CN106929491B - (s)-羰基还原酶异源聚合体及其在催化多苯环化合物的应用 - Google Patents
(s)-羰基还原酶异源聚合体及其在催化多苯环化合物的应用 Download PDFInfo
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Abstract
本发明公开了(S)‑羰基还原酶异源聚合体及其在催化多苯环化合物的应用,属于生物催化技术领域。本发明构建了(S)‑羰基还原酶异源聚合体重组菌、获得了异源聚合体SCR/SCR2和SCR2/SCR3,并将其应用于催化2,4‑二氯二苯甲酮和2‑萘乙酮。本发明的异源聚合体SCR2/SCR3能催化2,4‑二氯二苯甲酮,酶活可达4.42U/mg,而同源聚合体SCR、SCR2和SCR3没有检测到催化上述底物的酶活。本发明利用异源聚合体蛋白的表达和纯化有效拓展了(S)‑羰基还原酶系(SCR,SCR2和SCR3)的生物催化的底物谱,为多苯类化合物的生物催化提供了新型氧化还原酶。
Description
技术领域
本发明涉及(S)-羰基还原酶异源聚合体及其在催化多苯环化合物的应用,属于生物催化技术领域。
背景技术
手性化合物在医药、农药、激素、食品添加剂等精细化工品的生产上得到了越来越广泛的应用,如联苯化合物2,4-二氯二苯甲酮对南美锥虫病的寄生虫有抑制作用,是一种潜在的药用化合物。而化合物2-萘乙酮作为重要的药物中间体,利用生物法催化该化合物,从而获得各种重要的药物前体也具有很大的价值。
氧化还原酶催化的不对称还原反应常用于手性化合物的制备。目前常用的氧化还原酶大部分产自重组型的大肠杆菌。而立体选择性氧化还原酶能用于工业转化手性醇的种类和数量非常有限,尤其是生产(S)-型手性醇的具有anti-Prelog立体选择性氧化还原酶数量更少。因此有必要对现有的氧化还原酶进行改造以获得具有新功能的工程酶,以满足实际需要,获得具有更多功能的手性生物催化剂。
发明人在前期工作中构建得到了分别表达(S)-羰基还原酶基因簇(scr,scr2和scr3)基因的重组型大肠杆菌(E.coli),重组菌能够催化转化制备(S)-苯基乙二醇。(S)-羰基还原酶系基因在大肠杆菌(E.coli)中分别异源表达后,对单苯环类化合物具有较好的催化活性。但是(S)-羰基还原酶SCR、SCR2、SCR3的底物谱较窄,无法催化多苯环类化合物。如目前报道(S)-羰基还原酶SCR、SCR2、SCR3同源蛋白聚合体能催化直链潜手性醇和单苯环类潜手性醇化合物,但对于联苯类化合物2,4-二氯二苯甲酮和2-萘乙酮等重要的药物中间体几乎没有催化能力。
发明内容
为了解决上述问题,本发明得到了(S)-羰基还原酶的异源聚合体,提高了(S)-羰基还原酶系基因的底物谱。通过蛋白和蛋白的相互作用,异源羰基还原酶聚合体催化底物谱扩大,能够催化双苯环类化合物,如联苯化合物2,4-二氯二苯甲酮和2-萘乙酮。
本发明的第一个目的是提供一种能够催化多苯环类化合物的异源羰基还原酶聚合体蛋白,所述异源羰基还原酶聚合体蛋白是(S)-羰基还原酶的异源聚合体蛋白复合物。
在一种实施方式中,所述异源聚合体蛋白复合物是两个以上氨基酸序列不同的(S)-羰基还原酶形成的异源聚合体。
在一种实施方式中,所述(S)-羰基还原酶是来源于近平滑假丝酵母的(S)-羰基还原酶。
在一种实施方式中,所述(S)-羰基还原酶可以是:氨基酸序列如SEQ ID NO:1所示的SCR、氨基酸序列如SEQ ID NO:2所示的SCR2、氨基酸序列如SEQ ID NO:3所示的SCR3。
在一种实施方式中,所述(S)-羰基还原酶的编码基因可以是:scr(GenBank ID:DQ675534)、scr2(GenBank ID:GQ411433)、scr3(GenBank ID:FJ939564)。
在一种实施方式中,所述异源聚合体蛋白复合物是由SCR和SCR2形成的异源聚合体SCR/SCR2,或者是由SCR2和SCR3形成的异源聚合体SCR2/SCR3。
在一种实施方式中,所述异源聚合体蛋白复合物是将两个以上氨基酸序列不同的(S)-羰基还原酶在同一宿主中共表达而得到的。
本发明的第二个目的是提供所述异源聚合体蛋白复合物的制备方法。所述方法,是将两个以上氨基酸序列不同的(S)-羰基还原酶在同一宿主中共表达而得到的。
在一种实施方式中,所述方法,是将两个以上氨基酸序列不同的(S)-羰基还原酶的基因分别连接到表达载体上,然后转化到同一宿主中得到重组菌,重组菌共表达这两个以上的(S)-羰基还原酶基因,得到异源聚合体蛋白复合物。
在一种实施方式中,所述连接到表达载体上,包括每个基因单独连接到一个表达载体上,或者将多个基因连接到同一个表达载体上。
