr Leu Gly Pro Phe Thr Asn Val Asp Gly Pro His
145 150 155 160
His Asp Pro Pro Phe Thr Glu Asp Met Pro Arg Leu Gln Ile Gln Asn
165 170 175
Phe Tyr Tyr Thr Gln Glu Asp Val Leu Phe Glu Glu Ile Lys Lys Lys
180 185 190
Glu Gly Val Thr Trp Ser Ile His Arg Pro Asn Met Ile Phe Gly Phe
195 200 205
Ser Pro Tyr Ser Leu Met Asn Ile Val Gly Thr Leu Cys Val Tyr Ala
210 215 220
Ala Ile Cys Lys His Glu Gly Ser Pro Leu Met Phe Pro Gly Ser Lys
225 230 235 240
Lys Ala Trp Glu Gly Phe Met Thr Ala Ser Asp Ala Asp Leu Ile Ala
245 250 255
Glu Gln Gln Ile Trp Ala Ala Val Asp Pro Tyr Ala Lys Asn Glu Ala
260 265 270
Phe Asn Cys Asn Asn Ala Asp Ile Phe Lys Trp Lys His Leu Trp Lys
275 280 285
Ile Leu Ala Glu Gln Phe Gly Ile Glu Glu Tyr Gly Phe Glu Glu Gly
290 295 300
Lys Asn Leu Gly Leu Val Glu Met Met Lys Gly Lys Glu Arg Val Trp
305 310 315 320
Glu Glu Met Val Lys Glu Asn Gln Leu Leu Glu Lys Lys Leu Asp Glu
325 330 335
Val Gly Val Trp Trp Phe Ala Asp Val Ile Leu Gly Val Glu Gly Met
340 345 350
Ile Asp Ser Met Asn Lys Ser Lys Glu His Gly Phe Leu Gly Phe Arg
355 360 365
Asn Ser Asn Asn Ser Phe Ile Ser Trp Ile Asp Lys Tyr Lys Ala Phe
370 375 380
Lys Ile Val Pro
385
<210> 2
<211> 1164
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atgagctggt ggggcgccgg tgcgattggt gcggcgaaaa agaaactgga cgatgacgag 60
ccgacccaga gctacgagag tgttgcgctg atcatcggcg ttacgggcat cgttggcaac 120
agtctggcgg aaattctgcc actgagcgat acgctgggtg gcccgtggaa agtgtatggt 180
gttgcgaaac atccacgtcc aagctggaat gccgaccacc cgatcgacta catccagtgc 240
gacgtgagta acgccgatga tgcgcgcagc aaactgagcc cgctgaccga tgttacccac 300
gtgttttacg tgacgtggac caaccgcgaa agcgaaacgg aaaactgcga agcgaacggc 360
agcatgctgc gcaatgtgct gcgcgccgtt gtgccacatg ccccaaatct gcgccatgtg 420
tgtctgcaga ccggcacgaa acactatctg ggcccgttta cgaatgtgga tggcccacac 480
cacgacccac cgttcacgga agatatgccg cgcctccaga tccagaactt ctactacacc 540
caagaagatg tgctctttga ggagatcaag aaaaaagaag gcgtgacgtg gagcatccac 600
cgcccaaata tgatcttcgg cttcagcccg tacagtctga tgaatatcgt gggcacgctg 660
tgtgtgtacg ccgccatctg caaacatgag ggtagtccgc tgatgttccc gggcagtaaa 720
aaagcgtggg agggcttcat gaccgccagc gatgccgacc tcatcgccga acagcagatt 780
tgggcggccg tggacccgta tgccaagaac gaggcgttca actgcaacaa cgccgacatc 840
ttcaagtgga aacacctctg gaaaattctg gccgagcagt ttggcattga ggagtacggc 900
ttcgaagaag gcaagaacct cggtctggtg gagatgatga aaggcaagga acgcgtgtgg 960
gaggagatgg ttaaggaaaa ccagctgctc gagaaaaagc tcgacgaggt tggcgtttgg 1020
tggtttgcgg acgttattct gggtgttgag ggcatgatcg acagtatgaa taagagcaag 1080
gaacacggct ttctgggctt ccgcaacagc aacaacagct tcattagctg gatcgataag 1140
tataaagcct tcaaaattgt gccg 1164
<210> 3
<211> 388
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Met Ser Trp Trp Trp Ala Gly Ala Ile Gly Ala Ala Lys Lys Lys Leu
