CN111718913B - 一种酮还原酶多肽及其催化制备(s)-1-(2-碘-5-氟苯基)乙醇的方法 - Google Patents
一种酮还原酶多肽及其催化制备(s)-1-(2-碘-5-氟苯基)乙醇的方法 Download PDFInfo
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Abstract
本发明涉及一种酮还原酶多肽及其催化制备(S)‑1‑(2‑碘‑5‑氟苯基)乙醇的方法，属于基因工程技术用于制备医药中间体领域。该种酮还原酶能够转化的底物1‑(5‑氟‑2‑碘苯基)乙酮浓度达到200g/L以上，并且产物(S)‑1‑(2‑碘‑5‑氟苯基)乙醇的手性纯度能够达到99％以上，同时底物转化率在99％以上，且在转化过程中未用到有机溶剂。本方案能够使得底物完全、高效的转化成为目标产物，并且所制备的产物分离提纯简单，后处理成本低，整个工艺流程环境友好度高，原子利用率高。
Description
技术领域
本发明涉及一种医药中间体制备方法，尤其涉及酮还原酶多肽及其催化制备(S)-1-(2-碘-5-氟苯基)乙醇的方法
背景技术
劳拉替尼(Lorlatinib，CAS号：1454846-35-5)，结构式如式I，是辉瑞公司开发的一种新型、可逆、强效的小分子ALK和ROS1抑制剂，其对ALK已知的耐药突变均具有很强的抑制作用，因而被誉为第3代ALK抑制剂。于2018年11月2日被FDA批准上市，用于治疗ALK阳性的非小细胞肺癌。
(S)-1-(2-碘-5-氟苯基)乙醇(CAS号：1454847-96-1)，结构式如式II，是合成劳拉替尼的重要中间体。
目前合成(S)-1-(2-碘-5-氟苯基)乙醇的主要方法为化学法。
专利WO2017148325公开一种化学合成方法，以1-(5-氟-2-碘苯基)乙酮为原料，使用NaBH4作为催化还原剂，收率可达99％，但是易燃有毒，不适于工业化生产。
专利WO2014207606和CN104169286公开一种化学合成方法，以1-(5-氟-2-碘苯基)乙酮为原料，采用(-)DIPCl作为催化还原剂，需要将反应液冷却至-20℃至-30℃，且操作过程繁琐，收率仅为80％。
文献Organic Process Research&Development,21(9),1340-1348；2017报道了采用2,4-二酮葡萄糖酸(DkgA)酶制备(S)-1-(2-碘-5-氟苯基)乙醇的方法，该酶转化底物的浓度仅为66g/L,且该工艺采用了价格相对昂贵的辅酶NADPH，其用量达到底物重量的5％-10％，该方法底物浓度较低，生产成本较高。
因此，需要开发一种新的环境友好、收率高且适于工业化应用的技术制备高光学纯度的(S)-1-(2-碘-5-氟苯基)乙醇的方法，以满足药物研发与生产方面的市场需求。
发明内容
本发明的目的在于提供一种催化1-(5-氟-2-碘苯基)乙酮转换为(S)-1-(2-碘-5-氟苯基)乙醇的酮还原酶多肽；
本发明的另一个目的在于提供上述酮还原酶多肽的氨基酸序列；
本发明的另一个目的在于提供基因序列，此基因序列能够编码上述氨基酸序列的酮还原酶；
本发明的最终目的在于运用所制备的酮还原酶催化1-(5-氟-2-碘苯基)乙酮转换为(S)-1-(2-碘-5-氟苯基)乙醇，提高合成效率。
本发明技术方案采用如下路线：
路线中所采用的酮还原酶多肽(KRED)为SEQ ID No.1-10所记载的氨基酸序列；
进一步，上述酮还原酶多肽以酮还原酶酶粉、酮还原酶酶液、含酮还原酶的细胞等形式参与催化反应，用于催化1-(5-氟-2-碘苯基)乙酮为(S)-1-(2-碘-5-氟苯基)乙醇。
进一步，所述酮还原酶多肽催化1-(5-氟-2-碘苯基)乙酮转化为(S)-1-(2-碘-5-氟苯基)乙醇的工艺步骤包括：将1-(5-氟-2-碘苯基)乙酮、酮还原酶酶粉或含有该种酮还原酶的细胞、辅酶、缓冲液配置成混合溶液，反应得到产物。
进一步，反应时间为12～36h，优选为24h。
进一步，所述1-(5-氟-2-碘苯基)乙酮浓度为1～400g/L；酮还原酶酶粉浓度为1～10g/L或含有酮还原酶的细胞的浓度为10～100g/L。
进一步，所述含有酮还原酶的细胞选自基因工程改造的大肠杆菌。
进一步，所述辅酶选自NAD、NADH、NADP和NADPH中的任意一种或它们的组合。优选NADP+；添加辅酶的浓度为0.02～0.4g/L，优选为0.05～0.10g/L。
进一步，酮还原酶以酶粉形式、细胞破碎液或全细胞的形式参与反应，优选以酶粉形式参与反应。
进一步，所述缓冲液为磷酸钾缓冲液。
进一步，本技术方案中，所述酮还原酶为来源于Flavobacterium hercynium的突变体，是通过对Flavobacterium hercynium的酮还原酶的突变体文库进行筛选后得到的。来源于Flavobacterium hercynium的野生型酮还原酶在NCBI的登记号为WP_089048302.1。并且本专利中所有的氨基酸序列及其可能对应的基因序列均可通过商业化的全基因合成服务制得。
进一步，所述酮还原酶多肽由基因工程菌发酵得到，所述基因工程菌为大肠杆菌。
本发明的有益效果：本发明提供一种与现有技术不同的新的酮还原酶及其应用于催化1-(5-氟-2-碘苯基)乙酮为(S)-1-(2-碘-5-氟苯基)乙醇的方法，并给出了该种酮还原酶所对应的一种基因序列。该种酮还原酶能够转化的底物1-(5-氟-2-碘苯基)乙酮的浓度高达200g/L，手性纯度值达到99％以上，同时能确保底物转化率在99％以上，使得底物完全、高效的转化成为目标产物，同时不需用到有机溶剂，并且所制备的产物分离提纯简单，后处理成本低，整个工艺流程环境友好度高，原子利用率高。
附图说明
图1为反应底物1-(5-氟-2-碘苯基)乙酮的常规HPLC分析谱图。
图2为消旋体1-(2-碘-5-氟苯基)乙醇的手性HPLC分析谱图。
图3为(S)-1-(2-碘-5-氟苯基)乙醇标准品的手性HPLC分析谱图。
图4为实施例6产物的手性HPLC分析谱图。
图5为实施例7产物的手性HPLC分析谱图。
具体实施方式
下面结合具体实施例对本发明的技术内容作进一步的阐述，其目的是为了更好的理解本发明的内容，但本发明的保护范围不限于此。
实施例1酮还原酶筛选
使用尚科生物医药(上海)有限公司的酮还原酶(KRED)酶库对底物进行反应筛选，反应体系组分如下：0.1M pH6.0磷酸钾缓冲液，底物10g/L，NADP0.2g/L，葡萄糖10g/L，葡萄糖脱氢酶(葡萄糖脱氢酶)5g/L，KRED酶粉10g/L。于30℃反应24h，进行HPLC检测。结果显示，筛选得到的效果最好的酶为来自于Flavobacterium hercynium的酮还原酶野生型，转化率为50.5％，手性HPLC检测显示产物手性纯度为95％，S构型为优势构型。
实施例2构建酮还原酶的单点饱和突变体库
为了提高该酶催化的转化率和手性纯度，使用蛋白质工程的手段对该酶进行了定向进化。根据该酮还原酶的蛋白序列，通过计算机模拟结构，与底物进行对接，推测Q147、Y188，S200位点的氨基酸残基可能会与催化作用密切相关，于是对这些位点分别构建饱和突变体库。
设计相应的突变引物，利用全质粒PCR扩增反应，扩增出带有突变基因的质粒(载体为pET21a)。接着利用DpnI限制性内切酶对PCR产物进行重组质粒模板消化，再经过纯化之后，转化入大肠杆菌BL21(DE3)感受态，之后将其涂布于含有50ug/L Amp的LB平板上，在37℃的培养箱中倒置培养18h长出单克隆。
