本發(fā)明涉及一種腈水解酶突變體，特別涉及一種腈水解酶突變體及其編碼基因，以及其在制備光學(xué)活性2-芳基丙酸中的應(yīng)用。
背景技術(shù)：
：在有機(jī)合成中，氰基易作為“水穩(wěn)定負(fù)碳離子”引入有機(jī)化合物，而腈類化合物則是一種多用途的中間體，能轉(zhuǎn)化為胺、酰胺、羧酸和羧基化合物等。用微生物酶水解腈為酰胺和羧酸時(shí)，由于反應(yīng)條件溫和以及高純度的產(chǎn)物使之成為一種備受關(guān)注的方法，迄今在工業(yè)生產(chǎn)上已有許多成功的例子。近年來，頗有意義的是研究用微生物酶立體選擇性水解腈類制備光學(xué)活性2-芳基丙酸(2-APA)，光學(xué)活性2-APA可作為藥物活性基質(zhì)，特別是作為非甾體抗炎藥(NSAID)應(yīng)用。由于藥理臨床的研究表明2-APA類的NSAID的對映體中只有右旋的構(gòu)型顯示高的藥理活性，左旋構(gòu)型活性低或無作用。鑒于一般化學(xué)反應(yīng)所產(chǎn)生的手性中心總是得到等量的一對對映體混合物，稱之為外消旋體。欲得到光學(xué)純右旋2-APA，拆分外消旋體是主要方法之一。一般來說采用化學(xué)拆分需用手性衍生試劑，價(jià)格昂貴，且過程冗長，較難得到滿意的結(jié)果。而用微生物酶法拆分則可提供一個經(jīng)濟(jì)的選擇。酮洛芬(Ketoprofen，KP)又名酮基布洛芬、苯酮苯丙酸、優(yōu)布芬、優(yōu)洛芬或Profenid，化學(xué)名為α-甲基-3-苯甲?；揭宜?，是由法國化學(xué)家Farge、Messer和Moutounier于1967年開發(fā)的優(yōu)良的2-芳基丙酸類非甾體抗炎鎮(zhèn)痛藥物。其作用機(jī)制主要是通過抑制體內(nèi)環(huán)氧合酶(COXs)、脂氧化酶(LOXs)的生物活性，從而抑制致炎物質(zhì)前列腺素(PGs)、白三烯(LTs)的合成，具有對抗緩激肽釋放、清除羥自由基以及穩(wěn)定溶酶體膜活性，從而產(chǎn)生良好的解熱、鎮(zhèn)痛和抗炎作用，并增強(qiáng)其外周止痛效果。臨床研究表明，酮洛芬作為重要的非甾體抗炎藥物，與同類藥物相比，具有劑量小、療效高、耐受性好和毒副作用輕微等顯著優(yōu)點(diǎn)，成為治療類風(fēng)濕性關(guān)節(jié)炎、風(fēng)濕性關(guān)節(jié)炎、骨關(guān)節(jié)炎、強(qiáng)硬性脊椎炎以及痛風(fēng)的理想藥物，廣泛適用于治療痛經(jīng)、牙痛、術(shù)后疼痛、癌性疼痛和神經(jīng)炎、紅斑狼瘡、咽喉及支氣管炎等疾病，對軟組織受傷的治療效果更好。酮洛芬是2-芳基丙酸類非甾體抗炎藥(NSAIDs)，只有右旋體才具有抗炎抗風(fēng)濕和鎮(zhèn)痛作用，左旋體幾乎沒有藥理活性。酮洛芬的主要生產(chǎn)企業(yè)，國外除法國Rhone-Poulenc公司外，還有德國Sanofi-Aventis、意大利SIMS、Cosma和BidaChem、印度BECChemicals等公司；國內(nèi)西南合成制藥股份有限公司(原西南合成制藥廠)、中國科學(xué)院上海藥物研究所實(shí)驗(yàn)藥廠較早開展了酮洛芬的產(chǎn)品研制和工藝開發(fā)工作。目前化學(xué)合成酮洛芬的工藝路線已非常成熟，但是合成的酮洛芬缺乏手型選擇性。由于左旋酮洛芬沒有藥理活性，并且有很大副作用。因此，直接合成右旋酮洛芬或者將左旋酮洛芬轉(zhuǎn)變成右旋酮洛芬都具有巨大的經(jīng)濟(jì)效益。定點(diǎn)突變是指通過聚合酶鏈?zhǔn)椒磻?yīng)(PCR)等方法向目的DNA片段(可以是基因組，也可以是質(zhì)粒)中引入所需變化(通常是表征有利方向的變化)，包括堿基的添加、刪除、點(diǎn)突變等。定點(diǎn)突變能迅速、高效的提高DNA所表達(dá)的目的蛋白的性狀及表征，是基因研究工作中一種非常有用的手段。體外定點(diǎn)突變技術(shù)是當(dāng)前生物、醫(yī)學(xué)各領(lǐng)域研究中的一種重要實(shí)驗(yàn)手段，是改造、優(yōu)化基因的便捷方案，是探索啟動子調(diào)節(jié)位點(diǎn)的有效手段，也是研究蛋白質(zhì)結(jié)構(gòu)和功能之間的復(fù)雜關(guān)系的有力工具。定點(diǎn)突變改造糞產(chǎn)堿菌腈水解酶的研究具有極佳的理論和應(yīng)用價(jià)值。技術(shù)實(shí)現(xiàn)要素：本發(fā)明的目的是通過定點(diǎn)突變的方法對糞產(chǎn)堿菌腈水解酶基因進(jìn)行改造，使改造后的基因工程腈水解酶在酶活和手性選擇性方面有所提高，使其符合在催化生產(chǎn)右旋2-芳基丙酸工業(yè)化應(yīng)用的需求。本發(fā)明采用的技術(shù)方案是：本發(fā)明提供一種腈水解酶突變體，所述突變體包含SEQIDNO：1所示氨基酸序列中下述任何一個或多個氨基酸位點(diǎn)上的氨基酸取代，所述位點(diǎn)為135、41、192、120；其中，第135位的纈氨酸被突變?yōu)楸彼峄蚶野彼?；?1位的天冬氨酸被突變?yōu)楸彼?；?92位亮氨酸被突變?yōu)槔i氨酸；第120位的賴氨酸被突變?yōu)楫惲涟彼?。?yōu)選地，在一些實(shí)施例中，所述腈水解酶突變體包含SEQIDNO：1所示氨基酸序列中第135位的纈氨酸被突變?yōu)楸彼峄蚶野彼?。還包含下述任何一個或多個氨基酸位點(diǎn)上的氨基酸取代，所述位點(diǎn)選自41、134、120、82、192、319。優(yōu)選的第41位的天冬氨酸被突變?yōu)楸彼?，?34位的組氨酸被突變?yōu)槔i氨酸，第120位的賴氨酸被突變?yōu)楫惲涟彼?，?2位的丙氨酸被突變?yōu)樯彼?，?92位亮氨酸被突變?yōu)槔i氨酸，第319位的絲氨酸被突變?yōu)楫惲涟彼帷?yōu)選地，在一些實(shí)施例中，所述腈水解酶突變體包含SEQIDNO：1所示氨基酸序列中第135位的纈氨酸被突變?yōu)楸彼峄蚶野彼?。還包含下述任何一個或多個氨基酸位點(diǎn)上的氨基酸取代，所述位點(diǎn)選自41、192、120。優(yōu)選的第41位的天冬氨酸被突變?yōu)楸彼?，?92位亮氨酸被突變?yōu)槔i氨酸，第120位的賴氨酸被突變?yōu)楫惲涟彼?。?yōu)選地，在一些實(shí)施例中，所述腈水解酶突變體包含SEQIDNO：1所示氨基酸序列中第134位的組氨酸被突變?yōu)槔i氨酸，第135位的纈氨酸被突變?yōu)楸彼峄蚶野彼帷?yōu)選地，在一些實(shí)施例中，所述腈水解酶突變體包含SEQIDNO：1所示氨基酸序列中第134位的組氨酸被突變?yōu)槔i氨酸，第135位的纈氨酸被突變?yōu)楸彼峄蚶野彼帷＿€包含下述任何一個或多個氨基酸位點(diǎn)上的氨基酸取代，所述位點(diǎn)選自41、82、192、120、319，優(yōu)選的第41位的天冬氨酸被突變?yōu)楸彼幔?2位的丙氨酸被突變?yōu)樯彼?