在一种实施方式中,所述表达载体可以是以下任意一种或者多种:pET21、pET28、pETDuet。
在一种实施方式中,所述宿主可以是大肠杆菌。
在一种实施方式中,所述宿主为(E.coli BL21(DE3)。
在一种实施方式中,所述连接到表达载体上,还包括在(S)-羰基还原酶的基因前添加蛋白纯化标签。
在一种实施方式中,所述方法还包括共表达后的纯化步骤。
在一种实施方式中,所述方法具体包括:(1)获取两个氨基酸序列不同的(S)-羰基还原酶的基因;(2)将上一步得到的两个基因分别连接到不同的表达载体上,并进一步构建带有不同纯化标签的含有目的基因的两个重组质粒;(3)将两个重组质粒转化到同一宿主大肠杆菌中,得到共表达两个氨基酸序列不同的(S)-羰基还原酶的重组大肠杆菌;(4)重组菌发酵表达,纯化得到异源聚合体蛋白复合物。
在一种实施方式中,所述纯化标签为组氨酸标签或者修饰的链球菌抗生物素蛋白标签。
在一种实施方式中,所述纯化,是先收集菌体、破碎细胞得到胞内表达的蛋白,然后离心取上清,先利用Ni-NTAcolumn纯化得到带有组氨酸标签的蛋白,再将得到的带有组氨酸标签的蛋经过Streptrap HP column的纯化,即得到异源聚合体蛋白。因为异源聚合体蛋白既能亲和吸附Ni-NTAcolumn,又能亲和吸附Streptrap HP column。
在一种实施方式中,所述纯化,先利用蛋白纯化仪连接镍柱Ni-NTA纯化带有组氨酸标签的蛋白;由于所表达的这两个氨基酸序列不同的(S)-羰基还原酶会形成异源聚合体,因此在这一步中有部分带有strep标签的蛋白也会被Ni-NTA结合;接着将结合在Ni-NTA柱子上的蛋白用含有咪唑的缓冲洗脱,将获得的收集液流经已经用缓冲平衡好的Streptrap HP column上,最后用含有脱硫生物素的缓冲将目的蛋白洗脱,获得异源聚合体蛋白。
在一种实施方式中,所述方法具体是:将来源于近平滑假丝酵母的(S)-羰基还原酶SCR、SCR2、SCR3中的任意两种,分别连接到表达载体上并进一步构建带有不同纯化标签的含有目的基因的两个重组质粒,得到pET21-Strep-SCR、pET28-His-SCR2、pET28-His-SCR3、pET21-Strep-SCR2,然后将pET21-Strep-SCR与pET28-His-SCR2,或者pET28-His-SCR3与pET21-Strep-SCR2共同转化到同一宿主细胞大肠杆菌中,筛选具有双质粒的重组大肠杆菌;重组菌发酵表达,纯化得到异源聚合体蛋白复合物。
在一种实施方式中,所述发酵表达,是将重组大肠杆菌接种于LB培养基中过夜培养,然后转接于LB培养基中,于37℃,200rpm振荡培养至OD达到0.6-0.8时,加入一定浓度IPTG诱导12h。
本发明的第三个目的是提供表达所述异源聚合体蛋白复合物的重组菌。
在一种实施方式中,所述重组菌是将两个以上氨基酸序列不同的(S)-羰基还原酶的基因分别连接到表达载体上,然后共同转化到同一宿主中而得到的。
在一种实施方式中,所述宿主是大肠杆菌。
在一种实施方式中,所述宿主为(E.coli BL21(DE3)。
在一种实施方式中,所述表达载体可以是以下任意一种或者多种:pET21、pET28和pETDuet。
在一种实施方式中,所述连接到表达载体上,还包括在(S)-羰基还原酶的基因前添加蛋白纯化标签。
本发明的第四个目的是提供所述异源聚合体蛋白复合物的应用。
在一种实施方式中,所述应用,是应用于催化领域。
在一种实施方式中,所述应用,是应用于精细化工品生产领域。
在一种实施方式中,所述应用,是用于催化双苯环类化合物。
在一种实施方式中,所述双苯环类化合物包括但不限于2,4-二氯二苯甲酮、2-萘乙酮。
本发明的有益效果:
本发明通过共表达(S)-羰基还原酶基因簇中的两个以上(S)-羰基还原酶基因,获得了异源聚合体蛋白。本发明的异源聚合体蛋白具有新的性能,能够催化双苯环类化合物,且具有较好的催化活性。本发明的异源聚合体蛋白可以用于精细化工品生产领域。
本发明根据近平滑假丝酵母(C.parapsilosis)(S)-羰基还原酶基因簇序列scr(GenBank ID:DQ675534),scr2(GenBank ID:GQ411433)和scr3(GenBank ID:FJ939564),通过构建不同(S)-羰基还原酶的共表达菌株,并通过不同蛋白所带有的不同标签,利用pulldown实验纯化异源聚合体,从而获得大量的异源聚合体蛋白。利用所获得的异源聚合体蛋白复合物,测定联苯化合物2,4-二氯二苯甲酮和2-萘乙酮的活性,相比于同源聚合体基因scr,scr2和scr3,可以得到较高的羰基还原酶催化活性。本发明的异源聚合体蛋白SCR/SCR2和SCR2/SCR3对联苯化合物2,4-二氯二苯甲酮的酶活性分别为4.55U/mg、4.42U/mg,SCR/SCR2对联苯化合物2-萘乙酮的酶活性为2.43U/mg。