1 5 10 15
Asp Glu Asp Glu Pro Ser Gln Ser Phe Glu Ser Val Ala Leu Ile Ile
20 25 30
Gly Val Thr Gly Ile Val Gly Asn Ser Leu Ala Glu Ile Leu Pro Leu
35 40 45
Ser Asp Thr Pro Gly Gly Pro Trp Lys Val Tyr Gly Val Ala Lys His
50 55 60
Pro Arg Pro Thr Trp Asn Ala Asp His Pro Ile Asp Tyr Ile Gln Cys
65 70 75 80
Asp Val Ser Asp Ala Glu Asp Thr Arg Ser Lys Leu Ser Pro Leu Thr
85 90 95
Asp Val Thr His Val Phe Tyr Val Thr Trp Thr Asn Arg Glu Ser Glu
100 105 110
Ser Glu Asn Cys Glu Ala Asn Gly Ser Met Leu Arg Asn Val Leu Gln
115 120 125
Ala Ile Ile Pro Tyr Ala Pro Asn Leu Arg His Val Cys Leu Gln Thr
130 135 140
Gly Thr Lys His Tyr Leu Gly Pro Phe Thr Asn Val Asp Gly Pro Arg
145 150 155 160
His Asp Pro Pro Phe Thr Glu Asp Met Pro Arg Leu Gln Ile Gln Asn
165 170 175
Phe Tyr Tyr Thr Gln Glu Asp Ile Leu Phe Glu Glu Ile Lys Lys Ile
180 185 190
Glu Thr Val Thr Trp Ser Ile His Arg Pro Asn Met Ile Phe Gly Phe
195 200 205
Ser Pro Tyr Ser Leu Met Asn Ile Val Gly Thr Leu Cys Val Tyr Ala
210 215 220
Ala Ile Cys Lys His Glu Gly Ser Pro Leu Leu Phe Pro Gly Ser Lys
225 230 235 240
Lys Ala Trp Glu Gly Phe Met Thr Ala Ser Asp Ala Asp Leu Ile Ala
245 250 255
Glu Gln Gln Ile Trp Ala Ala Val Asp Pro Tyr Ala Lys Asn Glu Ala
260 265 270
Phe Asn Cys Asn Asn Ala Asp Ile Phe Lys Trp Lys His Leu Trp Lys
275 280 285
Ile Leu Ala Glu Gln Phe Gly Ile Glu Glu Tyr Gly Phe Glu Glu Gly
290 295 300
Lys Asn Leu Gly Leu Val Glu Met Met Lys Gly Lys Glu Arg Val Trp
305 310 315 320
Glu Glu Met Val Lys Glu Asn Gln Leu Gln Glu Lys Lys Leu Glu Glu
325 330 335
Val Gly Val Trp Trp Phe Ala Asp Val Ile Leu Gly Val Glu Gly Met
340 345 350
Ile Asp Ser Met Asn Lys Ser Lys Glu Tyr Gly Phe Leu Gly Phe Arg
355 360 365
Asn Ser Asn Asn Ser Phe Ile Ser Trp Ile Asp Lys Tyr Lys Ala Phe
370 375 380
Lys Ile Val Pro
385
<210> 4
<211> 1164
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atgagctggt ggtgggcagg tgcaattggt gccgccaaga agaagctgga tgaggatgaa 60
ccgagccaga gctttgaaag cgtggccctg atcatcggtg tgaccggcat cgttggcaat 120
agcctggccg aaatcctgcc gctgagcgat acccctggtg gtccgtggaa agtttatggt 180
gtggcaaaac atcctcgtcc gacctggaac gcagatcacc cgattgacta catccaatgc 240
gacgtgagcg atgcagaaga cacccgtagc aaactgagcc cgctgacaga tgtgacccac 300
gtgttctacg tgacctggac caaccgtgaa agcgagagcg aaaattgtga ggccaacggc 360
agcatgctgc gcaatgtgct gcaggcaatt atcccgtacg caccgaatct gcgtcacgtg 420
tgtctgcaga caggcaccaa gcattacctg ggcccgttta ccaacgttga tggccctcgc 480
catgatcctc cgtttaccga ggacatgccg cgcctgcaga tccagaattt ctactacacc 540
caagaagata ttctgtttga agaaatcaaa aagatcgaaa ccgtgacctg gagcatccac 600
cgcccgaaca tgatctttgg cttcagcccg tatagcctga tgaacatcgt gggcacactg 660
tgcgtgtacg cagccatctg caagcacgaa ggtagcccgc tgctgtttcc gggtagcaag 720