，?92位亮氨酸被突變?yōu)槔i氨酸，第120位的賴氨酸被突變?yōu)楫惲涟彼?，?19位的絲氨酸被突變?yōu)楫惲涟彼?。?yōu)選地，在一些實(shí)施例中，所述腈水解酶突變體包含SEQIDNO：1所示氨基酸序列中第134位的組氨酸被突變?yōu)槔i氨酸，第135位的纈氨酸被突變?yōu)楸彼峄蚶野彼?。還包含下述任何一個或多個氨基酸位點(diǎn)上的氨基酸取代，所述位點(diǎn)選自41、192、120，優(yōu)選的第41位的天冬氨酸被突變?yōu)楸彼?，?92位亮氨酸被突變?yōu)槔i氨酸，第120位的賴氨酸被突變?yōu)楫惲涟彼帷８鼉?yōu)選地，在一些實(shí)施例中，所述腈水解酶突變體，包含SEQIDNO：1所示氨基酸序列中選自以下氨基酸突變組成的組：1)V135A+D41A；2)V135A+K120I；3)V135A+L192V；4)H134V+V135A；5)H134V+V135A+L192V；6)H134V+V135A+L192V+D41A；7)H134V+V135A+L192V+K120I。本發(fā)明還涉及一種多核苷酸，其編碼以上所述的腈水解酶突變體。本發(fā)明還涉及一種包含上述多核苷酸的重組載體。本發(fā)明還提供一種由所述重組載體轉(zhuǎn)化制備的重組基因工程菌。此外，本發(fā)明還提供一種所述腈水解酶突變體在制備光學(xué)活性2-APA中的應(yīng)用。本發(fā)明通過定點(diǎn)突變的方式提供了一種腈水解酶突變體，該突變體與野生型腈水解酶相比具有更高的酶活和/或立體選擇性，該腈水解酶突變體在制備光學(xué)活性2-APA中的應(yīng)用，較化學(xué)合成法，能夠降低生產(chǎn)成本，提高生產(chǎn)效率，適應(yīng)工業(yè)化生產(chǎn)的需求。附圖說明附圖1實(shí)施例1野生型腈水解酶瓊脂糖凝膠電泳圖；附圖2實(shí)施例5腈水解酶突變體立體選擇性催化水解氰基酮洛芬；附圖3實(shí)施例5腈水解酶突變體瓊脂糖凝膠電泳圖；附圖4野生型腈水解酶三維結(jié)構(gòu)模擬圖。具體實(shí)施方式本發(fā)明的其他特點(diǎn)和優(yōu)越性可通過以下詳細(xì)的描述體現(xiàn)。但是，應(yīng)該理解的是，僅以示例性的方式給出詳細(xì)描述和具體實(shí)施例以表明本發(fā)明的實(shí)施方案，這是因?yàn)閷τ诒绢I(lǐng)域熟練技術(shù)人員從所述的詳細(xì)描述中，在本發(fā)明的實(shí)質(zhì)和范圍內(nèi)多種改變和改良是顯而易見的。術(shù)語定義本發(fā)明所述“2-芳基丙酸(2-APA)”是指具有式Ⅰ結(jié)構(gòu)的化合物，也被稱為芳基-2-丙酸。術(shù)語“芳基”可以是單環(huán)，雙環(huán)，和三環(huán)的碳環(huán)體系，其中，至少一個環(huán)體系是芳香族的，其中每一個環(huán)體系包含3-7個原子。具體來說，本文所述具有消炎作用的2-芳基丙酸類藥物包括但不限于如下舉例，例如酮洛芬(Ketoprofen)，甲氧基甲基萘乙酸(naproxene)、異丁苯丙酸(Ibrufen)、舒洛芬(Sutoprofen)、非諾洛芬(Fenoprofen)、苯惡洛芬(Bennoxaprofen)、卡布洛芬(Carprofen)、環(huán)洛芬(Cicloprofen)、氟比洛芬(Flurbiprofen)和氟洛芬(Fluprofen)等。本文所述“腈水解酶的活性”是指，腈水解酶催化水解腈為酰胺和羧酸，本文中優(yōu)選水解為羧酸。本文所述“酶活”或“轉(zhuǎn)化率”是指腈水解酶催化水解腈為羧酸的能力，或腈水解酶催化水解腈轉(zhuǎn)化為羧酸的比例。腈被催化水解為羧酸的比例越高，酶活越高，即轉(zhuǎn)化率越高，酶活越高。本文所述“光學(xué)活性2-芳基丙酸”，是指具有藥理活性的2-芳基丙酸，更進(jìn)一步的，為S(+)-2-APA。本文所述“ee％”意為對映體超量，即在手性合成中，生成目標(biāo)產(chǎn)物(某一種特定的立體異構(gòu)體)的百分含量減去副產(chǎn)物(另一種異構(gòu)體)的百分含量。例如，ee％為98％，即表示生成的目標(biāo)產(chǎn)物的含量為99％。本文所述“右旋ee％”＝(右旋％-左旋％)/(右旋％+左旋％)本文中所述的腈水解酶突變體，除文中提到的氨基酸取代，還包含普通意義上的保守氨基酸取代，所述“保守氨基酸取代”可包括天然氨基酸殘基被非天然殘基取代，使得對該位置上的氨基酸殘基的極性或電荷幾乎沒有或沒有影響。保守氨基酸取代還包括通常通過化學(xué)上的肽合成而不是通過在生物系統(tǒng)中的合成而摻入的非天然存在的氨基酸殘基。這些包括肽模擬物(peptidomimetics)和氨基酸部分的其它反轉(zhuǎn)形式或反向形式。常見的保守氨基酸取代見表A。表A氨基酸氨基酸取代AlanincD-Ala，Gly，Aib，β-Ala，L-Cys，D-CysArginincD-Arg，Lys，D-Lys，OrnD-OmAsparagineD-Asn.Asp，D-Asp，Glu，D-GluGln，D-GlnAsparticAcidD-Asp，D-Asn.Asn.Glu.D-Glu，Gln，D-GlnCysteineD-Cys，S-Me-Cys，Met，D-Met，Thr，D-Thr，L-Ser，D-SerGlutamincD-Gln，Asn，D-Asn，Glu，D-Glu，Asp，D-AspGlutamicAcidD-Glu，D-Asp，Asp，Asn，D-Asn，Gln，D-GlnGlycincAla，D-Ala，Pro，D-Pro，Aib，β-AlaIsoleucineD-Ile，Val，D-Val，leu，D-Leu，Met，D-MetLeucineVal，D-Val，Met，D-Met，D-Ile，D-Leu，lleLysincD-Lys，Arg，D-Arg，Orn，D-OmMethionineD-Met，S-Me-Cys，lle，D-Ile，Leu，D-Leu，Val，D-ValPhcnylalanincD-Phc，Tyr，D-Tyr，His，D-His，Trp，D-Trp腈水解酶突變體、制備方法及應(yīng)用NagasawaT等發(fā)現(xiàn)玫瑰色紅球菌(Rhodococcusrhodochrous)J1產(chǎn)生的腈水解酶既可以用作脂肪族腈，也可以作用于芳香族水解為相應(yīng)羧酸，此后，MaugerJ等分離出糞產(chǎn)堿桿菌(Alcaligenesfaecalis)JM3能夠?qū)⒎蓟译嫠鉃橄鄳?yīng)羧酸。