具体实施方案
异源聚合体蛋白对不同底物的酶活力测定方法:
(1)原理:辅酶NADPH被还原后,会引起340nm的吸光度的降低。因此可以通过测定反应过程中340nm处吸光度的变化来检测氧化还原酶的活力。
(2)测定还原酶活的条件:总反应体积为250uL,分别加入0.5mmol/LNADPH,5mmol/L底物2,4-二氯二苯甲酮或者2-萘乙酮。30℃水浴恒温2min,加入适量酶液后开始扫描340nm处吸光度的变化。
(3)计算方法与酶活力定义
酶活计算公式:酶活(U)=EW×V×103/(6220×0.15)
比活的计算公式:比活(U/mg)=酶活(U)/蛋白量(mg)
其中,EW:1min内340nm处吸光度的变化;V:反应液的体积(mL);6220:摩尔消光系数(Lmol-1cm-1);0.15:光程距离(cm)。
羰基还原酶酶活力的定义:在上述条件下,每分钟催化氧化1umol NADPH的酶量为1个酶活单位。
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此。
实施例1
近平滑假丝酵母(C.parapsilosis)CCTCC M203011的菌体培养,其生长培养基组成为:葡萄糖2%,酵母膏1%,蛋白胨2%。将近平滑假丝酵母接种于5mL试管中于28℃,200rpm振荡培养16-18h。
实施例2
近平滑假丝酵母(C.parapsilosis)基因组的提取:将实施例1培养的菌体于6,000rpm,5min下离心,用生理盐水洗涤两次后,收集细胞利用基因组DNA提取试剂盒GenomicDNA Mini Preparation Kit(Takara公司)提取基因组。
实施例3
从近平滑假丝酵母中调取目的基因序列,包括scr(氨基酸序列如SEQ ID NO:1所示),scr2(氨基酸序列如SEQ ID NO:2所示)和scr3(氨基酸序列如SEQ ID NO:3所示)。
合成两端引物(序列如SEQ ID NO:4~SEQ ID NO:11所示)
B-S-SCR-F1:atcggatccgtggtctcatcctcaatttgaaaagggttctatgggcgaaatcgaatctta
X-S-SCR-R1:tgactctcgagctatggacacgtgtatccacc
B-S-SCR2-F1:atcggatccgtggtctcatcctcaatttgaaaagggttctatgggcgaaatcgaatctta
X-S-SCR2-R1:tgactctcgagctatggacaagtgtaaccaccat
B-His-SCR2-F1:cgcggatccgaaaatttatatttccagagtatgggcgaaatcgaatctta
X-His-SCR2-R1:tgactctcgagctatggacacgtgtatccacc
E-His-SCR3-F1:ccggaattcgaaaatttatatttccagagtatgggcgaaatcgaatctta
X-His-SCR3-R1:gcccgctcgagctatggacaggtgaatccaccatc
实施例4
scr、scr2和scr3基因的获得:采用PCR反应体系50ul:1ul基因组,1ul引物(上下游分别1ul),PrimeSTAR HS premix 25ul,灭菌的超纯水22ul。PCR条件为:98℃热变性10s;98℃ 10s,52℃ 15s,72℃ 1min。经过30个循环后,最后72℃再延伸10min。利用3SSpinAgarose Gel DNAPurification Kit(上海申能博彩生物科技有限公司)纯化DNA片断。
利用引物B-S-SCR-F1和X-S-SCR-R1扩增得到了氮端具有修饰的链球菌抗生物素蛋白标签strep的scr基因,即strep-scr基因。利用引物B-S-SCR2-F1和X-S-SCR2-R1扩增得到了氮端具有修饰的链球菌抗生物素蛋白标签的strep-scr2基因。利用引物B-His-SCR2-F1和X-His-SCR2-R1扩增得到了氮端具有组氨酸标签的his-scr2基因。利用引物E-His-SCR3-F1和X-His-SCR3-R1扩增得到了氮端具有组氨酸标签的his-scr3基因。
实施例5
pET21-Strep-SCR、pET28-His-SCR2、pET28-His-SCR3和pET21-Strep-SCR2重组质粒的获得scr,scr2,scr3及空质粒pET21和pET28的酶切反应体系。