aaagcctggg agggctttat gacagcaagc gatgccgacc tgattgccga acagcagatt 780
tgggccgccg tggatccgta tgccaaaaac gaggccttca actgcaataa cgccgatatt 840
tttaaatgga aacatctgtg gaaaatcctg gccgagcagt ttggcatcga agaatacggc 900
ttcgaagaag gcaagaacct gggcctggtt gagatgatga aaggcaagga gcgcgtgtgg 960
gaagaaatgg ttaaggagaa ccagctgcag gagaaaaagc tggaggaagt gggtgtgtgg 1020
tggttcgccg atgtgatcct gggcgttgaa ggcatgatcg atagtatgaa taaaagcaag 1080
gaatatggct tcctgggctt tcgcaacagc aacaacagct ttattagctg gattgataaa 1140
tataaagcat ttaagattgt gcct 1164
<210> 5
<211> 389
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Met Ser Trp Trp Trp Ala Gly Ala Ile Gly Ala Ala Lys Lys Arg Leu
1 5 10 15
Glu Glu Asp Asp Ala Gln Pro Lys His Ser Ser Val Ala Leu Ile Val
20 25 30
Gly Val Thr Gly Ile Ile Gly Asn Ser Leu Ala Glu Ile Leu Pro Leu
35 40 45
Ala Asp Thr Pro Gly Gly Pro Trp Lys Val Tyr Gly Val Ala Lys His
50 55 60
Thr Arg Pro Ala Trp His Glu Asp Asn Pro Ile Asn Tyr Val Gln Cys
65 70 75 80
Asp Ile Ser Asp Pro Asp Asp Ser Gln Ala Lys Leu Ser Pro Leu Thr
85 90 95
Asp Val Thr His Val Phe Tyr Val Thr Trp Ala Asn Arg Ser Thr Glu
100 105 110
Gln Glu Asn Cys Glu Ala Asn Ser Lys Met Phe Arg Asn Val Leu Asp
115 120 125
Ala Val Ile Pro Asn Cys Pro Asn Leu Lys His Ile Ser Leu Gln Thr
130 135 140
Gly Arg Lys His Tyr Met Gly Pro Phe Glu Ser Tyr Gly Lys Ile Glu
145 150 155 160
Ser His Asp Pro Pro Tyr Thr Glu Asp Leu Pro Arg Leu Lys Tyr Met
165 170 175
Asn Phe Tyr Tyr Asp Leu Glu Asp Ile Met Leu Glu Glu Val Glu Lys
180 185 190
Lys Glu Gly Leu Thr Trp Ser Val His Arg Pro Gly Asn Ile Phe Gly
195 200 205
Phe Ser Pro Tyr Ser Met Met Asn Leu Val Gly Thr Leu Cys Val Tyr
210 215 220
Ala Ala Ile Cys Lys His Glu Gly Lys Val Leu Arg Phe Thr Gly Cys
225 230 235 240
Lys Ala Ala Trp Asp Gly Tyr Ser Asp Cys Ser Asp Ala Asp Leu Ile
245 250 255
Ala Glu His His Ile Trp Ala Ala Val Asp Pro Tyr Ala Lys Asn Glu
260 265 270
Ala Phe Asn Val Ser Asn Gly Asp Val Phe Lys Trp Lys His Phe Trp
275 280 285
Lys Val Leu Ala Glu Gln Phe Gly Val Gly Cys Gly Glu Tyr Glu Glu
290 295 300
Gly Val Asp Leu Lys Leu Gln Asp Leu Met Lys Gly Lys Glu Pro Val
305 310 315 320
Trp Glu Glu Ile Val Arg Glu Asn Gly Leu Thr Pro Thr Lys Leu Lys
325 330 335
Asp Val Gly Ile Trp Trp Phe Gly Asp Val Ile Leu Gly Asn Glu Cys
340 345 350
Phe Leu Asp Ser Met Asn Lys Ser Lys Glu His Gly Phe Leu Gly Phe
355 360 365
Arg Asn Ser Lys Asn Ala Phe Ile Ser Trp Ile Asp Lys Ala Lys Ala
370 375 380
Tyr Lys Ile Val Pro
385
<210> 6
<211> 1167
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
atgagctggt ggtgggccgg ggcgattggt gccgcaaaaa aacgtctgga agaagatgat 60
gcacagccga aacatagcag cgttgcactg attgttggtg ttaccggtat tattggtaat 120
agcctggcag aaattctgcc gctggcagat accccgggtg gtccgtggaa agtttatggt 180
