CN109055324A - 一种改进的酮还原酶及其应用 - Google Patents
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Abstract
本发明涉及一种改进的酮还原酶，属于生物催化合成技术领域。本发明所述酮还原酶突变体，所述酮还原酶突变体的氨基酸序列是SEQ ID NO:1所示氨基酸序列发生突变的氨基酸序列，发生突变的氨基酸序列突变位点分别为：第140位I突变为S，可选的突变点位至少还包括下列点位之一：第205位D突变为E、第125位H突变为Q、第329位N突变为S、第294位L突变为I、第150位D突变为Q、第36位P突变为S。本发明提供了一种酮还原酶及其应用，可以有效降低(4S)‑3‑[(5S)‑5‑(4‑氟苯基)‑5‑羟基戊酰基]‑4‑苯基‑1,3‑氧氮杂环戊烷‑2‑酮和依折麦布工业化生产的成本。
Description
技术领域
本发明属于生物催化合成技术领域，具体而言，涉及一种改进的酮还原酶及其在制备依折麦布手性中间体(4S)-3-[(5S)-5-(4-氟苯基)-5-羟基戊酰基]-4-苯基-1,3-氧氮杂环戊烷-2-酮的方法。
背景技术
依折麦布（Ezetimibe），化学名为：(3R,4S)-1-(4-氟苯基)-3-[(3S)-3-(4-氟苯基)-3-羟丙基]-4-(4-羟苯基)-2-氮杂环丁酮，是由先灵葆雅公司和默克共同开发并于2002年上市的第一个胆固醇选择性吸收抑制剂药物，商品名为Ezetrol（益适纯）。(4S)-3-[(5S)-5-(4-氟苯基)-5-羟基戊酰基]-4-苯基-1,3-氧氮杂环戊烷-2-酮作为依折麦布原料药合成中的关键手性中间体，目前已报道的制备工艺中最具优势的是利用羰基还原酶催化5-((4S)-2-氧代-4-苯基(1, 3-恶唑烷-3-基))-1-(4-氟苯基) 戊烷-1, 5-二酮进行制备，可以达到较高的光学纯度和转化率。
贺沁婷（CN201710285381.6）利用解淀粉芽孢杆菌（Bacillus amyloliquefaciens）发酵的羰基还原酶，将5-((4S)-2-氧代-4-苯基(1, 3-恶唑烷-3-基))-1-(4-氟苯基) 戊烷-1, 5-二酮在管氏反应器中循环反应生成(4S)-3-[(5S)-5-(4-氟苯基)-5-羟基戊酰基]-4-苯基-1,3-氧氮杂环戊烷-2-酮，底物转化率可达99.5%，产物纯度可达99.5%，但底物浓度最高仅为25g/L。郑国钧（CN201610067795.7）利用大肠杆菌全细胞催化5-((4S)-2-氧代-4-苯基(1, 3-恶唑烷-3-基))-1-(4-氟苯基) 戊烷-1, 5-二酮合成(4S)-3-[(5S)-5-(4-氟苯基)-5-羟基戊酰基]-4-苯基-1,3-氧氮杂环戊烷-2-酮，e.e.值可达99.5%，但转化率仅为70%。艾米丽-穆德弗（CN200980141486.8）以高加索酸奶乳杆菌（Lactobacillus kefir）和微小乳杆菌（Lactobacillus minor）中的天然酮还原酶进化出一系列突变体，得到了可以催化5-((4S)-2-氧代-4-苯基(1, 3-恶唑烷-3-基))-1-(4-氟苯基) 戊烷-1, 5-二酮合成(4S)-3-[(5S)-5-(4-氟苯基)-5-羟基戊酰基]-4-苯基-1,3-氧氮杂环戊烷-2-酮的醇脱氢酶，具体表现为100 g/L5-((4S)-2-氧代-4-苯基(1, 3-恶唑烷-3-基))-1-(4-氟苯基) 戊烷-1, 5-二酮，2 g/L酶粉，体系反应24 hr底物转化率≥98%，产物e.e.值≥99%。
贺沁婷利用B. amyloliquefaciens发酵的羰基还原酶催化5-((4S)-2-氧代-4-苯基(1, 3-恶唑烷-3-基))-1-(4-氟苯基) 戊烷-1, 5-二酮合成(4S)-3-[(5S)-5-(4-氟苯基)-5-羟基戊酰基]-4-苯基-1,3-氧氮杂环戊烷-2-酮中，虽然转化率和产物纯度较高，但底物浓度较低，不利于放大生产，生产成本也偏高。郑国钧使用的大肠杆菌全细胞催化工艺中，底物转化率较低，无法规模化使用。艾米丽-穆德弗的突变体酶催化效果有所提升，但仍存在酶用量大和转化效率不够高等问题，导致(4S)-3-[(5S)-5-(4-氟苯基)-5-羟基戊酰基]-4-苯基-1,3-氧氮杂环戊烷-2-酮的酶法生产成本偏高。
继续筛选不同生物来源的酮还原酶并借助蛋白质工程方法对酶分子进行改造获得更加高效的酮还原酶突变体是实现依折麦布手性中间体(4S)-3-[(5S)-5-(4-氟苯基)-5-羟基戊酰基]-4-苯基-1,3-氧氮杂环戊烷-2-酮酶法工业化生产的努力方向。
发明内容
本发明基于Thermoanaerobacterbrockii (T.brockii)天然酮还原酶而改进的酮还原酶及其编码基因，利用经分子体外进化的酮还原酶突变体催化前体酮合成依折麦布手性中间体。
技术方案
本发明一方面提供了一种改进的酮还原酶，即酮还原酶突变体，该酮还原酶突变体的酶活性和立体选择性均高于野生型酮还原酶。
本发明的技术方案是，一种酮还原酶突变体，其特征是，所述酮还原酶突变体的氨基酸序列是SEQ ID NO: 1所示氨基酸序列发生突变的氨基酸序列，发生突变的氨基酸序列突变位点分别为：第140位I突变为S，可选的突变点位至少还包括下列点位之一：第205位D突变为E、第125位H突变为Q、第329位N突变为S、第294位L突变为I、第150位D突变为Q、第36位P突变为S。
本发明所述酮还原酶突变体的氨基酸序列为：SEQ ID NO: 2、SEQ ID NO: 3、SEQID NO: 4、SEQ ID NO: 5、SEQ ID NO: 6或SEQ ID NO: 7所示的氨基酸序列。
根据本发明的一方面，提供了一种基因，该基因编码上述任一种酮还原酶突变体，基因的核苷酸序列为SEQ ID NO: 9、SEQ ID NO: 10、SEQ ID NO: 11、SEQ ID NO: 12、SEQID NO: 13或SEQ ID NO: 14。