实施例3突变体库的筛选
对Q147位点的突变体文库随机挑选178个单克隆进行96孔板振荡培养，对Y188位点的突变体文库随机挑选165个单克隆进行96孔板振荡培养，对S200位点的突变体文库随机挑选187个单克隆进行96孔板振荡培养。在无菌的96孔板中加入400uL的LB培养基，37℃培养约12h，按照10％的接种量，转接于第二个96孔板中培养，继续培养约3h，加入终浓度为0.1mM的IPTG诱导表达16h，诱导温度25℃。培养结束后离心弃上清，冻存于-20℃冰箱中待用。
对以上突变体文库进行反应筛选，反应体系组分如下：0.1M pH6.0磷酸钾缓冲液，底物100g/L，NADP 0.2g/L，葡萄糖100g/L，葡萄糖脱氢酶5g/L，用排枪吸取400uL上述溶液转入离心后的96孔板中，于30℃摇床反应24h，进行HPLC检测。结果显示，Q147L，Q147F，Q147I，Q147V，Y188C，Y188A，Y188I，S200A，S200W，S200Y，S200M突变体的转化率和手性选择性相对于野生型均有不同程度的提高。其中，Y188C，Y188A，Y188I的转化率和手性选择性最佳。
实施例4叠加突变体文库的构建和筛选
为了通过突变体的叠加效应让转化效果进一步提升，在Y188C，Y188A，Y188I单点突变的基础上，选取Q147L，Q147F，S200W，S200Y进行叠加突变，共构建了12个双突变和12个三突变。并进行反应筛选，体系同实施例3。结果显示Q147F/Y188C/S200W,Q147F/Y188C/S200Y,Q147F/Y188A/S200W,Q147F/Y188A/S200Y,Q147F/Y188I/S200W,Q147F/Y188I/S200Y,Q147L/Y188C/S200W,Q147L/Y188C/S200Y,Q147L/Y188I/S200W,Q147L/Y188I/S200Y这10个突变体可将底物完全转化，且手性纯度达99.9％。将底物浓度增加到200g/L，对这10个突变体进行进一步测试，结果显示依然可以实现全部转化。
实施例5酮还原酶突变体细胞/酶粉的制备
将突变体Q147F/Y188C/S200W接种至5mL含氨苄青霉素的LB试管培养基中活化培养(37℃培养12h)，按1％接种量转接活化培养物至400mL含氨苄青霉素的2YT液体培养基中，37℃培养OD至0.6-0.8，加入IPTG(终浓度0.1mM)于25℃诱导培养16h。离心收集菌体得到酮还原酶突变体细胞。若要制备酶粉以利于长期保存，则用40mL磷酸盐缓冲液(10mM，pH7.5)重悬20g菌体后，于均质机中均质破碎，离心收集上清，-20℃预冻后真空冷冻干燥48h后碾碎，即得酮还原酶突变体酶粉。
实施例6克级(S)-1-(2-碘-5-氟苯基)乙醇的制备
向250mL三口瓶中加入底物1-(5-氟-2-碘苯基)乙酮(10g)，葡萄糖(10g)，酮还原酶突变体1F3细胞4g、葡萄糖脱氢酶细胞1g、辅酶NADP+(4mg),采用0.1M磷酸钾缓冲液定容至80mL，30℃保温搅拌反应24h，同时TLC检测反应进程。反应结束后过滤，滤饼用60mL甲醇打浆，过滤,所得滤液减压浓缩即得产物。HPLC检测转化率及产物手性纯度值如附图4所示：底物转化率＝99.7％，S型产物手性纯度99.97％。
实施例7百克级(S)-1-(2-碘-5-氟苯基)乙醇的制备
向5000mL三口瓶中加入底物1-(5-氟-2-碘苯基)乙酮(200g)，葡萄糖(200g)，酮还原酶突变体细胞3B8 80g、葡萄糖脱氢酶细胞20g、辅酶NADPH(80mg),采用0.1M磷酸钾缓冲液定容至1600mL，30℃保温搅拌反应20h，同时TLC检测反应进程。反应结束后过滤，滤饼用1200mL甲醇打浆，过滤,所得滤液减压浓缩即得产物。HPLC检测转化率及产物手性纯度值如附图5所示：底物转化率＝99.7％，S型产物手性纯度99.95％。
实施例8公斤级(S)-1-(2-碘-5-氟苯基)乙醇的制备
向反应容器中加入底物1-(5-氟-2-碘苯基)乙酮(5.0Kg)，葡萄糖(5.0Kg)，酮还原酶突变体1F3细胞2.0Kg、葡萄糖脱氢酶细胞500g、辅酶NADP+(2g),采用0.1M磷酸钾缓冲液定容至20L，30℃保温搅拌反应24h，同时TLC检测反应进程。反应结束后过滤，滤饼用15L甲醇打浆，过滤,所得滤液减压浓缩即得产物。底物转化率＝99.7％，S型产物手性纯度99.96％。
序列表
<110> 尚科生物医药（上海）有限公司
<120> 一种酮还原酶及其催化制备(S)-1-(2-碘-5-氟苯基)乙醇的方法
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225 230 235 240
Pro Ile Ser Ala Gly Gly Thr Thr Ala Ile
245 250
<210> 3
<211> 250
<212> PRT
<213> Flavobacterium hercynium
<400> 3
Met Ser Leu Leu Gly Ala Leu Val Ala Pro Val Ser Gly Ala Gly Ser
1 5 10 15
Gly Ile Gly Ala Ala Val Ala Gly Ala Thr Ala Ala Gly Gly Ala Leu
20 25 30
Val Ile Val Ser Ala Ile Ala Val Gly His Gly Gly Gly Thr Val Leu
35 40 45
Ile Ile Ala Ala Ala Gly Gly Gly Ala Pro Pro Ile Leu Gly Ala Ser
50 55 60
Ser Ser Ala Ser Ala Ala Gly Ala Val Val Gly Gly Thr Val Ala Leu
65 70 75 80
Thr Gly Ala Leu Ala Ile Ala Cys Ala Ala Ala Gly Met Gly Gly Pro
85 90 95
Ala Leu Pro Thr Gly Gly Thr Ala Pro Gly Ala Thr Ala Ala Val Ile
100 105 110
Gly Leu Ala Leu Ala Gly Val Pro Thr Ala Cys Ala Thr Gly Leu Gly
115 120 125
Gly Met Gly Leu Ala Gly Gly Gly Ser Ile Val Ala Ile Ala Ser Ile
130 135 140
His Gly Pro Val Ala Ala Pro Leu Ser Ser Ala Thr Thr Ala Ser Leu
145 150 155 160
His Gly Val Val Gly Leu Thr Leu Ala Ile Ala Ala Gly Thr Ala Gly
165 170 175
Leu Ala Ile Ala Cys Ala Ala Val Gly Pro Gly Ala Ile Gly Thr Ala
180 185 190
Leu Leu Leu Ala Ala Leu Ala Thr Ser Ala Met Gly Ala Val Ala Ala
195 200 205
Leu Ser Pro Met Ala Ala Leu Gly Thr Ser Gly Gly Ile Ala Gly Leu