gttgcaaaac atacccgtcc ggcatggcat gaagataatc cgattaatta tgttcagtgt 240
gatattagcg atccggatga tagccaggca aaactgagcc cgctgaccga tgttacccat 300
gttttttatg ttacctgggc aaatcgtagc accgaacagg aaaattgtga agcaaatagc 360
aaaatgtttc gtaatgttct ggatgcagtt attccgaatt gtccgaatct gaaacatatt 420
agcctgcaga ccggtcgtaa acattatatg ggtccgtttg aaagctatgg taaaattgaa 480
agccatgatc cgccgtatac cgaagatctg ccgcgtctga aatatatgaa tttttattat 540
gatctggaag atattatgct ggaagaagtt gaaaaaaaag aaggtctgac ctggagcgtt 600
catcgtccgg gtaatatttt tggttttagc ccgtatagca tgatgaatct ggttggtacc 660
ctgtgtgttt atgcagcaat ttgtaaacat gaaggtaaag ttctgcgttt taccggttgt 720
aaagcagcat gggatggtta tagcgattgt agcgatgcag atctgattgc agaacatcat 780
atttgggcag cagttgatcc gtatgcaaaa aatgaagcat ttaatgttag caatggtgat 840
gtttttaaat ggaaacattt ttggaaagtt ctggcagaac agtttggtgt tggttgtggt 900
gaatatgaag aaggtgttga tctgaaactg caggatctga tgaaaggtaa agaaccggtt 960
tgggaagaaa ttgttcgtga aaatggtctg accccgacca aactgaaaga tgttggtatt 1020
tggtggtttg gtgatgttat tctgggtaat gaatgttttc tggatagcat gaataaaagc 1080
aaagaacatg gttttctggg ttttcgtaat agcaaaaatg catttattag ctggattgat 1140
aaagcaaaag catataaaat tgttccg 1167
<210> 7
<211> 388
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Met Ser Trp Trp Gly Ala Gly Ala Ile Gly Ala Ala Lys Lys Lys Leu
1 5 10 15
Asp Asp Asp Glu Pro Thr Gln Ser Tyr Glu Ser Val Ala Leu Ile Ile
20 25 30
Gly Val Thr Gly Ile Val Gly Asn Ser Leu Ala Glu Ile Leu Pro Leu
35 40 45
Ser Asp Thr Leu Gly Gly Pro Trp Lys Val Tyr Gly Val Ala Arg Arg
50 55 60
Pro Arg Pro Ser Trp Asn Ala Asp His Pro Ile Asp Tyr Ile Gln Cys
65 70 75 80
Asp Val Ser Asn Ala Asp Asp Ala Arg Ser Lys Leu Ser Pro Leu Thr
85 90 95
Asp Val Thr His Val Phe Tyr Val Thr Trp Thr Asn Arg Glu Ser Glu
100 105 110
Thr Glu Asn Cys Glu Ala Asn Gly Ser Met Leu Arg Asn Val Leu Arg
115 120 125
Ala Val Val Pro His Ala Pro Asn Leu Arg His Val Cys Leu Gln Thr
130 135 140
Gly Thr Lys His Tyr Leu Gly Pro Phe Thr Asn Val Asp Gly Pro His
145 150 155 160
His Asp Pro Pro Phe Thr Glu Asp Met Pro Arg Leu Gln Ile Gln Asn
165 170 175
Phe Tyr Tyr Thr Gln Glu Asp Val Leu Phe Glu Glu Ile Lys Lys Lys
180 185 190
Glu Gly Val Thr Trp Ser Ile His Arg Pro Asn Met Ile Phe Gly Phe
195 200 205
Ser Pro Tyr Ser Leu Met Asn Ile Val Gly Thr Leu Cys Val Tyr Ala
210 215 220
Ala Ile Cys Lys His Glu Gly Ser Pro Leu Met Phe Pro Gly Ser Lys
225 230 235 240
Lys Ala Trp Glu Gly Phe Met Thr Ala Ser Asp Ala Asp Leu Ile Ala
245 250 255
Glu Gln Gln Ile Trp Ala Ala Val Asp Pro Tyr Ala Lys Asn Glu Ala
260 265 270
Phe Asn Cys Asn Asn Ala Asp Ile Phe Lys Trp Lys His Leu Trp Lys
275 280 285
Ile Leu Ala Glu Gln Phe Gly Ile Glu Glu Tyr Gly Phe Glu Glu Gly
290 295 300
Lys Asn Leu Gly Leu Val Glu Met Met Lys Gly Lys Glu Arg Val Trp
305 310 315 320