根据本发明的另一方面，提供了一种重组质粒，重组质粒上含有上述任一种基因的核苷酸序列，进一步地，质粒为pET-28a(+)、pET-28b(+)、pET-28c(+)、pET-5b(+)、pET-15b、pET-24a(+)、pET-24c(+)、pET-24d(+)、pET-25b(+)、pET-27b(+)、pET-28c(+)、pET-29a(+)、pET-29b(+)、pET-29c(+)、pET-30b(+)、pET-30c(+)、pET-30 Xa/LIC、pET-30 EK/LIC、pET-31b(+)、pET-32b(+)、pET-32c(+)、pET-32 EK/LIC、pET-32 Xa/LIC、pET-33b(+)、pET-37b(+)、pET-39b(+)、pET-40b(+)、pET-41a(+)、pET-41b(+)、pET-42b(+)、pET-42c(+)、pET-43.1a(+)、pET-43.1b(+)、pET-43.1c(+)、pET-43.1 EK/LIC、pET-44a(+)、pET-44b(+)、pET-44c(+)、pET-44 EK/LIC、pET-45b(+)、pET-46 EK/LIC、pET-47b(+)、pET-48b(+)、pET-49b(+)、pET-51b(+)、pET-52b(+)、pQE30、pQE31、pQE32、pQE40、pBV220、pBV221、pCold-GST、pCold IV、pCold-GST或pTrcHis C。
根据本发明的再一方面，提供了一种宿主细胞，宿主细胞内含有上述任一种重组质粒，且所述宿主细胞包括原核细胞、酵母或真核细胞，原核细胞优选为大肠杆菌BL21(DE3)细胞。
根据本发明的又一方面，提供了一种生产(4S)-3-[(5S)-5-(4-氟苯基)-5-羟基戊酰基]-4-苯基-1,3-氧氮杂环戊烷-2-酮的方法，包括酮还原酶对5-((4S)-2-氧代-4-苯基(1, 3-恶唑烷-3-基))-1-(4-氟苯基) 戊烷-1, 5-二酮进行催化加氢以形成(4S)-3-[(5S)-5-(4-氟苯基)-5-羟基戊酰基]-4-苯基-1,3-氧氮杂环戊烷-2-酮的反应步骤，酮还原酶为上述任一种酮还原酶突变体。
【附图说明】
图1酮还原酶催化5-((4S)-2-氧代-4-苯基(1, 3-恶唑烷-3-基))-1-(4-氟苯基) 戊烷-1, 5-二酮的还原反应
图2本发明优选实施例6中酮还原酶突变体的蛋白电泳检测结果图：其中，1表示野生型酮还原酶母本；2表示I140S突变体；3表示I140S-D205E突变体；4表示H125Q-I140S-N329S突变体；5表示I140S-L294I突变体；6表示I140S-D150Q突变体；7表示P36S-I140S突变体。
有益效果：
应用本发明的技术方案，通过在SEQ ID NO: 1所示的野生型酮还原酶的基础上，采用随机突变的分子生物学方法对酮还原酶的基因进行突变，从而改变酶的氨基酸序列，实现酶结构和功能的改变，再通过定向筛选的方法，得到具有上述位点中的至少之一发生突变的酮还原酶。使用该酮还原酶突变体催化前体酮合成(4S)-3-[(5S)-5-(4-氟苯基)-5-羟基戊酰基]-4-苯基-1,3-氧氮杂环戊烷-2-酮，相比于野生型酮还原酶母本，酶活性提高为约200倍（实施例5），具有非常高的立体选择性和转化率，表现在100 g/L 5-((4S)-2-氧代-4-苯基(1, 3-恶唑烷-3-基))-1-(4-氟苯基) 戊烷-1, 5-二酮，1 g/L酶粉，体系反应16 hr，底物转化率≥99%，产物e.e.值≥99%。相比于现有公开的最优酶催化工艺（艾米丽-穆德弗（CN200980141486.8）100 g/L底物，2 g/L酶粉，反应体系24 hr底物转化率≥98%，产物e.e.值≥99%。），本发明提供了一种酶用量更低（1/2倍）、底物转化率（反应时间16 hr相比于24hr）更高的技术方案，可进一步降低(4S)-3-[(5S)-5-(4-氟苯基)-5-羟基戊酰基]-4-苯基-1,3-氧氮杂环戊烷-2-酮和依折麦布的工业生产成本，具有良好的工业应用价值。
【具体实施方式】
下面结合具体实施实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：获得T. brockii菌株的野生型酮还原酶母本重组质粒
通过NCBI Benbank核酸数据库获得T. brockii菌株的酮还原酶母本编码基因（GenBank: X64841.1），经密码子和酶切位点优化并委托服务商人工合成全长基因和两端的酶切位点，与pET28a(+)表达质粒分别进行酶切、切胶回收、连接和转化大肠杆菌BL21(DE3)感受态细胞，涂布于含有50 mg/L 硫酸卡那霉素的LB琼脂平皿中，37℃培养过夜。挑选数个单菌落至LB培养基（含50 mg/L 硫酸卡那霉素），37℃培养过夜后使用质粒提取试剂盒提取重组质粒，进行PCR和测序验证，获得阳性酮还原酶母本的重组质粒。
实施例2：对野生型酮还原酶母本基因进行随机突变
根据实施例1所述内容，以含T. brockii菌株野生型酮还原酶母本编码基因的重组质粒为模板，并根据T. brockii菌株的酮还原酶母本编码基因用Primer 5.0设计并合成两端引物（表1），使用易错PCR技术（物料及浓度见表2，反应条件见表3）获得含有大量碱基突变的线性基因片段，将这些PCR产物和pET28a(+)表达质粒分别进行酶切、切胶回收、连接和转化大肠杆菌BL21(DE3)感受态细胞，涂布于含有50 mg/L 硫酸卡那霉素的LB琼脂平皿中，37℃培养过夜。
表1 随机突变引物序列
表2 50 μL易错PCR物料体系
表3 易错PCR反应条件：
实施例3：酮还原酶突变体的克隆与表达
为了便于酮还原酶突变体的克隆和表达及鉴定，在其基因的5’和3’末端设计了兼容的限制内切酶位点，可以采用Nde I和Xho I限制性内切酶分别将目的基因和pET28a(+)（其它可以在大肠杆菌中表达蛋白质的表达质粒也可使用）同时进行酶切和DNA切胶回收，回收后的目的基因和质粒的较大片段用T4 DNA连接酶进行连接反应，将连接产物转化到大肠杆菌BL21(DE3)感受态细胞中，然后将转化后的感受态细胞涂布于含有50 mg/L硫酸卡那霉素的LB琼脂平皿中，37℃培养过夜。
挑取上述培养皿上长出的单菌落接种于含有50 mg/L硫酸卡那霉素的LB 液体培养基中，37℃振荡培养过夜，收集菌体进行质粒提取、PCR 鉴定和双酶切鉴定后，将正确的重组质粒命名为pET28a(+)-A-Z，并将含有正确的重组质粒的大肠杆菌进行后续的诱导表达。将上述菌液转接于500 mL含50 mg/L硫酸卡那霉素的LB 液体培养基中，37℃振荡培养至OD600＝0.6～0.7 时，加入IPTG 至终浓度分别为0.05～0.5 mM，在22～25℃下进行诱导表达12～16 hr后，取出菌液，6000 ×g 离心15 min收集菌体，于-20℃冻存备用。
实施例4：酮还原酶突变体的初筛