發(fā)明人在土壤中分離出一株糞產(chǎn)堿桿菌，并提取出野生型腈水解酶，發(fā)現(xiàn)其在制備光學(xué)活性2-APA中具有較低的轉(zhuǎn)化率且立體選擇性不佳。發(fā)明人通過對該腈水解酶的三維結(jié)構(gòu)進(jìn)行模擬(見圖4)，篩選出可能對催化水解起關(guān)鍵作用的25個位點(diǎn)：R127、V135、L167、V57、W164、S189、L192、K203、M206、S106、K131、P169、M314、L322、P330、I344、V12、D41、A82、K120、H134、V139、S319、W58、H134，這些位點(diǎn)所在的區(qū)域被稱為催化口袋，在與底物結(jié)合或立體選擇性方面可能存在較大影響，發(fā)明人對它們分別進(jìn)行定點(diǎn)突變，意外獲得7個有益的突變位點(diǎn)V135、L192、D41、A82、K120、S319、H134，其中包含這些位點(diǎn)突變的腈水解酶突變體，在催化轉(zhuǎn)化率和/或立體選擇性方面有所增強(qiáng)。在一些實(shí)施例中，本發(fā)明提供一種腈水解酶突變體，所述突變體包含SEQIDNO：1所示氨基酸序列中下述任何一個或多個氨基酸位點(diǎn)上的氨基酸取代，所述位點(diǎn)為135、41、192、120。其中第135位的纈氨酸被突變?yōu)楸彼峄蚶野彼?；?1位的天冬氨酸被突變?yōu)楸彼?；?92位亮氨酸被突變?yōu)槔i氨酸；第120位的賴氨酸被突變?yōu)楫惲涟彼?。在一些?shí)施例中，所述腈水解酶突變體包含SEQIDNO：1所示氨基酸序列中第135位的纈氨酸被突變?yōu)楸彼峄蚶野彼?。還包含下述任何一個或多個氨基酸位點(diǎn)上的氨基酸取代，所述位點(diǎn)選自41、134、120、82、192、319。優(yōu)選的第41位的天冬氨酸被突變?yōu)楸彼?，?34位的組氨酸被突變?yōu)槔i氨酸，第120位的賴氨酸被突變?yōu)楫惲涟彼幔?2位的丙氨酸被突變?yōu)樯彼?，?92位亮氨酸被突變?yōu)槔i氨酸，第319位的絲氨酸被突變?yōu)楫惲涟彼?。在一些?shí)施例中，所述腈水解酶突變體包含SEQIDNO：1所示氨基酸序列中第135位的纈氨酸被突變?yōu)楸彼峄蚶野彼?。還包含下述任何一個或多個氨基酸位點(diǎn)上的氨基酸取代，所述位點(diǎn)選自41、192、120。優(yōu)選的第41位的天冬氨酸被突變?yōu)楸彼?，?92位亮氨酸被突變?yōu)槔i氨酸，第120位的賴氨酸被突變?yōu)楫惲涟彼?。在一些?shí)施例中，所述腈水解酶突變體包含SEQIDNO：1所示氨基酸序列中第134位的組氨酸被突變?yōu)槔i氨酸，第135位的纈氨酸被突變?yōu)楸彼峄蚶野彼帷Ｔ谝恍?shí)施例中，所述腈水解酶突變體包含SEQIDNO：1所示氨基酸序列中第134位的組氨酸被突變?yōu)槔i氨酸，第135位的纈氨酸被突變?yōu)楸彼峄蚶野彼帷＿€包含下述任何一個或多個氨基酸位點(diǎn)上的氨基酸取代，所述位點(diǎn)選自41、82、192、120、319，優(yōu)選的第41位的天冬氨酸被突變?yōu)楸彼?，?2位的丙氨酸被突變?yōu)樯彼?，?92位亮氨酸被突變?yōu)槔i氨酸，第120位的賴氨酸被突變?yōu)楫惲涟彼幔?19位的絲氨酸被突變?yōu)楫惲涟彼?。在一些?shí)施例中，所述腈水解酶突變體包含SEQIDNO：1所示氨基酸序列中第134位的組氨酸被突變?yōu)槔i氨酸，第135位的纈氨酸被突變?yōu)楸彼峄蚶野彼?。還包含下述任何一個或多個氨基酸位點(diǎn)上的氨基酸取代，所述位點(diǎn)選自41、192、120，優(yōu)選的第41位的天冬氨酸被突變?yōu)楸彼?，?92位亮氨酸被突變?yōu)槔i氨酸，第120位的賴氨酸被突變?yōu)楫惲涟彼?。在一些?yōu)選實(shí)施例中，所述腈水解酶突變體，包含SEQIDNO：1所示氨基酸序列中選自以下氨基酸突變組成的組：1)V135A+D41A；2)V135A+K120I；3)V135A+L192V；4)H134V+V135A；5)H134V+V135A+L192V；6)H134V+V135A+L192V+D41A；7)H134V+V135A+L192V+K120I；本發(fā)明還涉及一種多核苷酸，其編碼以上所述的腈水解酶突變體。本發(fā)明還涉及一種包含上述多核苷酸的重組載體。本發(fā)明還提供一種由所述重組載體轉(zhuǎn)化制備的重組基因工程菌。此外，本發(fā)明還提供一種所述腈水解酶突變體制備光學(xué)活性2-APA中的應(yīng)用。本文中所用到的試劑均可通過市售得到。實(shí)施例實(shí)施例1野生型腈水解酶基因的克隆(1)野生型腈水解酶基因的獲得從工業(yè)廢水附近土壤中篩選出一株糞產(chǎn)堿桿菌HEC-AF001使用OMEGA細(xì)菌基因組試劑盒進(jìn)行總基因組DNA的提取，使用下述引物擴(kuò)增長約1.1kb大小的基因片段。HEC-AF001-F：CCCATATGCAGACAAGAAAAATC(SEQIDNO：17)HEC-AF001-R：CCAAGCTTTCAGGACGGTTCTTG(SEQIDNO：18)PCR反應(yīng)體系見表1表1成分體系(μl)5×PrimeSTARPCRHSBuffer10引物11引物21模板(<0.2μg)1dNTPs(2.5mM)4PrimeSTARPCRHSPolymerase0.5ddH2O50PCR擴(kuò)增程序：98℃，預(yù)變性20s；98℃，變性10s；60℃退火10s；72℃延伸60s；重復(fù)30個循環(huán)；72℃繼續(xù)延伸10min。PCR產(chǎn)物經(jīng)瓊脂糖凝膠電泳純化，利用瓊脂糖凝膠DNA回收試劑盒回收1100bp左右的目標(biāo)條帶(見圖1)。送廣州艾基生物公司測序，確認(rèn)是目的條帶，如此獲得一條完整的腈水解酶全長基因序列，命名為NIT1，全長1071bp，堿基序列如序列表中SEQIDNo：2所示，利用軟件對該基因序列進(jìn)行分析，得到如SEQIDNo.2所示的野生型腈水解酶氨基酸序列SEQIDNo：1。實(shí)施例2重組表達(dá)質(zhì)粒和重組表達(dá)轉(zhuǎn)化體的制備將實(shí)施例1所得的PCR產(chǎn)物在37℃用限制性內(nèi)切酶NdeI和HindⅢ雙酶切4h，經(jīng)瓊脂糖凝膠電泳純化，利用瓊脂糖凝膠DNA回收試劑盒回收目標(biāo)片段，其中含有正確的插入片段。將目標(biāo)片段與同樣經(jīng)NdeI和HindⅢ酶切后的質(zhì)粒pET28a混合，在T4DNA連接酶的作用下，16℃連接4h得到重組表達(dá)質(zhì)粒pET28a-NIT1。