210 215 220
Val Leu Pro Leu Ser Ser Ala Leu Ser Ser Pro Thr Thr Gly Ser Thr
225 230 235 240
Pro Ile Ser Ala Gly Gly Thr Thr Ala Ile
245 250
<210> 4
<211> 250
<212> PRT
<213> Flavobacterium hercynium
<400> 4
Met Ser Leu Leu Gly Ala Leu Val Ala Pro Val Ser Gly Ala Gly Ser
1 5 10 15
Gly Ile Gly Ala Ala Val Ala Gly Ala Thr Ala Ala Gly Gly Ala Leu
20 25 30
Val Ile Val Ser Ala Ile Ala Val Gly His Gly Gly Gly Thr Val Leu
35 40 45
Ile Ile Ala Ala Ala Gly Gly Gly Ala Pro Pro Ile Leu Gly Ala Ser
50 55 60
Ser Ser Ala Ser Ala Ala Gly Ala Val Val Gly Gly Thr Val Ala Leu
65 70 75 80
Thr Gly Ala Leu Ala Ile Ala Cys Ala Ala Ala Gly Met Gly Gly Pro
85 90 95
Ala Leu Pro Thr Gly Gly Thr Ala Pro Gly Ala Thr Ala Ala Val Ile
100 105 110
Gly Leu Ala Leu Ala Gly Val Pro Thr Ala Cys Ala Thr Gly Leu Gly
115 120 125
Gly Met Gly Leu Ala Gly Gly Gly Ser Ile Val Ala Ile Ala Ser Ile
130 135 140
His Gly Pro Val Ala Ala Pro Leu Ser Ser Ala Thr Thr Ala Ser Leu
145 150 155 160
His Gly Val Val Gly Leu Thr Leu Ala Ile Ala Ala Gly Thr Ala Gly
165 170 175
Leu Ala Ile Ala Cys Ala Ala Val Gly Pro Gly Ala Ile Gly Thr Ala
180 185 190
Leu Leu Leu Ala Ala Leu Ala Thr Ser Ala Met Gly Ala Val Ala Ala
195 200 205
Leu Ser Pro Met Ala Ala Leu Gly Thr Ser Gly Gly Ile Ala Gly Leu
210 215 220
Val Leu Pro Leu Ser Ser Ala Leu Ser Ser Pro Thr Thr Gly Ser Thr
225 230 235 240
Pro Ile Ser Ala Gly Gly Thr Thr Ala Ile
245 250
<210> 5
<211> 250
<212> PRT
<213> Flavobacterium hercynium
<400> 5
Met Ser Leu Leu Gly Ala Leu Val Ala Pro Val Ser Gly Ala Gly Ser
1 5 10 15
Gly Ile Gly Ala Ala Val Ala Gly Ala Thr Ala Ala Gly Gly Ala Leu
20 25 30
Val Ile Val Ser Ala Ile Ala Val Gly His Gly Gly Gly Thr Val Leu
35 40 45
Ile Ile Ala Ala Ala Gly Gly Gly Ala Pro Pro Ile Leu Gly Ala Ser
50 55 60
Ser Ser Ala Ser Ala Ala Gly Ala Val Val Gly Gly Thr Val Ala Leu
65 70 75 80
Thr Gly Ala Leu Ala Ile Ala Cys Ala Ala Ala Gly Met Gly Gly Pro
85 90 95
Ala Leu Pro Thr Gly Gly Thr Ala Pro Gly Ala Thr Ala Ala Val Ile
100 105 110
Gly Leu Ala Leu Ala Gly Val Pro Thr Ala Cys Ala Thr Gly Leu Gly
115 120 125
Gly Met Gly Leu Ala Gly Gly Gly Ser Ile Val Ala Ile Ala Ser Ile
130 135 140
His Gly Pro Val Ala Ala Pro Leu Ser Ser Ala Thr Thr Ala Ser Leu
145 150 155 160
His Gly Val Val Gly Leu Thr Leu Ala Ile Ala Ala Gly Thr Ala Gly
165 170 175
Leu Ala Ile Ala Cys Ala Ala Val Gly Pro Gly Ile Ile Gly Thr Ala
180 185 190
Leu Leu Leu Ala Ala Leu Ala Thr Ser Ala Met Gly Ala Val Ala Ala
195 200 205
Leu Ser Pro Met Ala Ala Leu Gly Thr Ser Gly Gly Ile Ala Gly Leu
210 215 220
Val Leu Pro Leu Ser Ser Ala Leu Ser Ser Pro Thr Thr Gly Ser Thr
225 230 235 240
Pro Ile Ser Ala Gly Gly Thr Thr Ala Ile
245 250
<210> 6
<211> 250
<212> PRT
<213> Flavobacterium hercynium
<400> 6
Met Ser Leu Leu Gly Ala Leu Val Ala Pro Val Ser Gly Ala Gly Ser
1 5 10 15
Gly Ile Gly Ala Ala Val Ala Gly Ala Thr Ala Ala Gly Gly Ala Leu
20 25 30
Val Ile Val Ser Ala Ile Ala Val Gly His Gly Gly Gly Thr Val Leu
35 40 45
Ile Ile Ala Ala Ala Gly Gly Gly Ala Pro Pro Ile Leu Gly Ala Ser
50 55 60
Ser Ser Ala Ser Ala Ala Gly Ala Val Val Gly Gly Thr Val Ala Leu
65 70 75 80
Thr Gly Ala Leu Ala Ile Ala Cys Ala Ala Ala Gly Met Gly Gly Pro
85 90 95