根据实施例2和实施例3所述内容，挑取上述LB琼脂培养基上的单克隆菌落接种于96深孔板中，每孔预先加入1 mL含有50 mg/L 硫酸卡那霉素的LB培养基，于37℃，220 rpm振荡培养3 hr后，加入一定量的诱导剂异丙基-β-D-硫代半乳糖苷（IPTG，终浓度为0.05 mM），25℃，220 rpm诱导培养16 hr，4000 ×g离心20 min收集菌细胞，倒掉离心上清液后使用相同体积的100 mM磷酸盐缓冲液（pH 7.0）冲悬菌细胞。通过室温-零下80℃反复5次冻融裂解菌细胞后，10℃，8000 ×g离心30 min获得上清液，即酮还原酶突变体粗酶液，用酶标仪进行活性初筛。向96孔板中加入25 μL含有5 mg/mL还原型烟酰胺腺嘌呤二核苷酸（NADH）和2 mM硫酸镁的磷酸盐缓冲液（100 mM，pH=7.0），125 μL 5-((4S)-2-氧代-4-苯基(1, 3-恶唑烷-3-基))-1-(4-氟苯基) 戊烷-1, 5-二酮溶液（10 mg/mL溶于异丙醇），于340 nm进行本底检测，然后分别向各孔中加入已经制备好的母本和突变体酶液100 μL，并立即于30℃检测340nm处吸光光度值的变化。
酶活计算公式：酶活（U/mL）=（△A×60×V1）/（6.22×t×V2）
△A：反应过程中的吸光光度值变化量；
V1：反应体系的总体积；
6.22：NADH消光系数；
t：△A的检测时间；
V2：加入的酶液体积。
实施例5：酮还原酶突变体的复筛
将实施例4中酶活高于母本的突变体菌株以0.01%接种量接种于500 mL含有50 mg/L硫酸卡那霉素的LB培养基，于37℃，220 rpm振荡培养5~6 hr，加入一定量的诱导剂异丙基-β-D-硫代半乳糖苷（IPTG，终浓度为0.05 mM），25℃，220 rpm诱导培养16 hr，7000 ×g离心收集菌体。使用100 mM磷酸盐缓冲液（pH 7.0）冲悬菌细胞后用超声波破碎仪破碎细胞，10℃，10000 ×g离心20 min获得上清液，即酮还原酶突变体粗酶液。向10 mL反应瓶中加入0.05 g主原料5-((4S)-2-氧代-4-苯基(1, 3-恶唑烷-3-基))-1-(4-氟苯基) 戊烷-1, 5-二酮和0.5 mL 异丙醇，振荡至原料溶解，加入4 mL磷酸盐缓冲液（100 mM ，pH =7.0），主原料均匀分散于缓冲液中。加入2 mg NAD+，20 mg甲酸铵，10 mg辅酶（FDH，甲酸脱氢酶）和0.5mL上述突变体粗酶液，体系pH=7.0，并于40℃保温16 hr后，薄层色谱跟踪，选取转化产品点明显、主原料点不明显的体系进行甲基叔丁基醚萃取，静置分液，取有机相进行HPLC分析。
选取催化活性优于母本的突变体进行测序，分析突变位点，复测催化活性确定突变体I140S（SEQ ID NO: 2）、I140S-D205E（SEQ ID NO: 3）、H125Q-I140S-N329S（SEQ IDNO: 4）、I140S-L294I（SEQ ID NO:5）、I140S-D150Q（SEQ ID NO: 6）和P36S-I140S（SEQ IDNO: 7）的催化活性及立体选择性均比本方案母本显著提高，复筛反应结果如表4所示。从表4可见，六种酮还原酶突变体对5-((4S)-2-氧代-4-苯基(1, 3-恶唑烷-3-基))-1-(4-氟苯基) 戊烷-1, 5-二酮的催化效率为野生型酮还原酶母本的约200倍，且产物有更高的e.e.值。
表4酮还原酶母本与突变体酶法制备(4S)-3-[(5S)-5-(4-氟苯基)-5-羟基戊酰基]-4-苯基-1,3-氧氮杂环戊烷-2-酮活性比较
| SEQ ID NO: | 突变位点 | 酶量<sup>*</sup> / wt | 转化率 / % | e.e. / % |
| 1 | 母本 | 20 | 10.2 | 78.0 |
| 1 | 母本 | 50 | 40.1 | 76.8 |
| 2 | I140S突变体 | 1 | 99.5 | 99.7 |
| 3 | I140S-D205E突变体 | 1 | 99.2 | 99.8 |
| 4 | H125Q-I140S-N329S突变体 | 1 | 99.8 | 99.8 |
| 5 | I140S-L294I突变体 | 1 | 99.1 | 99.8 |
| 6 | I140S-D150Q突变体 | 1 | 99.6 | 99.7 |
| 7 | P36S-I140S突变体 | 1 | 99.5 | 99.5 |
注：表4中的*指转化1 g底物所需各酮还原酶突变体重组细胞的湿重。1wt 指转化1 g主原料需要1 g酮还原酶突变体重组湿细胞。
实施例6：酮还原酶突变体酶粉制备
将实施例5中酶活高于母本的突变体菌株分别以0.01%接种量接种于（500 mL/瓶*10瓶/突变体）含有50 mg/L 硫酸卡那霉素的LB培养基，于37℃，220 rpm振荡培养5~6 hr，加入一定量的诱导剂异丙基-β-D-硫代半乳糖苷（IPTG，终浓度为0.05 mM），25℃，220 rpm诱导培养16 hr，7000 ×g离心收集菌体。所得25~30 g菌细胞使用100 mM磷酸盐缓冲液（pH7.0）冲悬菌细胞后用高压均质机破碎细胞，10℃，10000 ×g离心20 min获得上清液，使用真空冷冻干燥机-25℃将其冻干制成3~5 g酶粉，即酮还原酶突变体酶粉。0.1%（w/v）各突变体酶粉溶液SDS-PAGE电泳检测见图2。
实施例7：SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6和SEQ ID NO:7所示的酮还原酶突变体在(4S)-3-[(5S)-5-(4-氟苯基)-5-羟基戊酰基]-4-苯基-1,3-氧氮杂环戊烷-2-酮制备中的应用
向500mL反应瓶中加入20 g主原料5-((4S)-2-氧代-4-苯基(1, 3-恶唑烷-3-基))-1-(4-氟苯基) 戊烷-1, 5-二酮，100 mL异丙醇，振荡至原料完全溶解，加入100 mL磷酸盐缓冲液（100mM，pH 7.0，含2 mM 硫酸镁）；加入20 mg氧化型烟酰胺腺嘌呤二核苷酸（NAD+），20.6 g 甲酸铵，0.1 g辅酶（FDH，即甲酸脱氢酶）和实例5中的0.2 g酮还原酶突变体酶粉，体系pH＝7.0，并于40±2℃保温16 h；用200mL乙酸乙酯终止反应，并用125 g硅藻土过滤体系，200 mL乙酸乙酯萃取两次，静置分液，有机相经浓缩得到粗品。反应转化率和产物e.e.值检测结果见表5。
表5酮还原酶突变体酶法制备(4S)-3-[(5S)-5-(4-氟苯基)-5-羟基戊酰基]-4-苯基-1,3-氧氮杂环戊烷-2-酮
| SEQ ID NO: | 突变位点 | 酶量/ g | 转化率 / % | e.e. / % |
| 2 | I140S突变体 | 0.2 | 99.5 | 99.7 |
| 3 | I140S-D205E突变体 | 0.2 | 99.2 | 99.8 |
| 4 | H125Q-I140S-N329S突变体 | 0.2 | 99.8 | 99.8 |