將上述重組表達(dá)質(zhì)粒轉(zhuǎn)化到E.coliTop10感受態(tài)細(xì)胞中。在含有卡那霉素的抗性平板(培養(yǎng)基成分LB培養(yǎng)基，蛋白胨10g/L，酵母膏5g/L，氯化鈉10g/L和瓊脂2％，抗生素含量50mg/L)上對陽性重組體進(jìn)行篩選，挑取單克隆，培養(yǎng)重組菌株，待質(zhì)粒擴(kuò)增后提取質(zhì)粒，經(jīng)測序驗(yàn)證正確后，重新轉(zhuǎn)化至E.coliBL21(DE3)感受態(tài)細(xì)胞中。轉(zhuǎn)化液涂布到含有卡那霉素(50mg/L)的LB平板上，37℃倒置培養(yǎng)過夜，獲得陽性重組大腸桿菌E.coliBL21(DE3)/pET28a-NIT1，命名為HEC-NIT1。實(shí)施例3重組腈水解酶的表達(dá)將實(shí)施例2所得的重組大腸桿菌，接種至含卡那霉素(50mg/L)的LB培養(yǎng)基中，37℃振蕩培養(yǎng)過夜。按2‰(v/v)的接種量接入裝有50mlLB培養(yǎng)基的250ml三角燒瓶中，置37℃、180rpm搖床震蕩培養(yǎng)。當(dāng)培養(yǎng)液的OD600達(dá)到0.8時(shí)，加入終濃度為0.5mmol/L的IPTG進(jìn)行誘導(dǎo)。30℃誘導(dǎo)8h后，將培養(yǎng)液離心，收集細(xì)胞，得0.3g濕細(xì)胞。實(shí)施例4利用NIT1重組腈水解酶催化水解氰基酮洛芬酶催化反應(yīng)：分別將實(shí)施例4得到的腈水解酶濕菌體加入至含25ml底物溶液(工作濃度為1mM，稱取0.235g氰基酮洛芬，使用甲醇90ml、1M磷酸鉀緩沖液(pH7.8)90ml、H2O720ml進(jìn)行溶解)的三角瓶中，于30℃、250rpm條件下反應(yīng)48h，取出三角瓶，分別加入10ml甲醇溶液使氰基酮洛芬底物完全溶解，8000rpm、10min離心，棄除菌渣，收集上清。反應(yīng)體系后處理：將得到的上清液分別用旋轉(zhuǎn)蒸發(fā)儀于40℃條件下?lián)]除甲醇，用1MHCl調(diào)溶液pH至2～3左右；加入等體積的乙酸乙酯、混合均勻，收集乙酸乙酯相；取乙酸乙酯層吹干，取200μl90％正己烷(異丙醇)復(fù)溶，過0.45um濾膜，待用。HPLC檢測轉(zhuǎn)化率：色譜柱，WatersXBridgeC18；柱溫，30℃；流動相，0.02MNaClO4-乙腈，結(jié)果見表5。HPLC檢測手性：：色譜柱，ChiralND柱；柱溫，30℃；流動相，正己烷(0.15％TFA)：異丙醇＝93:7，檢測結(jié)果見表5。實(shí)施例5：野生型腈水解酶定向改造參考野生型腈水解酶基因序列(SEQIDNO：2)，設(shè)計(jì)并合成兩條寡核苷酸引物，并采用未突變的重組質(zhì)粒，通過PCR擴(kuò)增的方法獲得表2中腈水解酶突變體：表2涉及到的引物見表3表3PCR反應(yīng)體系見表4表4成分體系(μl)PrimeSTARMaxPremix25引物12引物22模板(約300ng)3滅菌水Upto50PCR程序條件設(shè)定為：98℃預(yù)變性5min；98℃變性20s，60℃退火20s，72℃延伸2min，30個循環(huán)；72℃終延伸5min。1％核酸電泳檢測結(jié)果見圖3。對擴(kuò)增獲得的PCR產(chǎn)物進(jìn)行醇沉處理，再使用DpnⅠ對模板進(jìn)行消化處理，最后使用DNA膠回收試劑盒進(jìn)行割膠回收。將回收處理得到的產(chǎn)物電轉(zhuǎn)化至電感受態(tài)細(xì)胞E.coliBL21(DE3)中，轉(zhuǎn)移細(xì)胞至含kana抗性(終濃度為50μg/ml)平板在37℃條件下培養(yǎng)過夜獲得大量轉(zhuǎn)化子。擴(kuò)培后進(jìn)行測序驗(yàn)證，驗(yàn)證正確的轉(zhuǎn)化子即為基因工程腈水解酶產(chǎn)生菌。將基因工程腈水解酶產(chǎn)生菌接入50ml/250ml的液體LB培養(yǎng)基中(含kana抗性，終濃度為50μg/ml)，37℃培養(yǎng)OD600至0.8左右時(shí)，使用IPTG在30℃條件下進(jìn)行誘導(dǎo)培養(yǎng)6h，收菌。菌體在離心機(jī)最大轉(zhuǎn)速中4℃離心10min收集蛋白。測定蛋白的酶轉(zhuǎn)化率及手性選擇性(方法與實(shí)施例4)相同，測定結(jié)果見表5。表5突變情況轉(zhuǎn)化率(酶活)右旋ee％值野生型18％8.9％D41A90％86％A82W84％77％K120I89％85％V135A90％90％L192V96％67％S319I82％70％V135A、D41A93％91％V135A、K120I92％92％V135A、L192V98％80％V135Y65％70％H134V、V135A93％98％H134V、V135A、L192V96％99％H134V、V135A、L192V、D41A99％99％H134V、V135A、L192V、K120I98％99％結(jié)果分析：在單一位點(diǎn)突變中，D41A、A82W、K120I、V135A、V135Y、L192V、S319I顯示出優(yōu)于野生型腈水解酶的酶活或立體選擇性，L192V在提高酶活方面最優(yōu)，V135A在立體選擇性方面最優(yōu)，在多位點(diǎn)突變中，135、192、41、120也表現(xiàn)出有益的效果。在三位點(diǎn)突變組合中加入第四個突變位點(diǎn)H134，可以使效果進(jìn)一步提高，根據(jù)三維立體結(jié)構(gòu)推測H134V、V135A位點(diǎn)的突變，破壞有利于R型底物結(jié)合的因素，降低對R型催化活力。His134可能在R型底物優(yōu)勢構(gòu)象的定位中具有重要作用，His134的咪唑基與底物苯環(huán)間可能存在π-π相互作用或陽離子-π相互作用，不利于左旋產(chǎn)物的形成。