Ala Leu Pro Thr Gly Gly Thr Ala Pro Gly Ala Thr Ala Ala Val Ile
100 105 110
Gly Leu Ala Leu Ala Gly Val Pro Thr Ala Cys Ala Thr Gly Leu Gly
115 120 125
Gly Met Gly Leu Ala Gly Gly Gly Ser Ile Val Ala Ile Ala Ser Ile
130 135 140
His Gly Pro Val Ala Ala Pro Leu Ser Ser Ala Thr Thr Ala Ser Leu
145 150 155 160
His Gly Val Val Gly Leu Thr Leu Ala Ile Ala Ala Gly Thr Ala Gly
165 170 175
Leu Ala Ile Ala Cys Ala Ala Val Gly Pro Gly Ile Ile Gly Thr Ala
180 185 190
Leu Leu Leu Ala Ala Leu Ala Thr Ser Ala Met Gly Ala Val Ala Ala
195 200 205
Leu Ser Pro Met Ala Ala Leu Gly Thr Ser Gly Gly Ile Ala Gly Leu
210 215 220
Val Leu Pro Leu Ser Ser Ala Leu Ser Ser Pro Thr Thr Gly Ser Thr
225 230 235 240
Pro Ile Ser Ala Gly Gly Thr Thr Ala Ile
245 250
<210> 7
<211> 250
<212> PRT
<213> Flavobacterium hercynium
<400> 7
Met Ser Leu Leu Gly Ala Leu Val Ala Pro Val Ser Gly Ala Gly Ser
1 5 10 15
Gly Ile Gly Ala Ala Val Ala Gly Ala Thr Ala Ala Gly Gly Ala Leu
20 25 30
Val Ile Val Ser Ala Ile Ala Val Gly His Gly Gly Gly Thr Val Leu
35 40 45
Ile Ile Ala Ala Ala Gly Gly Gly Ala Pro Pro Ile Leu Gly Ala Ser
50 55 60
Ser Ser Ala Ser Ala Ala Gly Ala Val Val Gly Gly Thr Val Ala Leu
65 70 75 80
Thr Gly Ala Leu Ala Ile Ala Cys Ala Ala Ala Gly Met Gly Gly Pro
85 90 95
Ala Leu Pro Thr Gly Gly Thr Ala Pro Gly Ala Thr Ala Ala Val Ile
100 105 110
Gly Leu Ala Leu Ala Gly Val Pro Thr Ala Cys Ala Thr Gly Leu Gly
115 120 125
Gly Met Gly Leu Ala Gly Gly Gly Ser Ile Val Ala Ile Ala Ser Ile
130 135 140
His Gly Leu Val Ala Ala Pro Leu Ser Ser Ala Thr Thr Ala Ser Leu
145 150 155 160
His Gly Val Val Gly Leu Thr Leu Ala Ile Ala Ala Gly Thr Ala Gly
165 170 175
Leu Ala Ile Ala Cys Ala Ala Val Gly Pro Gly Cys Ile Gly Thr Ala
180 185 190
Leu Leu Leu Ala Ala Leu Ala Thr Ser Ala Met Gly Ala Val Ala Ala
195 200 205
Leu Ser Pro Met Ala Ala Leu Gly Thr Ser Gly Gly Ile Ala Gly Leu
210 215 220
Val Leu Pro Leu Ser Ser Ala Leu Ser Ser Pro Thr Thr Gly Ser Thr
225 230 235 240
Pro Ile Ser Ala Gly Gly Thr Thr Ala Ile
245 250
<210> 8
<211> 250
<212> PRT
<213> Flavobacterium hercynium
<400> 8
Met Ser Leu Leu Gly Ala Leu Val Ala Pro Val Ser Gly Ala Gly Ser
1 5 10 15
Gly Ile Gly Ala Ala Val Ala Gly Ala Thr Ala Ala Gly Gly Ala Leu
20 25 30
Val Ile Val Ser Ala Ile Ala Val Gly His Gly Gly Gly Thr Val Leu
35 40 45
Ile Ile Ala Ala Ala Gly Gly Gly Ala Pro Pro Ile Leu Gly Ala Ser
50 55 60
Ser Ser Ala Ser Ala Ala Gly Ala Val Val Gly Gly Thr Val Ala Leu
65 70 75 80
Thr Gly Ala Leu Ala Ile Ala Cys Ala Ala Ala Gly Met Gly Gly Pro
85 90 95
Ala Leu Pro Thr Gly Gly Thr Ala Pro Gly Ala Thr Ala Ala Val Ile
100 105 110
Gly Leu Ala Leu Ala Gly Val Pro Thr Ala Cys Ala Thr Gly Leu Gly
115 120 125
Gly Met Gly Leu Ala Gly Gly Gly Ser Ile Val Ala Ile Ala Ser Ile
130 135 140
His Gly Leu Val Ala Ala Pro Leu Ser Ser Ala Thr Thr Ala Ser Leu
145 150 155 160
His Gly Val Val Gly Leu Thr Leu Ala Ile Ala Ala Gly Thr Ala Gly
165 170 175
Leu Ala Ile Ala Cys Ala Ala Val Gly Pro Gly Cys Ile Gly Thr Ala
180 185 190
Leu Leu Leu Ala Ala Leu Ala Thr Ser Ala Met Gly Ala Val Ala Ala
195 200 205
Leu Ser Pro Met Ala Ala Leu Gly Thr Ser Gly Gly Ile Ala Gly Leu
210 215 220
Val Leu Pro Leu Ser Ser Ala Leu Ser Ser Pro Thr Thr Gly Ser Thr
225 230 235 240
Pro Ile Ser Ala Gly Gly Thr Thr Ala Ile