| 5 | I140S-L294I突变体 | 0.2 | 99.1 | 99.7 |
| 6 | I140S-D150Q突变体 | 0.2 | 99.6 | 99.4 |
| 7 | P36S-I140S突变体 | 0.2 | 99.5 | 99.5 |
表5结果表明，六条突变体（I140S突变体（SEQ ID NO: 2）、I140S-D205E突变体（SEQ IDNO: 3）、H125Q-I140S-N329S突变体（SEQ ID NO: 4）、I140S-L294I突变体（SEQ ID NO:5）、I140S-D150Q突变体（SEQ ID NO: 6）和P36S-I140S突变体（SEQ ID NO: 7））在酶催化5-((4S)-2-氧代-4-苯基(1, 3-恶唑烷-3-基))-1-(4-氟苯基) 戊烷-1, 5-二酮合成(4S)-3-[(5S)-5-(4-氟苯基)-5-羟基戊酰基]-4-苯基-1, 3-氧氮杂环戊烷-2-酮中，即100 g/L底物和1 g/L酶粉的反应体系中，反应16 hr，转化率即可达到99.0%以上，产物e.e.值均达到99.0%以上，筛选出的六条酮还原酶突变体在酶法制备 (4S)-3-[(5S)-5-(4-氟苯基)-5-羟基戊酰基]-4-苯基-1, 3-氧氮杂环戊烷-2-酮中均表现出了极高的立体选择性和高效性。
序列表
<110> 迪沙药业集团（天津）药物研究有限公司；迪沙药业集团有限公司
<120> 一种改进的酮还原酶及其在依折麦布手性中间体制备上的应用
<160> 14
<170> PatentIn version 3.3
<210> SEQ NO ID：1
<211> 352
<212> PRT
<213> Thermoanaerobacter brockii野生型
<400> 1
Met Lys Gly Phe Ala Met Leu Ser Ile Gly Lys Val Gly Trp Ile Glu
1 5 10 15
Lys Glu Lys Pro Ala Pro Gly Pro Phe Asp Ala Ile Val Arg Pro Leu
20 25 30
Ala Val Ala Pro Cys Thr Ser Asp Ile His Thr Val Phe Glu Gly Ala
35 40 45
Ile Gly Glu Arg His Asn Met Ile Leu Gly His Glu Ala Val Gly Glu
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Val Val Glu Val Gly Ser Glu Val Lys Asp Phe Lys Pro Gly Asp Arg
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Val Val Val Pro Ala Ile Thr Pro Asp Trp Arg Thr Ser Glu Val Gln
85 90 95
Arg Gly Tyr His Gln His Ser Gly Gly Met Leu Ala Gly Trp Lys Phe
100 105 110
Ser Asn Val Lys Asp Gly Val Phe Gly Glu Phe Phe His Val Asn Asp
115 120 125
Ala Asp Met Asn Leu Ala His Leu Pro Lys Glu Ile Pro Leu Glu Ala
130 135 140
Ala Val Met Ile Pro Asp Met Met Thr Thr Gly Phe His Gly Ala Glu
145 150 155 160
Leu Ala Asp Ile Glu Leu Gly Ala Thr Val Ala Val Leu Gly Ile Gly
165 170 175
Pro Val Gly Leu Met Ala Val Ala Gly Ala Lys Leu Arg Gly Ala Gly
180 185 190
Arg Ile Ile Ala Val Gly Ser Arg Pro Val Cys Val Asp Ala Ala Lys
195 200 205
Tyr Tyr Gly Ala Thr Asp Ile Val Asn Tyr Lys Asp Gly Pro Ile Glu
210 215 220
Ser Gln Ile Met Asn Leu Thr Glu Gly Lys Gly Val Asp Ala Ala Ile
225 230 235 240
Ile Ala Gly Gly Asn Ala Asp Ile Met Ala Thr Ala Val Lys Ile Val
245 250 255
Lys Pro Gly Gly Thr Ile Ala Asn Val Asn Tyr Phe Gly Glu Gly Glu
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Val Leu Pro Val Pro Arg Leu Glu Trp Gly Cys Gly Met Ala His Lys
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Thr Ile Lys Gly Gly Leu Cys Pro Gly Gly Arg Leu Arg Met Glu Arg
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Leu Ile Asp Leu Val Phe Tyr Lys Arg Val Asp Pro Ser Lys Leu Val
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Thr His Val Phe Arg Gly Phe Asp Asn Ile Glu Lys Ala Phe Met Leu
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Met Lys Asp Lys Pro Lys Asp Leu Ile Lys Pro Val Val Ile Leu Ala
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<210> SEQ NO ID：2
<211> 352
<212> PRT
<213> I140S突变体
<400> 2