利用BamH I、Xho I酶切pET21和strep-scr基因,酶切产物纯化后进行连接、转化、验证,得到阳性质粒pET21-Strep-SCR。利用BamH I、Xho I酶切pET21和strep-scr2基因,酶切产物纯化后进行连接、转化、验证,得到阳性质粒pET21-Strep-SCR2。利用BamH I、Xho I酶切pET28和his-scr2基因,酶切产物纯化后进行连接、转化、验证,得到阳性质粒pET28-his-SCR2。利用BamH I、Xho I酶切pET28和his-scr3基因,酶切产物纯化后进行连接、转化、验证,得到阳性质粒pET28-his-SCR3。
实施例6
异源表达(S)-羰基还原酶和(S)-羰基还原酶2,以及(S)-羰基还原酶2和(S)-羰基还原酶3的大肠杆菌重组菌的获得:
利用质粒提取试剂盒Plasmidmini Kit I(200)(OMEGA)从E.coli JM109中提取质粒pET21-Strep-SCR、pET21-Strep-SCR2、pET28-His-SCR2和pET28-His-SCR3。
在100μLE.coli BL21(DE3)感受态细胞悬液中分别加入0.5μLpET21-Strep-SCR和pET28-His-SCR2,同时在另外一管100μLE.coli BL21(DE3)感受态细胞悬液中分别加入0.5μLpET21-Strep-SCR2和pET28-His-SCR3。轻轻混匀,冰浴中静置30min。转入42℃水浴中,热击90s。快速转移至冰浴冷却2min。每管中加入700μL LB液体培养基,37℃ 100rpm摇床温育培养1h。培养后菌液5,000rpm离心2min,弃上清600μL,剩余菌液混匀后涂布到含有100μg/mL氨苄青霉素和50μg/mL卡那霉素LB双抗性平板上,37℃倒置培养过夜。次日挑选单菌落进行试管培养,筛选得到具有双质粒的重组大肠杆菌E.coli/pET21-Strep-SCR/pET28-His-SCR2和E.coli/pET21-Strep-SCR2/pET28-His-SCR3。
实施例7
重组菌株大肠杆菌E.coli/pET21-Strep-SCR/pET28-His-SCR2和E.coli/pET21-Strep-SCR2/pET28-His-SCR3的培养和菌体获得:
重组菌株培养基(LB)组成为:1%氯化钠、1%蛋白胨、0.5%酵母膏。重组型的大肠杆菌培养还需添加100μg/mL的氨苄青霉素和50μg/mL卡那霉素。
挑取阳性克隆单菌落接种于5mL含氨苄青霉素(100μg/mL)和卡那霉素(50μg/mL)的LB试管培养基中,于37℃,200rpm振荡培养过夜。取3mL培养液转接于150mL LB培养基中,于37℃,200rpm振荡培养至OD达到0.6-0.8时,加入IPTG的浓度为0.1、0.2、0.5和1.0mM,30℃诱导12h。培养后的重组型大肠杆菌细胞于12,000rpm离心10min并用生理盐水洗涤三次后收集。
实施例8
重组菌株大肠杆菌E.coli/pET21-Strep-SCR/pET28-His-SCR2和E.coli/pET21-Strep-SCR2/pET28-His-SCR3的细胞破碎:将收集好并洗涤干净的大肠杆菌利用Ni-NTAcolumn的纯化缓冲A液(20mM Tris,150NaCl,pH 8.0)以1:20的比例稀释并悬浮,然后利用磁力搅拌器把菌体充分打散,接着利用超声破碎仪(Sonic)破碎细胞(破碎2s,停4s,工作时间30min,工作强度40%),释放胞内表达的(S)-羰基还原酶系。利用冷冻离心机将细胞破碎液以12,000rpm离心40min,然后将破碎液上清过0.22um的水系滤膜,并转移至干净的离心管,作为蛋白纯化的样品备用。
实施例9
异源聚合体蛋白SCR/SCR2和SCR2/SCR3的Ni-NTAcolumn纯化:
将过膜后的细胞破碎上清液先用Ni-NTA纯化。利用AKTAPurifer进行样品的纯化,样品流经柱子后,先用平衡液冲洗柱子,直到紫外吸收值降低并保持平衡,接着先用40mM的咪唑缓冲液洗杂带,然后再用200mM咪唑缓冲液将带有组氨酸标签的蛋白复合物洗脱并接收相关样品。
实施例10
异源聚合体蛋白SCR/SCR2和SCR2/SCR3的Streptrap HP column纯化:将Ni-NTA纯化后收集的样品继续使用Streptrap HP column纯化。利用AKTAPurifer进行样品的纯化,样品流经柱子后,先用平衡液冲洗柱子,直到紫外吸收值降低并保持平衡,接着先用2.5mM的脱硫生物素缓冲液将带有Strep标签的蛋白复合物洗脱并接收。
实施例11