SEQUENCELISTING<110>東莞東陽光藥物研發(fā)有限公司;黃石世星藥業(yè)有限責(zé)任公司<120>腈水解酶突變體及其編碼基因和應(yīng)用<130>2017<160>34<170>PatentInversion3.5<210>1<211>356<212>PRT<213>Alcaligenesfaecalis<400>1MetGlnThrArgLysIleValArgAlaAlaAlaValGlnAlaAlaSer151015ProAsnTyrAspLeuAlaAlaGlyValAspLysThrIleGluLeuAla202530ArgGlnAlaArgAspGluGlyCysAspLeuIleValPheGlyGluThr354045TrpLeuProGlyTyrProPheHisValTrpLeuGlyAlaProAlaTrp505560SerLeuLysTyrSerAlaArgTyrTyrAlaAsnSerLeuSerLeuAsp65707580SerAlaGluPheGlnArgIleAlaGlnAlaAlaArgThrLeuGlyIle859095PheIleAlaLeuGlyTyrSerGluArgSerGlyGlySerLeuTyrLeu100105110GlyGlnCysLeuIleAspAspLysGlyGluMetLeuTrpSerArgArg115120125LysLeuLysProThrHisValGluArgThrValPheGlyGluGlyTyr130135140AlaArgAspLeuIleValSerAspThrGluLeuGlyArgValGlyAla145150155160LeuCysCysTrpGluHisLeuSerProLeuSerLysTyrAlaLeuTyr165170175SerGlnHisGluAlaIleHisIleAlaAlaTrpProSerPheSerLeu180185190TyrSerGluGlnAlaHisAlaLeuSerAlaLysValAsnMetAlaAla195200205SerGlnIleTyrSerValGluGlyGlnCysPheThrIleAlaAlaSer210215220SerValValThrGlnGluThrLeuAspMetLeuGluValGlyGluHis225230235240AsnAlaSerLeuLeuThrValGlyGlyGlySerSerMetIlePheAla245250255ProAspGlyArgThrLeuAlaProTyrLeuProHisAspAlaGluGly260265270LeuIleIleAlaAspLeuAsnMetGluGluIleAlaPheAlaLysAla275280285IleAsnAspProAlaGlyHisTyrSerLysProGluAlaThrArgLeu290295300ValLeuAspLeuGlyHisArgGluProMetThrArgValHisSerGln305310315320SerLeuIleLysGluGluAlaCysGluProLeuThrProSerThrIle325330335AlaProValAlaIleSerGlnIleGlnGluSerAspThrLeuLeuVal340345350GlnGluProSer355<210>2<211>1071<212>DNA<213>Alcaligenesfaecalis<400>2atgcagacaagaaaaatcgtccgggcagccgccgtacaggccgcctctcccaactacgat60ctggcagcaggggttgataaaaccattgagctggctcgtcaggcccgcgatgagggctgc120gacctgatcgtgtttggtgaaacctggctaccaggctaccccttccacgtctggttgggc180gcaccggcctggtcgctgaaatacagtgcccgctactatgccaactcgctctcgctggac240agtgcggaatttcaacgcattgcccaggccgcacggaccttgggcattttcattgccttg300ggctatagcgagcgcagtggtggcagcctgtatctgggccaatgcctgattgacgacaaa360ggcgagatgctgtggtcgcgccgcaaacttaaacccacacatgtcgagcgcaccgtgttt420ggtgaaggctatgcccgtgatctgattgtgtccgacaccgagctgggccgcgtcggtgcg480ctgtgctgctgggagcacctgtctcccttgagcaagtacgcgctgtactcccagcacgaa540gccattcacattgccgcctggccgtctttttcgctgtacagtgaacaggcccatgctctc600agcgccaaagtaaacatggctgcctcgcaaatctattcggtcgaaggccaatgctttacc660atcgccgccagcagtgtggtcactcaagagacgctggacatgctggaagtgggagagcac720aacgcctccttattgaccgtgggcggtggcagttccatgatttttgcgccagacggacgc780acactggccccctacctgccgcacgatgccgaaggcctgatcattgccgacctgaacatg840gaagaaattgccttcgccaaggcaatcaacgaccccgcaggtcactattccaaacctgag900gctacccgtttggtactggatttggggcaccgtgagcccatgacacgggttcactcccaa960agcctgatcaaggaagaggcctgcgaaccactcacacccagtacgattgcaccagttgcc1020atcagccagatccaggaatcggacacactgctggtgcaagaaccgtcctga1071<210>3<211>356<212>PRT<213>人工序列<400>3MetGlnThrArgLysIleValArgAlaAlaAlaValGlnAlaAlaSer151015ProAsnTyrAspLeuAlaAlaGlyValAspLysThrIleGluLeuAla202530ArgGlnAlaArgAspGluGlyCysAlaLeuIleValPheGlyGluThr354045TrpLeuProGlyTyrProPheHisValTrpLeuGlyAlaProAlaTrp505560SerLeuLysTyrSerAlaArgTyrTyrAlaAsnSerLeuSerLeuAsp65707580SerAlaGluPheGlnArgIleAlaGlnAlaAlaArgThrLeuGlyIle859095PheIleAlaLeuGlyTyrSerGluArgSerGlyGlySerLeuTyrLeu100105110GlyGlnCysLeuIleAspAspLysGlyGluMetLeuTrpSerArgArg115120125LysLeuLysProThrHisValGluArgThrValPheGlyGluGlyTyr130135140AlaArgAspLeuIleValSerAspThrGluLeuGlyArgValGlyAla145150155160LeuCysCysTrpGluHisLeuSerProLeuSerLysTyrAlaLeuTyr165170175SerGlnHisGluAlaIleHisIleAlaAlaTrpProSerPheSerLeu180185190TyrSerGluGlnAlaHisAlaLeuSerAlaLysValAsnMetAlaAla195200