Met Lys Gly Phe Ala Met Leu Ser Ile Gly Lys Val Gly Trp Ile Glu
1 5 10 15
Lys Glu Lys Pro Ala Pro Gly Pro Phe Asp Ala Ile Val Arg Pro Leu
20 25 30
Ala Val Ala Pro Cys Thr Ser Asp Ile His Thr Val Phe Glu Gly Ala
35 40 45
Ile Gly Glu Arg His Asn Met Ile Leu Gly His Glu Ala Val Gly Glu
50 55 60
Val Val Glu Val Gly Ser Glu Val Lys Asp Phe Lys Pro Gly Asp Arg
65 70 75 80
Val Val Val Pro Ala Ile Thr Pro Asp Trp Arg Thr Ser Glu Val Gln
85 90 95
Arg Gly Tyr His Gln His Ser Gly Gly Met Leu Ala Gly Trp Lys Phe
100 105 110
Ser Asn Val Lys Asp Gly Val Phe Gly Glu Phe Phe His Val Asn Asp
115 120 125
Ala Asp Met Asn Leu Ala His Leu Pro Lys Glu Ser Pro Leu Glu Ala
130 135 140
Ala Val Met Ile Pro Asp Met Met Thr Thr Gly Phe His Gly Ala Glu
145 150 155 160
Leu Ala Asp Ile Glu Leu Gly Ala Thr Val Ala Val Leu Gly Ile Gly
165 170 175
Pro Val Gly Leu Met Ala Val Ala Gly Ala Lys Leu Arg Gly Ala Gly
180 185 190
Arg Ile Ile Ala Val Gly Ser Arg Pro Val Cys Val Asp Ala Ala Lys
195 200 205
Tyr Tyr Gly Ala Thr Asp Ile Val Asn Tyr Lys Asp Gly Pro Ile Glu
210 215 220
Ser Gln Ile Met Asn Leu Thr Glu Gly Lys Gly Val Asp Ala Ala Ile
225 230 235 240
Ile Ala Gly Gly Asn Ala Asp Ile Met Ala Thr Ala Val Lys Ile Val
245 250 255
Lys Pro Gly Gly Thr Ile Ala Asn Val Asn Tyr Phe Gly Glu Gly Glu
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Val Leu Pro Val Pro Arg Leu Glu Trp Gly Cys Gly Met Ala His Lys
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Thr Ile Lys Gly Gly Leu Cys Pro Gly Gly Arg Leu Arg Met Glu Arg
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Leu Ile Asp Leu Val Phe Tyr Lys Arg Val Asp Pro Ser Lys Leu Val
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Thr His Val Phe Arg Gly Phe Asp Asn Ile Glu Lys Ala Phe Met Leu
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Met Lys Asp Lys Pro Lys Asp Leu Ile Lys Pro Val Val Ile Leu Ala
340 345 350
<210> SEQ NO ID：3
<211> 352
<212> PRT
<213> I140S-D205E突变体
<400> 3
Met Lys Gly Phe Ala Met Leu Ser Ile Gly Lys Val Gly Trp Ile Glu
1 5 10 15
Lys Glu Lys Pro Ala Pro Gly Pro Phe Asp Ala Ile Val Arg Pro Leu
20 25 30
Ala Val Ala Pro Cys Thr Ser Asp Ile His Thr Val Phe Glu Gly Ala
35 40 45
Ile Gly Glu Arg His Asn Met Ile Leu Gly His Glu Ala Val Gly Glu
50 55 60
Val Val Glu Val Gly Ser Glu Val Lys Asp Phe Lys Pro Gly Asp Arg
65 70 75 80
Val Val Val Pro Ala Ile Thr Pro Asp Trp Arg Thr Ser Glu Val Gln
85 90 95
Arg Gly Tyr His Gln His Ser Gly Gly Met Leu Ala Gly Trp Lys Phe
100 105 110
Ser Asn Val Lys Asp Gly Val Phe Gly Glu Phe Phe His Val Asn Asp
115 120 125
Ala Asp Met Asn Leu Ala His Leu Pro Lys Glu Ser Pro Leu Glu Ala
130 135 140
Ala Val Met Ile Pro Asp Met Met Thr Thr Gly Phe His Gly Ala Glu
145 150 155 160
Leu Ala Asp Ile Glu Leu Gly Ala Thr Val Ala Val Leu Gly Ile Gly
165 170 175
Pro Val Gly Leu Met Ala Val Ala Gly Ala Lys Leu Arg Gly Ala Gly
180 185 190