异源聚合体蛋白SCR/SCR2和SCR2/SCR3对联苯化合物2,4-二氯二苯甲酮的酶活测定。测定还原酶活的条件:总反应体积为250uL,分别加入0.5mmol/LNADPH,5mmol/L底物2,4-二氯二苯甲酮。30℃水浴恒温2min,加入适量酶液后开始扫描340nm处吸光度的变化。最后计算得SCR/SCR2酶活性为4.55U/mg、SCR2/SCR3酶活性为4.42U/mg,而同源聚合体SCR、SCR2和SCR3并未测到任何活性。
实施例12
异源聚合体蛋白SCR/SCR2和SCR2/SCR3对联苯化合物2-萘乙酮的酶活测定。测定还原酶活的条件:总反应体积为250uL,分别加入0.5mmol/LNADPH,5mmol/L底物2-萘乙酮。30℃水浴恒温2min,加入适量酶液后开始扫描340nm处吸光度的变化。最后计算得SCR/SCR2酶活性为2.43U/mg,而同源聚合体SCR、SCR2和SCR3并未测到任何活性。
此外,发明人采用类似的方法,得到了异源聚合体蛋白SCR2/SCR3和SCR/SCR3,结果显示,这两种异源聚合体蛋白也具有一定的催化双苯环类化合物的活性。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
序列表
<110> 江南大学
<120> (S)-羰基还原酶异源聚合体及其在催化多苯环化合物的应用
<160> 11
<170> PatentIn version 3.3
<210> 1
<211> 279
<212> PRT
<213> SCR
<400> 1
Met Gly Glu Ile Glu Ser Tyr Cys Asn Lys Glu Leu Gly Pro Leu Pro
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Ala Val Ala Glu Ala Tyr Ala Gln Ala Gly Ala Asp Val Ala Ile Trp
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Tyr Asn Ser His Pro Ala Asp Glu Lys Ala Glu His Leu Gln Lys Thr
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Tyr Gly Val His Ser Lys Ala Tyr Lys Cys Asn Ile Ser Asp Pro Lys
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Ser Val Glu Glu Thr Ile Ser Gln Gln Glu Lys Asp Phe Gly Thr Ile
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Asp Val Phe Val Ala Asn Ala Gly Val Thr Trp Thr Gln Gly Pro Glu
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Ile Asp Val Asp Asn Tyr Asp Ser Trp Asn Lys Ile Ile Ser Val Asp
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Leu Asn Gly Val Tyr Tyr Cys Ser His Asn Ile Gly Lys Ile Phe Lys
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Lys Asn Gly Lys Gly Ser Leu Ile Ile Thr Ser Ser Ile Ser Gly Lys
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Ile Val Asn Ile Pro Gln Leu Gln Ala Pro Tyr Asn Thr Ala Lys Ala
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Ala Cys Thr His Leu Ala Lys Ser Leu Ala Ile Glu Trp Ala Pro Phe
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Ala Arg Val Asn Thr Ile Ser Pro Gly Tyr Ile Asp Thr Asp Ile Thr