Glu Glu Met Val Lys Glu Asn Gln Leu Leu Glu Lys Lys Leu Asp Glu
325 330 335
Val Gly Val Trp Trp Phe Ala Asp Val Ile Leu Gly Val Glu Gly Met
340 345 350
Ile Asp Ser Met Asn Lys Ser Lys Glu His Gly Phe Leu Gly Phe Arg
355 360 365
Asn Ser Asn Asn Ser Phe Ile Ser Trp Ile Asp Lys Tyr Lys Ala Phe
370 375 380
Lys Ile Val Pro
385
<210> 8
<211> 1164
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atgagctggt ggggcgccgg tgcgattggt gcggcgaaaa agaaactgga cgatgacgag 60
ccgacccaga gctacgagag tgttgcgctg atcatcggcg ttacgggcat cgttggcaac 120
agtctggcgg aaattctgcc actgagcgat acgctgggtg gcccgtggaa agtgtatggt 180
gttgcgcgtc gcccacgtcc aagctggaat gccgaccacc cgatcgacta catccagtgc 240
gacgtgagta acgccgatga tgcgcgcagc aaactgagcc cgctgaccga tgttacccac 300
gtgttttacg tgacgtggac caaccgcgaa agcgaaacgg aaaactgcga agcgaacggc 360
agcatgctgc gcaatgtgct gcgcgccgtt gtgccacatg ccccaaatct gcgccatgtg 420
tgtctgcaga ccggcacgaa acactatctg ggcccgttta cgaatgtgga tggcccacac 480
cacgacccac cgttcacgga agatatgccg cgcctccaga tccagaactt ctactacacc 540
caagaagatg tgctctttga ggagatcaag aaaaaagaag gcgtgacgtg gagcatccac 600
cgcccaaata tgatcttcgg cttcagcccg tacagtctga tgaatatcgt gggcacgctg 660
tgtgtgtacg ccgccatctg caaacatgag ggtagtccgc tgatgttccc gggcagtaaa 720
aaagcgtggg agggcttcat gaccgccagc gatgccgacc tcatcgccga acagcagatt 780
tgggcggccg tggacccgta tgccaagaac gaggcgttca actgcaacaa cgccgacatc 840
ttcaagtgga aacacctctg gaaaattctg gccgagcagt ttggcattga ggagtacggc 900
ttcgaagaag gcaagaacct cggtctggtg gagatgatga aaggcaagga acgcgtgtgg 960
gaggagatgg ttaaggaaaa ccagctgctc gagaaaaagc tcgacgaggt tggcgtttgg 1020
tggtttgcgg acgttattct gggtgttgag ggcatgatcg acagtatgaa taagagcaag 1080
gaacacggct ttctgggctt ccgcaacagc aacaacagct tcattagctg gatcgataag 1140
tataaagcct tcaaaattgt gccg 1164
<210> 9
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
aaaaacatat gagctggtgg ggcgccggtg 30
<210> 10
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
aaaaaaagct tcggcacaat tttgaaggct t 31
<210> 11
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
ccagcttgga cgtggnnnnn ncgcaacacc atacac 36
<210> 12
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
ggtgtatggt gttgcgnnnn nnccacgtcc aagctgg 37
<210> 13
<211> 388
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Met Ser Trp Trp Trp Ala Gly Ala Ile Gly Ala Ala Lys Lys Lys Leu
1 5 10 15
Asp Glu Asp Glu Pro Ser Gln Ser Phe Glu Ser Val Ala Leu Ile Ile
20 25 30
Gly Val Thr Gly Ile Val Gly Asn Ser Leu Ala Glu Ile Leu Pro Leu
35 40 45
Ser Asp Thr Pro Gly Gly Pro Trp Lys Val Tyr Gly Val Ala Arg Arg
50 55 60
Pro Arg Pro Thr Trp Asn Ala Asp His Pro Ile Asp Tyr Ile Gln Cys
65 70 75 80
Asp Val Ser Asp Ala Glu Asp Thr Arg Ser Lys Leu Ser Pro Leu Thr
85 90 95