245 250
<210> 9
<211> 250
<212> PRT
<213> Flavobacterium hercynium
<400> 9
Met Ser Leu Leu Gly Ala Leu Val Ala Pro Val Ser Gly Ala Gly Ser
1 5 10 15
Gly Ile Gly Ala Ala Val Ala Gly Ala Thr Ala Ala Gly Gly Ala Leu
20 25 30
Val Ile Val Ser Ala Ile Ala Val Gly His Gly Gly Gly Thr Val Leu
35 40 45
Ile Ile Ala Ala Ala Gly Gly Gly Ala Pro Pro Ile Leu Gly Ala Ser
50 55 60
Ser Ser Ala Ser Ala Ala Gly Ala Val Val Gly Gly Thr Val Ala Leu
65 70 75 80
Thr Gly Ala Leu Ala Ile Ala Cys Ala Ala Ala Gly Met Gly Gly Pro
85 90 95
Ala Leu Pro Thr Gly Gly Thr Ala Pro Gly Ala Thr Ala Ala Val Ile
100 105 110
Gly Leu Ala Leu Ala Gly Val Pro Thr Ala Cys Ala Thr Gly Leu Gly
115 120 125
Gly Met Gly Leu Ala Gly Gly Gly Ser Ile Val Ala Ile Ala Ser Ile
130 135 140
His Gly Leu Val Ala Ala Pro Leu Ser Ser Ala Thr Thr Ala Ser Leu
145 150 155 160
His Gly Val Val Gly Leu Thr Leu Ala Ile Ala Ala Gly Thr Ala Gly
165 170 175
Leu Ala Ile Ala Cys Ala Ala Val Gly Pro Gly Ile Ile Gly Thr Ala
180 185 190
Leu Leu Leu Ala Ala Leu Ala Thr Ser Ala Met Gly Ala Val Ala Ala
195 200 205
Leu Ser Pro Met Ala Ala Leu Gly Thr Ser Gly Gly Ile Ala Gly Leu
210 215 220
Val Leu Pro Leu Ser Ser Ala Leu Ser Ser Pro Thr Thr Gly Ser Thr
225 230 235 240
Pro Ile Ser Ala Gly Gly Thr Thr Ala Ile
245 250
<210> 10
<211> 250
<212> PRT
<213> Flavobacterium hercynium
<400> 10
Met Ser Leu Leu Gly Ala Leu Val Ala Pro Val Ser Gly Ala Gly Ser
1 5 10 15
Gly Ile Gly Ala Ala Val Ala Gly Ala Thr Ala Ala Gly Gly Ala Leu
20 25 30
Val Ile Val Ser Ala Ile Ala Val Gly His Gly Gly Gly Thr Val Leu
35 40 45
Ile Ile Ala Ala Ala Gly Gly Gly Ala Pro Pro Ile Leu Gly Ala Ser
50 55 60
Ser Ser Ala Ser Ala Ala Gly Ala Val Val Gly Gly Thr Val Ala Leu
65 70 75 80
Thr Gly Ala Leu Ala Ile Ala Cys Ala Ala Ala Gly Met Gly Gly Pro
85 90 95
Ala Leu Pro Thr Gly Gly Thr Ala Pro Gly Ala Thr Ala Ala Val Ile
100 105 110
Gly Leu Ala Leu Ala Gly Val Pro Thr Ala Cys Ala Thr Gly Leu Gly
115 120 125
Gly Met Gly Leu Ala Gly Gly Gly Ser Ile Val Ala Ile Ala Ser Ile
130 135 140
His Gly Leu Val Ala Ala Pro Leu Ser Ser Ala Thr Thr Ala Ser Leu
145 150 155 160
His Gly Val Val Gly Leu Thr Leu Ala Ile Ala Ala Gly Thr Ala Gly
165 170 175
Leu Ala Ile Ala Cys Ala Ala Val Gly Pro Gly Ile Ile Gly Thr Ala
180 185 190
Leu Leu Leu Ala Ala Leu Ala Thr Ser Ala Met Gly Ala Val Ala Ala
195 200 205
Leu Ser Pro Met Ala Ala Leu Gly Thr Ser Gly Gly Ile Ala Gly Leu
210 215 220
Val Leu Pro Leu Ser Ser Ala Leu Ser Ser Pro Thr Thr Gly Ser Thr
225 230 235 240
Pro Ile Ser Ala Gly Gly Thr Thr Ala Ile
245 250
<210> 11
<211> 753
<212> DNA
<213> Flavobacterium hercynium
<400> 11
atgtcacttt tagaaaacaa agttgctttt gtatccggag ccggttcagg aattggtcgt 60
gctgtcgcag aagcgtatgc tcgtgaagga gcaaaagtaa tagtatccga tattaatgta 120
gaacacggac aggaaacagt aaagataatt gcagcaaatg gcggagaagc tttttttatt 180
aaaggagatt cgtcaagtgc aagcgataac caaagagtag tacaggaaac agtagcaaaa 240
tacggtcgac ttgatattgc ctgtaacaat gcaggaatgg gtggtccggc caaaccaaca 300
ggagaatatg atcccgaagc ttgggatcgc gttattggtt tgaatttaaa tggtgttttt 360
tatgcctgtc gttaccaatt agaacaaatg gaaaaaaacg gtggaggaag catagtaaat 420
attgcttcga ttcacggatt tgttgcagca cctcttagtt cagcttatac tgcttcaaaa 480
catggagttg ttggattaac caaaaatatt gctgctgagt atgcacaaaa aaatattcgt 540