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Asp Phe Ala Ser Lys Asp Met Lys Ala Lys Trp Trp Gln Leu Thr Pro
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Leu Gly Arg Glu Gly Leu Thr Gln Glu Leu Val Gly Gly Tyr Leu Tyr
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Leu Ala Ser Asn Ala Ser Thr Phe Thr Thr Gly Ser Asp Val Val Ile
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Asp Gly Gly Tyr Thr Cys Pro
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<210> 2
<211> 279
<212> PRT
<213> SCR2
<400> 2
Met Gly Glu Ile Glu Ser Tyr Cys Asn Lys Glu Leu Gly Pro Leu Pro
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Lys Gly Lys Val Ala Ser Val Thr Gly Ser Ser Gly Gly Ile Gly Trp
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Ala Val Ala Glu Ala Tyr Ala Gln Ala Gly Ala Asp Val Ala Ile Trp
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Tyr Asn Ser His Pro Ala Asp Glu Lys Ala Glu His Leu Gln Lys Thr
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Tyr Gly Val Arg Ser Lys Ala Tyr Lys Cys Asn Ile Ser Asp Pro Lys
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Ser Val Glu Glu Thr Ile Ser Gln Gln Glu Lys Asp Phe Gly Thr Ile
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Asp Val Phe Val Ala Asn Ala Gly Val Pro Trp Thr Glu Gly Pro Glu
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Ile Asn Val Asp Asn Tyr Asp Ser Trp Asn Lys Ile Ile Asn Leu Asp
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Leu Asn Gly Val Tyr Tyr Cys Ala His Thr Val Gly Lys Ile Phe Lys
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Lys Asn Gly Lys Gly Ser Leu Val Ile Thr Ser Ser Met Ser Gly Thr
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Ile Val Asn Val Pro Gln Leu Gln Ala Ala Tyr Asn Ala Ala Lys Ala
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<210> 3
<211> 279
<212> PRT
<213> SCR3
<400> 3
Met Gly Glu Ile Glu Ser Tyr Cys Asn Lys Glu Leu Gly Pro Leu Pro
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Ala Val Ala Glu Ala Tyr Ala Gln Ala Gly Ala Asp Val Ala Ile Trp