Asp Val Thr His Val Phe Tyr Val Thr Trp Thr Asn Arg Glu Ser Glu
100 105 110
Ser Glu Asn Cys Glu Ala Asn Gly Ser Met Leu Arg Asn Val Leu Gln
115 120 125
Ala Ile Ile Pro Tyr Ala Pro Asn Leu Arg His Val Cys Leu Gln Thr
130 135 140
Gly Thr Lys His Tyr Leu Gly Pro Phe Thr Asn Val Asp Gly Pro Arg
145 150 155 160
His Asp Pro Pro Phe Thr Glu Asp Met Pro Arg Leu Gln Ile Gln Asn
165 170 175
Phe Tyr Tyr Thr Gln Glu Asp Ile Leu Phe Glu Glu Ile Lys Lys Ile
180 185 190
Glu Thr Val Thr Trp Ser Ile His Arg Pro Asn Met Ile Phe Gly Phe
195 200 205
Ser Pro Tyr Ser Leu Met Asn Ile Val Gly Thr Leu Cys Val Tyr Ala
210 215 220
Ala Ile Cys Lys His Glu Gly Ser Pro Leu Leu Phe Pro Gly Ser Lys
225 230 235 240
Lys Ala Trp Glu Gly Phe Met Thr Ala Ser Asp Ala Asp Leu Ile Ala
245 250 255
Glu Gln Gln Ile Trp Ala Ala Val Asp Pro Tyr Ala Lys Asn Glu Ala
260 265 270
Phe Asn Cys Asn Asn Ala Asp Ile Phe Lys Trp Lys His Leu Trp Lys
275 280 285
Ile Leu Ala Glu Gln Phe Gly Ile Glu Glu Tyr Gly Phe Glu Glu Gly
290 295 300
Lys Asn Leu Gly Leu Val Glu Met Met Lys Gly Lys Glu Arg Val Trp
305 310 315 320
Glu Glu Met Val Lys Glu Asn Gln Leu Gln Glu Lys Lys Leu Glu Glu
325 330 335
Val Gly Val Trp Trp Phe Ala Asp Val Ile Leu Gly Val Glu Gly Met
340 345 350
Ile Asp Ser Met Asn Lys Ser Lys Glu Tyr Gly Phe Leu Gly Phe Arg
355 360 365
Asn Ser Asn Asn Ser Phe Ile Ser Trp Ile Asp Lys Tyr Lys Ala Phe
370 375 380
Lys Ile Val Pro
385
<210> 14
<211> 389
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Met Ser Trp Trp Trp Ala Gly Ala Ile Gly Ala Ala Lys Lys Arg Leu
1 5 10 15
Glu Glu Asp Asp Ala Gln Pro Lys His Ser Ser Val Ala Leu Ile Val
20 25 30
Gly Val Thr Gly Ile Ile Gly Asn Ser Leu Ala Glu Ile Leu Pro Leu
35 40 45
Ala Asp Thr Pro Gly Gly Pro Trp Lys Val Tyr Gly Val Ala Arg Arg
50 55 60
Thr Arg Pro Ala Trp His Glu Asp Asn Pro Ile Asn Tyr Val Gln Cys
65 70 75 80
Asp Ile Ser Asp Pro Asp Asp Ser Gln Ala Lys Leu Ser Pro Leu Thr
85 90 95
Asp Val Thr His Val Phe Tyr Val Thr Trp Ala Asn Arg Ser Thr Glu
100 105 110
Gln Glu Asn Cys Glu Ala Asn Ser Lys Met Phe Arg Asn Val Leu Asp
115 120 125
Ala Val Ile Pro Asn Cys Pro Asn Leu Lys His Ile Ser Leu Gln Thr
130 135 140
Gly Arg Lys His Tyr Met Gly Pro Phe Glu Ser Tyr Gly Lys Ile Glu
145 150 155 160
Ser His Asp Pro Pro Tyr Thr Glu Asp Leu Pro Arg Leu Lys Tyr Met
165 170 175
Asn Phe Tyr Tyr Asp Leu Glu Asp Ile Met Leu Glu Glu Val Glu Lys
180 185 190
Lys Glu Gly Leu Thr Trp Ser Val His Arg Pro Gly Asn Ile Phe Gly
195 200 205
Phe Ser Pro Tyr Ser Met Met Asn Leu Val Gly Thr Leu Cys Val Tyr
210 215 220
Ala Ala Ile Cys Lys His Glu Gly Lys Val Leu Arg Phe Thr Gly Cys
225 230 235 240
Lys Ala Ala Trp Asp Gly Tyr Ser Asp Cys Ser Asp Ala Asp Leu Ile
245 250 255
Ala Glu His His Ile Trp Ala Ala Val Asp Pro Tyr Ala Lys Asn Glu