tgcaatgctg taggacctgg ttgtattgaa accgctttat taaaagataa tctggattgg 600
agtgcgatgg aagctgtagc ggcaaaatca ccaatgaacc gtttggggac atcagaagaa 660
attgcggaat tggttttgtt tttaagttct gataaatctt cttttacaac gggaagttat 720
tttatttcag acggaggtta tacggcaata taa 753
<210> 12
<211> 753
<212> DNA
<213> Flavobacterium hercynium
<400> 12
atgtcacttt tagaaaacaa agttgctttt gtatccggag ccggttcagg aattggtcgt 60
gctgtcgcag aagcgtatgc tcgtgaagga gcaaaagtaa tagtatccga tattaatgta 120
gaacacggac aggaaacagt aaagataatt gcagcaaatg gcggagaagc tttttttatt 180
aaaggagatt cgtcaagtgc aagcgataac caaagagtag tacaggaaac agtagcaaaa 240
tacggtcgac ttgatattgc ctgtaacaat gcaggaatgg gtggtccggc caaaccaaca 300
ggagaatatg atcccgaagc ttgggatcgc gttattggtt tgaatttaaa tggtgttttt 360
tatgcctgtc gttaccaatt agaacaaatg gaaaaaaacg gtggaggaag catagtaaat 420
attgcttcga ttcacggatt tgttgcagca cctcttagtt cagcttatac tgcttcaaaa 480
catggagttg ttggattaac caaaaatatt gctgctgagt atgcacaaaa aaatattcgt 540
tgcaatgctg taggacctgg ttgtattgaa accgctttat taaaagataa tctggattat 600
agtgcgatgg aagctgtagc ggcaaaatca ccaatgaacc gtttggggac atcagaagaa 660
attgcggaat tggttttgtt tttaagttct gataaatctt cttttacaac gggaagttat 720
tttatttcag acggaggtta tacggcaata taa 753
<210> 13
<211> 753
<212> DNA
<213> Flavobacterium hercynium
<400> 13
atgtcacttt tagaaaacaa agttgctttt gtatccggag ccggttcagg aattggtcgt 60
gctgtcgcag aagcgtatgc tcgtgaagga gcaaaagtaa tagtatccga tattaatgta 120
gaacacggac aggaaacagt aaagataatt gcagcaaatg gcggagaagc tttttttatt 180
aaaggagatt cgtcaagtgc aagcgataac caaagagtag tacaggaaac agtagcaaaa 240
tacggtcgac ttgatattgc ctgtaacaat gcaggaatgg gtggtccggc caaaccaaca 300
ggagaatatg atcccgaagc ttgggatcgc gttattggtt tgaatttaaa tggtgttttt 360
tatgcctgtc gttaccaatt agaacaaatg gaaaaaaacg gtggaggaag catagtaaat 420
attgcttcga ttcacggatt tgttgcagca cctcttagtt cagcttatac tgcttcaaaa 480
catggagttg ttggattaac caaaaatatt gctgctgagt atgcacaaaa aaatattcgt 540
tgcaatgctg taggacctgg tgcgattgaa accgctttat taaaagataa tctggattgg 600
agtgcgatgg aagctgtagc ggcaaaatca ccaatgaacc gtttggggac atcagaagaa 660
attgcggaat tggttttgtt tttaagttct gataaatctt cttttacaac gggaagttat 720
tttatttcag acggaggtta tacggcaata taa 753
<210> 14
<211> 753
<212> DNA
<213> Flavobacterium hercynium
<400> 14
atgtcacttt tagaaaacaa agttgctttt gtatccggag ccggttcagg aattggtcgt 60
gctgtcgcag aagcgtatgc tcgtgaagga gcaaaagtaa tagtatccga tattaatgta 120
gaacacggac aggaaacagt aaagataatt gcagcaaatg gcggagaagc tttttttatt 180
aaaggagatt cgtcaagtgc aagcgataac caaagagtag tacaggaaac agtagcaaaa 240
tacggtcgac ttgatattgc ctgtaacaat gcaggaatgg gtggtccggc caaaccaaca 300
ggagaatatg atcccgaagc ttgggatcgc gttattggtt tgaatttaaa tggtgttttt 360
tatgcctgtc gttaccaatt agaacaaatg gaaaaaaacg gtggaggaag catagtaaat 420
attgcttcga ttcacggatt tgttgcagca cctcttagtt cagcttatac tgcttcaaaa 480
catggagttg ttggattaac caaaaatatt gctgctgagt atgcacaaaa aaatattcgt 540
tgcaatgctg taggacctgg tgcgattgaa accgctttat taaaagataa tctggattat 600
agtgcgatgg aagctgtagc ggcaaaatca ccaatgaacc gtttggggac atcagaagaa 660
attgcggaat tggttttgtt tttaagttct gataaatctt cttttacaac gggaagttat 720
tttatttcag acggaggtta tacggcaata taa 753
<210> 15
<211> 753
<212> DNA
<213> Flavobacterium hercynium