205SerGlnIleTyrSerValGluGlyGlnCysPheThrIleAlaAlaSer210215220Ser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LeuIleAspAspLysGlyGluMetLeuTrpSerArgArg115120125LysLeuLysProThrHisAlaGluArgThrValPheGlyGluGlyTyr130135140AlaArgAspLeuIleValSerAspThrGluLeuGlyArgValGlyAla145150155160LeuCysCysTrpGluHisLeuSerProLeuSerLysTyrAlaLeuTyr165170175SerGlnHisGluAlaIleHisIleAlaAlaTrpProSerPheSerVal180185190TyrSerGluGlnAlaHisAlaLeuSerAlaLysValAsnMetAlaAla195200205SerGlnIleTyrSerValGluGlyGlnCysPheThrIleAlaAlaSer210215220SerValValThrGlnGluThrLeuAspMetLeuGluValGlyGluHis225230235240AsnAlaSerLeuLeuThrValGlyGlyGlySerSerMetIlePheAla245250255ProAspGlyArgThrLeuAlaProTyrLeuProHisAspAlaGluGly260265270LeuIleIleAlaAspLeuAsnMetGluGluIleAlaPheAlaLysAla275280285IleAsnAspProAlaGlyHisTyrSerLysProGluAlaThrArgLeu290295300ValLeuAspLeuGlyHisArgGluProMetThrArgValHisSerGln305310315320SerLeuIleLysGluGluAlaCysGluProLeuThrProSerThrIle325330335AlaProValAlaIleSerGlnIleGlnGluSerAspThrLeuLeuVal340345350GlnGluProSer355<210>12<211>356<212>PRT<213>人工序列<400>12MetGlnThrArgLysIleValArgAlaAlaAlaValGlnAlaAlaSer151015ProAsnTyrAspLeuAlaAlaGlyValAspLysThrIleGluLeuAla202530ArgGlnAlaArgAspGluGlyCysAspLeuIleValPheGlyGluThr354045TrpLeuProGlyTyrProPheHisValTrpLeuGlyAlaProAlaTrp505560SerLeuLysTyrSerAlaArgTyrTyrAlaAsnSerLeuSerLeuAsp65707580SerAlaGluPheGlnArgIleAlaGlnAlaAlaArgThrLeuGlyIle859095PheIleAlaLeuGlyTyrSerGluArgSerGlyGlySerLeuTyrLeu100105110GlyGlnCysLeuIleAspAspLysGlyGluMetLeuTrpSerArgArg115120125LysLeuLysProThrHisTyrGluArgThrValPheGlyGluGlyTyr130135140AlaArgAspLeuIleValSerAspThrGluLeuGlyArgValGlyAla145150155160LeuCysCysTrpGluHisLeuSerProLeuSerLysTyrAlaLeuTyr165170175SerGlnHisGluAlaIleHisIleAlaAlaTrpProSerPheSerLeu180185190TyrSerGluGlnAlaHisAlaLeuSerAlaLysValAsnMetAlaAla195200205SerGlnIleTyrSerValGluGlyGlnCysPheThrIleAlaAlaSer210215220SerValValThrGlnGluThrLeuAspMetLeuGluValGlyGluHis225230235240AsnAlaSerLeuLeuThrValGlyGlyGlySerSerMetIlePheAla245250255ProAspGlyArgThrLeuAlaProTyrLeuProHisAspAlaGluGly260265270LeuIleIleAlaAspLeuAsnMetGluGluIleAlaPheAlaLysAla275280285IleAsnAspProAlaGlyHisTyrSerLysProGluAlaThrArgLeu290295300ValLeuAspLeuGlyHisArgGluProMetThrArgValHisSerGln305310315320SerLeuIleLysGluGluAlaCysGluProLeuThrProSerThrIle325330335AlaProValAlaIleSerGlnIleGlnGluSerAspThrLeuLeuVal340345350GlnGluProSer355<210>13<211>356<212>PRT<213>人工序列<400>13MetGlnThrArgLysIleValArgAlaAlaAlaValGlnAlaAlaSer151015ProAsnTyrAspLeuAlaAlaGlyValAspLysThrIleGluLeuAla202530ArgGlnAlaArgAspGluGlyCysAspLeuIleValPheGlyGluThr354045TrpLeuProGlyTyrProPheHisValTrpLeuGlyAlaProAlaTrp505560SerLeuLysTyrSerAlaArgTyrTyrAlaAsnSerLeuSerLeuAsp65707580SerAlaGluPheGlnArgIleAlaGlnAlaAlaArgThrLeuGlyIle859095PheIleAlaLeuGlyTyrSerGluArgSerGlyGlySerLeuTyrLeu100105110GlyGlnCysLeuIleAspAspLysGlyGluMetLeuTrpSerArgArg115120125LysLeuLysProThrValAlaGluArgThrValPheGlyGluGlyTyr130135140AlaArgAspLeuIleValSerAspThrGluLeuGlyArgValGlyAla145150155160LeuCysCysTrpGluHisLeuSerProLeuSerLysTyrAlaLeuTyr165170175SerGlnHisGluAlaIleHisIleAlaAlaTrpProSerPheSerLeu180185190