260 265 270
Ala Phe Asn Val Ser Asn Gly Asp Val Phe Lys Trp Lys His Phe Trp
275 280 285
Lys Val Leu Ala Glu Gln Phe Gly Val Gly Cys Gly Glu Tyr Glu Glu
290 295 300
Gly Val Asp Leu Lys Leu Gln Asp Leu Met Lys Gly Lys Glu Pro Val
305 310 315 320
Trp Glu Glu Ile Val Arg Glu Asn Gly Leu Thr Pro Thr Lys Leu Lys
325 330 335
Asp Val Gly Ile Trp Trp Phe Gly Asp Val Ile Leu Gly Asn Glu Cys
340 345 350
Phe Leu Asp Ser Met Asn Lys Ser Lys Glu His Gly Phe Leu Gly Phe
355 360 365
Arg Asn Ser Lys Asn Ala Phe Ile Ser Trp Ile Asp Lys Ala Lys Ala
370 375 380
Tyr Lys Ile Val Pro
385
<210> 15
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
aaaaacatat gagctggtgg tgggcaggtg 30
<210> 16
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
aaaaaaagct taggcacaat cttaaatg 28
<210> 17
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
aaaaacatat gagctggtgg tgggccggg 29
<210> 18
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
aaaaaaagct tcggaacaat tttatatgc 29
<210> 19
<211> 1164
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
atgagctggt ggtgggcagg tgcaattggt gccgccaaga agaagctgga tgaggatgaa 60
ccgagccaga gctttgaaag cgtggccctg atcatcggtg tgaccggcat cgttggcaat 120
agcctggccg aaatcctgcc gctgagcgat acccctggtg gtccgtggaa agtttatggt 180
gtggcacgtc gccctcgtcc gacctggaac gcagatcacc cgattgacta catccaatgc 240
gacgtgagcg atgcagaaga cacccgtagc aaactgagcc cgctgacaga tgtgacccac 300
gtgttctacg tgacctggac caaccgtgaa agcgagagcg aaaattgtga ggccaacggc 360
agcatgctgc gcaatgtgct gcaggcaatt atcccgtacg caccgaatct gcgtcacgtg 420
tgtctgcaga caggcaccaa gcattacctg ggcccgttta ccaacgttga tggccctcgc 480
catgatcctc cgtttaccga ggacatgccg cgcctgcaga tccagaattt ctactacacc 540
caagaagata ttctgtttga agaaatcaaa aagatcgaaa ccgtgacctg gagcatccac 600
cgcccgaaca tgatctttgg cttcagcccg tatagcctga tgaacatcgt gggcacactg 660
tgcgtgtacg cagccatctg caagcacgaa ggtagcccgc tgctgtttcc gggtagcaag 720
aaagcctggg agggctttat gacagcaagc gatgccgacc tgattgccga acagcagatt 780
tgggccgccg tggatccgta tgccaaaaac gaggccttca actgcaataa cgccgatatt 840
tttaaatgga aacatctgtg gaaaatcctg gccgagcagt ttggcatcga agaatacggc 900
ttcgaagaag gcaagaacct gggcctggtt gagatgatga aaggcaagga gcgcgtgtgg 960
gaagaaatgg ttaaggagaa ccagctgcag gagaaaaagc tggaggaagt gggtgtgtgg 1020
tggttcgccg atgtgatcct gggcgttgaa ggcatgatcg atagtatgaa taaaagcaag 1080
gaatatggct tcctgggctt tcgcaacagc aacaacagct ttattagctg gattgataaa 1140
tataaagcat ttaagattgt gcct 1164
<210> 20
<211> 1167
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
atgagctggt ggtgggccgg ggcgattggt gccgcaaaaa aacgtctgga agaagatgat 60
gcacagccga aacatagcag cgttgcactg attgttggtg ttaccggtat tattggtaat 120
agcctggcag aaattctgcc gctggcagat accccgggtg gtccgtggaa agtttatggt 180
gttgcacgtc gtacccgtcc ggcatggcat gaagataatc cgattaatta tgttcagtgt 240