<400> 15
atgtcacttt tagaaaacaa agttgctttt gtatccggag ccggttcagg aattggtcgt 60
gctgtcgcag aagcgtatgc tcgtgaagga gcaaaagtaa tagtatccga tattaatgta 120
gaacacggac aggaaacagt aaagataatt gcagcaaatg gcggagaagc tttttttatt 180
aaaggagatt cgtcaagtgc aagcgataac caaagagtag tacaggaaac agtagcaaaa 240
tacggtcgac ttgatattgc ctgtaacaat gcaggaatgg gtggtccggc caaaccaaca 300
ggagaatatg atcccgaagc ttgggatcgc gttattggtt tgaatttaaa tggtgttttt 360
tatgcctgtc gttaccaatt agaacaaatg gaaaaaaacg gtggaggaag catagtaaat 420
attgcttcga ttcacggatt tgttgcagca cctcttagtt cagcttatac tgcttcaaaa 480
catggagttg ttggattaac caaaaatatt gctgctgagt atgcacaaaa aaatattcgt 540
tgcaatgctg taggacctgg tattattgaa accgctttat taaaagataa tctggattgg 600
agtgcgatgg aagctgtagc ggcaaaatca ccaatgaacc gtttggggac atcagaagaa 660
attgcggaat tggttttgtt tttaagttct gataaatctt cttttacaac gggaagttat 720
tttatttcag acggaggtta tacggcaata taa 753
<210> 16
<211> 753
<212> DNA
<213> Flavobacterium hercynium
<400> 16
atgtcacttt tagaaaacaa agttgctttt gtatccggag ccggttcagg aattggtcgt 60
gctgtcgcag aagcgtatgc tcgtgaagga gcaaaagtaa tagtatccga tattaatgta 120
gaacacggac aggaaacagt aaagataatt gcagcaaatg gcggagaagc tttttttatt 180
aaaggagatt cgtcaagtgc aagcgataac caaagagtag tacaggaaac agtagcaaaa 240
tacggtcgac ttgatattgc ctgtaacaat gcaggaatgg gtggtccggc caaaccaaca 300
ggagaatatg atcccgaagc ttgggatcgc gttattggtt tgaatttaaa tggtgttttt 360
tatgcctgtc gttaccaatt agaacaaatg gaaaaaaacg gtggaggaag catagtaaat 420
attgcttcga ttcacggatt tgttgcagca cctcttagtt cagcttatac tgcttcaaaa 480
catggagttg ttggattaac caaaaatatt gctgctgagt atgcacaaaa aaatattcgt 540
tgcaatgctg taggacctgg tattattgaa accgctttat taaaagataa tctggattat 600
agtgcgatgg aagctgtagc ggcaaaatca ccaatgaacc gtttggggac atcagaagaa 660
attgcggaat tggttttgtt tttaagttct gataaatctt cttttacaac gggaagttat 720
tttatttcag acggaggtta tacggcaata taa 753
<210> 17
<211> 753
<212> DNA
<213> Flavobacterium hercynium
<400> 17
atgtcacttt tagaaaacaa agttgctttt gtatccggag ccggttcagg aattggtcgt 60
gctgtcgcag aagcgtatgc tcgtgaagga gcaaaagtaa tagtatccga tattaatgta 120
gaacacggac aggaaacagt aaagataatt gcagcaaatg gcggagaagc tttttttatt 180
aaaggagatt cgtcaagtgc aagcgataac caaagagtag tacaggaaac agtagcaaaa 240
tacggtcgac ttgatattgc ctgtaacaat gcaggaatgg gtggtccggc caaaccaaca 300
ggagaatatg atcccgaagc ttgggatcgc gttattggtt tgaatttaaa tggtgttttt 360
tatgcctgtc gttaccaatt agaacaaatg gaaaaaaacg gtggaggaag catagtaaat 420
attgcttcga ttcacggact ggttgcagca cctcttagtt cagcttatac tgcttcaaaa 480
catggagttg ttggattaac caaaaatatt gctgctgagt atgcacaaaa aaatattcgt 540
tgcaatgctg taggacctgg ttgtattgaa accgctttat taaaagataa tctggattgg 600
agtgcgatgg aagctgtagc ggcaaaatca ccaatgaacc gtttggggac atcagaagaa 660
attgcggaat tggttttgtt tttaagttct gataaatctt cttttacaac gggaagttat 720
tttatttcag acggaggtta tacggcaata taa 753
<210> 18
<211> 753
<212> DNA
<213> Flavobacterium hercynium
<400> 18
atgtcacttt tagaaaacaa agttgctttt gtatccggag ccggttcagg aattggtcgt 60
gctgtcgcag aagcgtatgc tcgtgaagga gcaaaagtaa tagtatccga tattaatgta 120
gaacacggac aggaaacagt aaagataatt gcagcaaatg gcggagaagc tttttttatt 180
aaaggagatt cgtcaagtgc aagcgataac caaagagtag tacaggaaac agtagcaaaa 240
tacggtcgac ttgatattgc ctgtaacaat gcaggaatgg gtggtccggc caaaccaaca 300