Arg Ile Ile Ala Val Gly Ser Arg Pro Val Cys Val Glu Ala Ala Lys
195 200 205
Tyr Tyr Gly Ala Thr Asp Ile Val Asn Tyr Lys Asp Gly Pro Ile Glu
210 215 220
Ser Gln Ile Met Asn Leu Thr Glu Gly Lys Gly Val Asp Ala Ala Ile
225 230 235 240
Ile Ala Gly Gly Asn Ala Asp Ile Met Ala Thr Ala Val Lys Ile Val
245 250 255
Lys Pro Gly Gly Thr Ile Ala Asn Val Asn Tyr Phe Gly Glu Gly Glu
260 265 270
Val Leu Pro Val Pro Arg Leu Glu Trp Gly Cys Gly Met Ala His Lys
275 280 285
Thr Ile Lys Gly Gly Leu Cys Pro Gly Gly Arg Leu Arg Met Glu Arg
290 295 300
Leu Ile Asp Leu Val Phe Tyr Lys Arg Val Asp Pro Ser Lys Leu Val
305 310 315 320
Thr His Val Phe Arg Gly Phe Asp Asn Ile Glu Lys Ala Phe Met Leu
325 330 335
Met Lys Asp Lys Pro Lys Asp Leu Ile Lys Pro Val Val Ile Leu Ala
340 345 350
<210> SEQ NO ID：4
<211> 352
<212> PRT
<213> H125Q-I140S-N329S突变体
<400> 4
Met Lys Gly Phe Ala Met Leu Ser Ile Gly Lys Val Gly Trp Ile Glu
1 5 10 15
Lys Glu Lys Pro Ala Pro Gly Pro Phe Asp Ala Ile Val Arg Pro Leu
20 25 30
Ala Val Ala Pro Cys Thr Ser Asp Ile His Thr Val Phe Glu Gly Ala
35 40 45
Ile Gly Glu Arg His Asn Met Ile Leu Gly His Glu Ala Val Gly Glu
50 55 60
Val Val Glu Val Gly Ser Glu Val Lys Asp Phe Lys Pro Gly Asp Arg
65 70 75 80
Val Val Val Pro Ala Ile Thr Pro Asp Trp Arg Thr Ser Glu Val Gln
85 90 95
Arg Gly Tyr His Gln His Ser Gly Gly Met Leu Ala Gly Trp Lys Phe
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Ser Asn Val Lys Asp Gly Val Phe Gly Glu Phe Phe Gln Val Asn Asp
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Ala Asp Met Asn Leu Ala His Leu Pro Lys Glu Ser Pro Leu Glu Ala
130 135 140
Ala Val Met Ile Pro Asp Met Met Thr Thr Gly Phe His Gly Ala Glu
145 150 155 160
Leu Ala Asp Ile Glu Leu Gly Ala Thr Val Ala Val Leu Gly Ile Gly
165 170 175
Pro Val Gly Leu Met Ala Val Ala Gly Ala Lys Leu Arg Gly Ala Gly
180 185 190
Arg Ile Ile Ala Val Gly Ser Arg Pro Val Cys Val Asp Ala Ala Lys
195 200 205
Tyr Tyr Gly Ala Thr Asp Ile Val Asn Tyr Lys Asp Gly Pro Ile Glu
210 215 220
Ser Gln Ile Met Asn Leu Thr Glu Gly Lys Gly Val Asp Ala Ala Ile
225 230 235 240
Ile Ala Gly Gly Asn Ala Asp Ile Met Ala Thr Ala Val Lys Ile Val
245 250 255
Lys Pro Gly Gly Thr Ile Ala Asn Val Asn Tyr Phe Gly Glu Gly Glu
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Val Leu Pro Val Pro Arg Leu Glu Trp Gly Cys Gly Met Ala His Lys
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Thr Ile Lys Gly Gly Leu Cys Pro Gly Gly Arg Leu Arg Met Glu Arg
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Leu Ile Asp Leu Val Phe Tyr Lys Arg Val Asp Pro Ser Lys Leu Val
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Thr His Val Phe Arg Gly Phe Asp Ser Ile Glu Lys Ala Phe Met Leu
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Met Lys Asp Lys Pro Lys Asp Leu Ile Lys Pro Val Val Ile Leu Ala
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<210> SEQ NO ID：5
<211> 352
<212> PRT