TyrSerGluGlnAlaHisAlaLeuSerAlaLysValAsnMetAlaAla195200205SerGlnIleTyrSerValGluGlyGlnCysPheThrIleAlaAlaSer210215220SerValValThrGlnGluThrLeuAspMetLeuGluValGlyGluHis225230235240AsnAlaSerLeuLeuThrValGlyGlyGlySerSerMetIlePheAla245250255ProAspGlyArgThrLeuAlaProTyrLeuProHisAspAlaGluGly260265270LeuIleIleAlaAspLeuAsnMetGluGluIleAlaPheAlaLysAla275280285IleAsnAspProAlaGlyHisTyrSerLysProGluAlaThrArgLeu290295300ValLeuAspLeuGlyHisArgGluProMetThrArgValHisSerGln305310315320SerLeuIleLysGluGluAlaCysGluProLeuThrProSerThrIle325330335AlaProValAlaIleSerGlnIleGlnGluSerAspThrLeuLeuVal340345350GlnGluProSer355<210>14<211>356<212>PRT<213>人工序列<400>14MetGlnThrArgLysIleValArgAlaAlaAlaValGlnAlaAlaSer151015ProAsnTyrAspLeuAlaAlaGlyValAspLysThrIleGluLeuAla202530ArgGlnAlaArgAspGluGlyCysAspLeuIleValPheGlyGluThr354045TrpLeuProGlyTyrProPheHisValTrpLeuGlyAlaProAlaTrp505560SerLeuLysTyrSerAlaArgTyrTyrAlaAsnSerLeuSerLeuAsp65707580SerAlaGluPheGlnArgIleAlaGlnAlaAlaArgThrLeuGlyIle859095PheIleAlaLeuGlyTyrSerGluArgSerGlyGlySerLeuTyrLeu100105110GlyGlnCysLeuIleAspAspLysGlyGluMetLeuTrpSerArgArg115120125LysLeuLysProThrValAlaGluArgThrValPheGlyGluGlyTyr130135140AlaArgAspLeuIleValSerAspThrGluLeuGlyArgValGlyAla145150155160LeuCysCysTrpGluHisLeuSerProLeuSerLysTyrAlaLeuTyr165170175SerGlnHisGluAlaIleHisIleAlaAlaTrpProSerPheSerVal180185190TyrSerGluGlnAlaHisAlaLeuSerAlaLysValAsnMetAlaAla195200205SerGlnIleTyrSerValGluGlyGlnCysPheThrIleAlaAlaSer210215220SerValValThrGlnGluThrLeuAspMetLeuGluValGlyGluHis225230235240AsnAlaSerLeuLeuThrValGlyGlyGlySerSerMetIlePheAla245250255ProAspGlyArgThrLeuAlaProTyrLeuProHisAspAlaGluGly260265270LeuIleIleAlaAspLeuAsnMetGluGluIleAlaPheAlaLysAla275280285IleAsnAspProAlaGlyHisTyrSerLysProGluAlaThrArgLeu290295300ValLeuAspLeuGlyHisArgGluProMetThrArgValHisSerGln305310315320SerLeuIleLysGluGluAlaCysGluProLeuThrProSerThrIle325330335AlaProValAlaIleSerGlnIleGlnGluSerAspThrLeuLeuVal340345350GlnGluProSer355<210>15<211>356<212>PRT<213>人工序列<400>15MetGlnThrArgLysIleValArgAlaAlaAlaValGlnAlaAlaSer151015ProAsnTyrAspLeuAlaAlaGlyValAspLysThrIleGluLeuAla202530ArgGlnAlaArgAspGluGlyCysAlaLeuIleValPheGlyGluThr354045TrpLeuProGlyTyrProPheHisValTrpLeuGlyAlaProAlaTrp505560SerLeuLysTyrSerAlaArgTyrTyrAlaAsnSerLeuSerLeuAsp65707580SerAlaGluPheGlnArgIleAlaGlnAlaAlaArgThrLeuGlyIle859095PheIleAlaLeuGlyTyrSerGluArgSerGlyGlySerLeuTyrLeu100105110GlyGlnCysLeuIleAspAspLysGlyGluMetLeuTrpSerArgArg115120125LysLeuLysProThrValAlaGluArgThrValPheGlyGluGlyTyr130135140AlaArgAspLeuIleValSerAspThrGluLeuGlyArgValGlyAla145150155160LeuCysCysTrpGluHisLeuSerProLeuSerLysTyrAlaLeuTyr165170175SerGlnHisGluAlaIleHisIleAlaAlaTrpProSerPheSerVal180185190TyrSerGluGlnAlaHisAlaLeuSerAlaLysValAsnMetAlaAla195200205SerGlnIleTyrSerValGluGlyGlnCysPheThrIleAlaAlaSer210215220SerValValThrGlnGluThrLeuAspMetLeuGluValGlyGluHis225230235240AsnAlaSerLeuLeuThrValGlyGlyGlySerSerMetIlePheAla245250255ProAspGlyArgThrLeuAlaProTyrLeuProHisAspAlaGluGly260265270LeuIleIleAlaAspLeuAsnMetGluGluIleAlaPheAlaLysAla275280285IleAsnAspProAlaGlyHisTyrSerLysProGluAlaThrArgLeu290295300ValLeuAspLeuGlyHisArgGluProMetThrArgValHisSerGln305310315320SerLeuIleLysGluGluAlaCysGluProLeuThrProSerThrIle325330335AlaProValAlaIleSerGlnIleGlnGluSerAspThrLeuLeuVal340345350GlnGluProSer355<210>16<211>356<212>PRT<213>人工序列<400>16MetGlnThrArgLysIleValArgAlaAlaAlaValGlnAlaAlaSer151015ProAsnTyrAspLeuAlaAlaGlyValAspLysThrIleGluLeuAla202530ArgGlnAlaArgAspGluGlyCysAspLeuIleValPheGlyGluThr354045TrpLeuProGlyTyrProPheHisValTrpLeuGlyAlaProAlaTrp505560SerLeuLysTyrSerAlaArgTyrTyrAlaAsnSerLeuSerLeuAsp65707580SerAlaGluPheGlnArgIleAlaGlnAlaAlaArgThrLeuGlyIle859095PheIleAlaLeuGlyTyrSerGluArgSerGlyGlySerLeuTyrLeu100105110GlyGlnCysLeuIleAspAspIleGlyGluMetLeuTrpSerArgArg115120125LysLeuLysProThrValAlaGluArgThrValPheGlyGluGlyTyr130135140AlaArgAspLeuIleValSerAspThrGluLeuGlyArgValGlyAla145150155160LeuCysCysTrpGluHisLeuSerProLeuSerLysTyrAlaLeuTyr165170175SerGlnHisGluAlaIleHisIleAlaAlaTrpProSerPheSerVal180185190TyrSerGluGlnAlaHisAlaLeuSerAlaLysValAsnMetAlaAla195200205SerGlnIleTyrSerValGluGlyGlnCysPheThrIleAlaAlaSer210215220SerValValThrGlnGluThrLeuAspMetLeuGluValGlyGluHis225230235240AsnAlaSerLeuLeuThrValGlyGlyGlySerSerMetIlePheAla245250255ProAspGlyArgThrLeuAlaProTyrLeuProHisAspAlaGluGly260265270LeuIleIleAlaAspLeuAsnMetGluGluIleAlaPheAlaLysAla275280285IleAsnAspProAlaGlyHisTyrSerLysProGluAlaThrArgLeu290295300ValLeuAspLeuGlyHisArgGluProMetThrArgValHisSerGln305310315320SerLeuIleLysGluGluAlaCysGluProLeuThrProSerThrIle325330335AlaProValAlaIleSerGlnIleGlnGluSerAspThrLeuLeuVal340345350GlnGluProSer355<210>17<211>23<212>DNA<213>人工序列<400>17cccatatgcagacaagaaaaatc23<210>18<211>23<212>DNA<213>人工序列<400>18ccaagctttcaggacggttcttg23<210>19<211>46<212>DNA<213>人工序列<400>19cgcaaacttaaacccacacatgcggagcgcaccgtgtttggtgaag46<210>20<211>46<212>DNA<213>人工序列<400>20cttcaccaaacacggtgcgctccgcatgtgtgggtttaagtttgcg46<210>21<211>46<212>DNA<213>人工序列<400>21cgcaaacttaaacccacacattatgagcgcaccgtgtttggtgaag46<210>22<211>46<212>DNA<213>人工序列<400>22cttcaccaaacacggtgcgctcataatgtgtgggtttaagtttgcg46<210>23<211>44<212>DNA<213>人工序列<400>23ccgcaaacttaaacccacagtggcggagcgcaccgtgtttggtg44<210>24<211>44<212>DNA<213>人工序列<400>24caccaaacacggtgcgctccgccactgtgggtttaagtttgcgg44<210>25<211>41<212>DNA<213>人工序列<400>25cgcctggccgtctttttcggtgtacagtgaacaggcccatg41<210>26<211>41<212>DNA<213>人工序列<400>26catgggcctgttcactgtacaccgaaaaagacggccaggcg41<210>27<211>45<212>DNA<213>人工序列<400>27caggcccgcgatgagggctgcgcactgatcgtgtttggtgaaacc45<210>28<211>46<212>DNA<213>人工序列<400>28ggtttcaccaaacacgatcagttgcgcagccctcatcgcgggcctg46<210>29<211>45<212>DNA<213>人工序列<400>29aactcgctctcgctggacagttgggaatttcaacgcattgcccag45<210>30<211>45<212>DNA<213>人工序列<400>30ctgggcaatgcgttgaaattcccaactgtccagcgagagcgagtt45<210>31<211>45<212>DNA<213>人工序列<400>31ggccaatgcctgattgacgacatcggcgagatgctgtggtcgcgc45<210>32<211>45<212>DNA<213>人工序列<400>32gcgcgaccacagcatctcgccgatgtcgtcaatcaggcattggcc45<210>33<211>45<212>DNA<213>人工序列<400>33gagcccatgacacgggttcacatccaaagcctgatcaaggaagag45<210>34<211>45<212>DNA<213>人工序列<400>34ctcttccttgatcaggctttggatgtgaacccgtgtcatgggctc45當(dāng)前第1頁1 2 3