ggagaatatg atcccgaagc ttgggatcgc gttattggtt tgaatttaaa tggtgttttt 360
tatgcctgtc gttaccaatt agaacaaatg gaaaaaaacg gtggaggaag catagtaaat 420
attgcttcga ttcacggact ggttgcagca cctcttagtt cagcttatac tgcttcaaaa 480
catggagttg ttggattaac caaaaatatt gctgctgagt atgcacaaaa aaatattcgt 540
tgcaatgctg taggacctgg ttgtattgaa accgctttat taaaagataa tctggattat 600
agtgcgatgg aagctgtagc ggcaaaatca ccaatgaacc gtttggggac atcagaagaa 660
attgcggaat tggttttgtt tttaagttct gataaatctt cttttacaac gggaagttat 720
tttatttcag acggaggtta tacggcaata taa 753
<210> 19
<211> 753
<212> DNA
<213> Flavobacterium hercynium
<400> 19
atgtcacttt tagaaaacaa agttgctttt gtatccggag ccggttcagg aattggtcgt 60
gctgtcgcag aagcgtatgc tcgtgaagga gcaaaagtaa tagtatccga tattaatgta 120
gaacacggac aggaaacagt aaagataatt gcagcaaatg gcggagaagc tttttttatt 180
aaaggagatt cgtcaagtgc aagcgataac caaagagtag tacaggaaac agtagcaaaa 240
tacggtcgac ttgatattgc ctgtaacaat gcaggaatgg gtggtccggc caaaccaaca 300
ggagaatatg atcccgaagc ttgggatcgc gttattggtt tgaatttaaa tggtgttttt 360
tatgcctgtc gttaccaatt agaacaaatg gaaaaaaacg gtggaggaag catagtaaat 420
attgcttcga ttcacggact ggttgcagca cctcttagtt cagcttatac tgcttcaaaa 480
catggagttg ttggattaac caaaaatatt gctgctgagt atgcacaaaa aaatattcgt 540
tgcaatgctg taggacctgg tattattgaa accgctttat taaaagataa tctggattgg 600
agtgcgatgg aagctgtagc ggcaaaatca ccaatgaacc gtttggggac atcagaagaa 660
attgcggaat tggttttgtt tttaagttct gataaatctt cttttacaac gggaagttat 720
tttatttcag acggaggtta tacggcaata taa 753
<210> 20
<211> 753
<212> DNA
<213> Flavobacterium hercynium
<400> 20
atgtcacttt tagaaaacaa agttgctttt gtatccggag ccggttcagg aattggtcgt 60
gctgtcgcag aagcgtatgc tcgtgaagga gcaaaagtaa tagtatccga tattaatgta 120
gaacacggac aggaaacagt aaagataatt gcagcaaatg gcggagaagc tttttttatt 180
aaaggagatt cgtcaagtgc aagcgataac caaagagtag tacaggaaac agtagcaaaa 240
tacggtcgac ttgatattgc ctgtaacaat gcaggaatgg gtggtccggc caaaccaaca 300
ggagaatatg atcccgaagc ttgggatcgc gttattggtt tgaatttaaa tggtgttttt 360
tatgcctgtc gttaccaatt agaacaaatg gaaaaaaacg gtggaggaag catagtaaat 420
attgcttcga ttcacggact ggttgcagca cctcttagtt cagcttatac tgcttcaaaa 480
catggagttg ttggattaac caaaaatatt gctgctgagt atgcacaaaa aaatattcgt 540
tgcaatgctg taggacctgg tattattgaa accgctttat taaaagataa tctggattat 600
agtgcgatgg aagctgtagc ggcaaaatca ccaatgaacc gtttggggac atcagaagaa 660
attgcggaat tggttttgtt tttaagttct gataaatctt cttttacaac gggaagttat 720
tttatttcag acggaggtta tacggcaata taa 753
Claims (7)
1.一种工程酮还原酶多肽，该多肽衍生自野生型Flavobacteriumhercynium酮还原酶，所述酮还原酶多肽的基因核苷酸序列选自SEQ ID No.11-20所示基因序列。
2.一种酮还原酶多肽催化制备(S)-1-(2-碘-5-氟苯基)乙醇的方法，其特征在于：1-(5-氟-2-碘苯基)乙酮在酮还原酶多肽催化作用下，转化为(S)-1-(2-碘-5-氟苯基)乙醇，所述酮还原酶多肽的基因核苷酸序列选自SEQ ID No.11-20所示基因序列。
3.如权利要求2所述的酮还原酶多肽催化制备(S)-1-(2-碘-5-氟苯基)乙醇的方法，其特征在于：1-(5-氟-2-碘苯基)乙酮在酮还原酶催化作用下，转化为(S)-1-(2-碘-5-氟苯基)乙醇，步骤包括：将酮还原酶酶粉或含有酮还原酶的细胞和1-(5-氟-2-碘苯基)乙酮、辅酶、缓冲溶液配制成混合溶液，反应得到产物。
4.如权利要求3所述的酮还原酶多肽催化制备(S)-1-(2-碘-5-氟苯基)乙醇的方法，其特征在于：反应时间为12～36h。
5.如权利要求3所述的酮还原酶多肽催化制备(S)-1-(2-碘-5-氟苯基)乙醇的方法，其特征在于：所述辅酶选自NADP和NADPH中的任意一种或它们的组合。
6.如权利要求3所述的酮还原酶多肽催化制备(S)-1-(2-碘-5-氟苯基)乙醇的方法，其特征在于：所述缓冲溶液为磷酸钾缓冲液。
7.如权利要求3所述的酮还原酶多肽催化制备(S)-1-(2-碘-5-氟苯基)乙醇的方法，其特征在于：所述含有酮还原酶的细胞选自基因工程改造的大肠杆菌。
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