<213> I140S-L294I突变体
<400> 5
Met Lys Gly Phe Ala Met Leu Ser Ile Gly Lys Val Gly Trp Ile Glu
1 5 10 15
Lys Glu Lys Pro Ala Pro Gly Pro Phe Asp Ala Ile Val Arg Pro Leu
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Ala Val Ala Pro Cys Thr Ser Asp Ile His Thr Val Phe Glu Gly Ala
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Ile Gly Glu Arg His Asn Met Ile Leu Gly His Glu Ala Val Gly Glu
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Val Val Glu Val Gly Ser Glu Val Lys Asp Phe Lys Pro Gly Asp Arg
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Val Val Val Pro Ala Ile Thr Pro Asp Trp Arg Thr Ser Glu Val Gln
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Arg Gly Tyr His Gln His Ser Gly Gly Met Leu Ala Gly Trp Lys Phe
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Ser Asn Val Lys Asp Gly Val Phe Gly Glu Phe Phe His Val Asn Asp
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Ala Asp Met Asn Leu Ala His Leu Pro Lys Glu Ser Pro Leu Glu Ala
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Ala Val Met Ile Pro Asp Met Met Thr Thr Gly Phe His Gly Ala Glu
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Leu Ala Asp Ile Glu Leu Gly Ala Thr Val Ala Val Leu Gly Ile Gly
165 170 175
Pro Val Gly Leu Met Ala Val Ala Gly Ala Lys Leu Arg Gly Ala Gly
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Arg Ile Ile Ala Val Gly Ser Arg Pro Val Cys Val Asp Ala Ala Lys
195 200 205
Tyr Tyr Gly Ala Thr Asp Ile Val Asn Tyr Lys Asp Gly Pro Ile Glu
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Ser Gln Ile Met Asn Leu Thr Glu Gly Lys Gly Val Asp Ala Ala Ile
225 230 235 240
Ile Ala Gly Gly Asn Ala Asp Ile Met Ala Thr Ala Val Lys Ile Val
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Lys Pro Gly Gly Thr Ile Ala Asn Val Asn Tyr Phe Gly Glu Gly Glu
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Val Leu Pro Val Pro Arg Leu Glu Trp Gly Cys Gly Met Ala His Lys
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Thr Ile Lys Gly Gly Lle Cys Pro Gly Gly Arg Leu Arg Met Glu Arg
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Leu Ile Asp Leu Val Phe Tyr Lys Arg Val Asp Pro Ser Lys Leu Val
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Thr His Val Phe Arg Gly Phe Asp Asn Ile Glu Lys Ala Phe Met Leu
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Met Lys Asp Lys Pro Lys Asp Leu Ile Lys Pro Val Val Ile Leu Ala
340 345 350
<210> SEQ NO ID：6
<211> 352
<212> PRT
<213> I140S-D150Q突变体
<400> 6
Met Lys Gly Phe Ala Met Leu Ser Ile Gly Lys Val Gly Trp Ile Glu
1 5 10 15
Lys Glu Lys Pro Ala Pro Gly Pro Phe Asp Ala Ile Val Arg Pro Leu
20 25 30
Ala Val Ala Pro Cys Thr Ser Asp Ile His Thr Val Phe Glu Gly Ala
35 40 45
Ile Gly Glu Arg His Asn Met Ile Leu Gly His Glu Ala Val Gly Glu
50 55 60
Val Val Glu Val Gly Ser Glu Val Lys Asp Phe Lys Pro Gly Asp Arg
65 70 75 80