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Tyr Asn Ser His Pro Ala Asp Glu Lys Ala Glu His Leu Gln Lys Thr
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<210> 4
<211> 60
<212> DNA
<213> 人工序列
<400> 4
atcggatccg tggtctcatc ctcaatttga aaagggttct atgggcgaaa tcgaatctta 60
<210> 5
<211> 32
<212> DNA
<213> 人工序列
<400> 5
tgactctcga gctatggaca cgtgtatcca cc 32
<210> 6
<211> 60
<212> DNA
<213> 人工序列
<400> 6
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<212> DNA
<213> 人工序列
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<210> 8
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<212> DNA
<213> 人工序列
<400> 8
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<210> 9
<211> 32
<212> DNA
<213> 人工序列
<400> 9
tgactctcga gctatggaca cgtgtatcca cc 32
<210> 10
<211> 50
<212> DNA
<213> 人工序列
<400> 10
ccggaattcg aaaatttata tttccagagt atgggcgaaa tcgaatctta 50
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<211> 35
<212> DNA
<213> 人工序列
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Claims (14)
1.一种能够催化多苯环类化合物的异源羰基还原酶聚合体蛋白,其特征在于,所述异源羰基还原酶聚合体蛋白是(S)-羰基还原酶的异源聚合体;所述(S)-羰基还原酶是氨基酸序列为SEQ ID NO:1所示的SCR、氨基酸序列为SEQ ID NO:2所示的SCR2或者氨基酸序列为SEQ ID NO:3所示的SCR3;所述异源聚合体是由SCR和SCR2形成的异源聚合体SCR/SCR2,或者是由SCR2和SCR3形成的异源聚合体SCR2/SCR3。
2.根据权利要求1所述的异源羰基还原酶聚合体蛋白,其特征在于,所述异源聚合体蛋白是将两个氨基酸序列不同的(S)-羰基还原酶在同一宿主中共表达而得到的。
3.表达权利要求1所述异源羰基还原酶聚合体蛋白的重组菌。
4.表达权利要求2所述异源羰基还原酶聚合体蛋白的重组菌。
5.根据权利要求3所述的重组菌,其特征在于,所述重组菌是将两个氨基酸序列不同的(S)-羰基还原酶的基因分别连接到表达载体上,然后共同转化到同一宿主中而得到的。
6.根据权利要求4所述的重组菌,其特征在于,所述重组菌是将两个氨基酸序列不同的(S)-羰基还原酶的基因分别连接到表达载体上,然后共同转化到同一宿主中而得到的。
7.根据权利要求5或6所述的重组菌,其特征在于,所述宿主是大肠杆菌。
8.根据权利要求5或6所述的重组菌,其特征在于,所述宿主为E. coli BL21 (DE3)。
9.根据权利要求7所述的重组菌,其特征在于,所述表达载体是以下任意一种或者两种:pET21、pET28和pETDuet。
10.根据权利要求8所述的重组菌,其特征在于,所述表达载体是以下任意一种或者两种:pET21、pET28和pETDuet。
11.权利要求1或2所述的异源羰基还原酶聚合体蛋白在催化2,4-二氯二苯甲酮、2-萘乙酮中的应用。
12.权利要求3~6,9~10任一所述的重组菌在催化2,4-二氯二苯甲酮、2-萘乙酮中的应用。
13.权利要求7所述的重组菌在催化2,4-二氯二苯甲酮、2-萘乙酮中的应用。
14.权利要求8所述的重组菌在催化2,4-二氯二苯甲酮、2-萘乙酮中的应用。
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