gatattagcg atccggatga tagccaggca aaactgagcc cgctgaccga tgttacccat 300
gttttttatg ttacctgggc aaatcgtagc accgaacagg aaaattgtga agcaaatagc 360
aaaatgtttc gtaatgttct ggatgcagtt attccgaatt gtccgaatct gaaacatatt 420
agcctgcaga ccggtcgtaa acattatatg ggtccgtttg aaagctatgg taaaattgaa 480
agccatgatc cgccgtatac cgaagatctg ccgcgtctga aatatatgaa tttttattat 540
gatctggaag atattatgct ggaagaagtt gaaaaaaaag aaggtctgac ctggagcgtt 600
catcgtccgg gtaatatttt tggttttagc ccgtatagca tgatgaatct ggttggtacc 660
ctgtgtgttt atgcagcaat ttgtaaacat gaaggtaaag ttctgcgttt taccggttgt 720
aaagcagcat gggatggtta tagcgattgt agcgatgcag atctgattgc agaacatcat 780
atttgggcag cagttgatcc gtatgcaaaa aatgaagcat ttaatgttag caatggtgat 840
gtttttaaat ggaaacattt ttggaaagtt ctggcagaac agtttggtgt tggttgtggt 900
gaatatgaag aaggtgttga tctgaaactg caggatctga tgaaaggtaa agaaccggtt 960
tgggaagaaa ttgttcgtga aaatggtctg accccgacca aactgaaaga tgttggtatt 1020
tggtggtttg gtgatgttat tctgggtaat gaatgttttc tggatagcat gaataaaagc 1080
aaagaacatg gttttctggg ttttcgtaat agcaaaaatg catttattag ctggattgat 1140
aaagcaaaag catataaaat tgttccg 1167
Claims (11)
1.一种具有甾体5β还原酶活性的甾体5β还原酶变体,所述甾体5β还原酶变体的氨基酸序列包括两个相邻位点的RR突变为相邻的KH。
2.根据权利要求1所述的甾体5β还原酶变体,其中所述甾体5β还原酶变体包含R63K和R64H突变,能够利用NADH为辅酶,且具有选自以下的氨基酸序列:
1)SEQ ID NO:1、SEQ ID NO:3或SEQ ID NO:5所示氨基酸序列;
2)SEQ ID NO:1、SEQ ID NO:3或SEQ ID NO:5所示氨基酸序列经过取代、缺失或添加一个或多个氨基酸而得到的氨基酸序列;或
3)与SEQ ID NO:1、SEQ ID NO:3或SEQ ID NO:5具有69%以上序列同一性的氨基酸序列;
优选地,所述甾体5β还原酶变体能够利用NADPH和NADH为辅酶。
3.根据权利要求1或2所述的甾体5β还原酶变体,其中所述甾体5β还原酶变体催化底物所获得的产物5位的氢构象为β型。
4.根据权利要求1-3中任一项所述的甾体5β还原酶变体,其中所述甾体5β还原酶变体的氨基酸序列如SEQ ID NO:1、SEQ ID NO:3或SEQ ID NO:5所示。
5.一种分离的多核苷酸,其编码权利要求1-4中任一项所述的甾体5β还原酶变体;优选地,所述多核苷酸序列如SEQ ID NO:2、SEQ ID NO:4或SEQ ID NO:6所示。
6.一种表达载体,其包含权利要求5所述的多核苷酸;优选地,所述表达载体为真核或原核生物的染色体;优选地,所述表达载体选自真核表达载体或原核表达载体;优选地,所述表达载体为质粒,优选为pcDNA3.1或pET26b(+)。
7.一种宿主细胞,其包含权利要求6所述的表达载体;
优选地,所述宿主细胞选自真核细胞或原核细胞;
更优选地,所述真核细胞是真菌细胞,更进一步优选为酵母菌;
更优选地,所述原核细胞选自大肠杆菌、分枝杆菌、假单胞菌、红球菌、节杆菌、枯草杆菌或放线菌细胞;更进一步优选为大肠杆菌T7 express或BL21(DE3)细胞。
8.一种甾体5β还原酶组合物,其包含权利要求1-4中任一项所述的甾体5β还原酶变体。
9.一种制备权利要求1-4中任一项所述的甾体5β还原酶变体的方法,所述方法包括以下步骤:
(1)在有助于生产所述甾体5β还原酶变体的条件下培养权利要求6所述的宿主细胞,以及
(2)从得到的培养液中获得所述甾体5β还原酶变体。
10.权利要求1-4中任一项所述的甾体5β还原酶变体、权利要求5所述的多核苷酸、权利要求6所述的表达载体、权利要求7所述的宿主细胞或权利要求8所述的组合物在制备5β-氢的甾体中的用途;
优选地,所述5β-氢的甾体为5β-孕甾烷-3,20-二酮、20-羟甲基-5β-孕甾-3-酮或胆酸类化合物;
更优选地,所述胆酸类化合物为具有去氧胆酸骨架结构的化合物,例如脱氧胆酸。
11.一种用于制备5β-氢的甾体的方法,其包括使用权利要求1-4中任一项所述的甾体5β还原酶变体、权利要求5所述的多核苷酸、权利要求6所述的表达载体、权利要求7所述的宿主细胞或权利要求8所述的组合物制备5β-氢的甾体;优选地,所述5β-氢的甾体为5β-孕甾烷-3,20-二酮、20-羟甲基-5β-孕甾-3-酮或胆酸类化合物;更优选地,所述胆酸类化合物为具有去氧胆酸骨架结构的化合物,例如脱氧胆酸。
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CN108048416A (zh) * | 2017-12-25 | 2018-05-18 | 吉林凯莱英医药化学有限公司 | 改进的酮还原酶突变体及其制备方法和应用 |
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