Val Val Val Pro Ala Ile Thr Pro Asp Trp Arg Thr Ser Glu Val Gln
85 90 95
Arg Gly Tyr His Gln His Ser Gly Gly Met Leu Ala Gly Trp Lys Phe
100 105 110
Ser Asn Val Lys Asp Gly Val Phe Gly Glu Phe Phe His Val Asn Asp
115 120 125
Ala Asp Met Asn Leu Ala His Leu Pro Lys Glu Ser Pro Leu Glu Ala
130 135 140
Ala Val Met Ile Pro Glu Met Met Thr Thr Gly Phe His Gly Ala Glu
145 150 155 160
Leu Ala Asp Ile Glu Leu Gly Ala Thr Val Ala Val Leu Gly Ile Gly
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Pro Val Gly Leu Met Ala Val Ala Gly Ala Lys Leu Arg Gly Ala Gly
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Arg Ile Ile Ala Val Gly Ser Arg Pro Val Cys Val Asp Ala Ala Lys
195 200 205
Tyr Tyr Gly Ala Thr Asp Ile Val Asn Tyr Lys Asp Gly Pro Ile Glu
210 215 220
Ser Gln Ile Met Asn Leu Thr Glu Gly Lys Gly Val Asp Ala Ala Ile
225 230 235 240
Ile Ala Gly Gly Asn Ala Asp Ile Met Ala Thr Ala Val Lys Ile Val
245 250 255
Lys Pro Gly Gly Thr Ile Ala Asn Val Asn Tyr Phe Gly Glu Gly Glu
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Val Leu Pro Val Pro Arg Leu Glu Trp Gly Cys Gly Met Ala His Lys
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Thr Ile Lys Gly Gly Leu Cys Pro Gly Gly Arg Leu Arg Met Glu Arg
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Leu Ile Asp Leu Val Phe Tyr Lys Arg Val Asp Pro Ser Lys Leu Val
305 310 315 320
Thr His Val Phe Arg Gly Phe Asp Asn Ile Glu Lys Ala Phe Met Leu
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Met Lys Asp Lys Pro Lys Asp Leu Ile Lys Pro Val Val Ile Leu Ala
340 345 350
<210> SEQ NO ID：7
<211> 352
<212> PRT
<213> P36S-I140S突变体
<400> 7
Met Lys Gly Phe Ala Met Leu Ser Ile Gly Lys Val Gly Trp Ile Glu
1 5 10 15
Lys Glu Lys Pro Ala Pro Gly Pro Phe Asp Ala Ile Val Arg Pro Leu
20 25 30
Ala Val Ala Ser Cys Thr Ser Asp Ile His Thr Val Phe Glu Gly Ala
35 40 45
Ile Gly Glu Arg His Asn Met Ile Leu Gly His Glu Ala Val Gly Glu
50 55 60
Val Val Glu Val Gly Ser Glu Val Lys Asp Phe Lys Pro Gly Asp Arg
65 70 75 80
Val Val Val Pro Ala Ile Thr Pro Asp Trp Arg Thr Ser Glu Val Gln
85 90 95
Arg Gly Tyr His Gln His Ser Gly Gly Met Leu Ala Gly Trp Lys Phe
100 105 110
Ser Asn Val Lys Asp Gly Val Phe Gly Glu Phe Phe His Val Asn Asp
115 120 125
Ala Asp Met Asn Leu Ala His Leu Pro Lys Glu Ser Pro Leu Glu Ala
130 135 140
Ala Val Met Ile Pro Asp Met Met Thr Thr Gly Phe His Gly Ala Glu
145 150 155 160
Leu Ala Asp Ile Glu Leu Gly Ala Thr Val Ala Val Leu Gly Ile Gly
165 170 175
Pro Val Gly Leu Met Ala Val Ala Gly Ala Lys Leu Arg Gly Ala Gly
180 185 190
Arg Ile Ile Ala Val Gly Ser Arg Pro Val Cys Val Asp Ala Ala Lys
195 200 205
Tyr Tyr Gly Ala Thr Asp Ile Val Asn Tyr Lys Asp Gly Pro Ile Glu
210 215 220
Ser Gln Ile Met Asn Leu Thr Glu Gly Lys Gly Val Asp Ala Ala Ile
225 230 235 240
Ile Ala Gly Gly Asn Ala Asp Ile Met Ala Thr Ala Val Lys Ile Val
245 250 255
Lys Pro Gly Gly Thr Ile Ala Asn Val Asn Tyr Phe Gly Glu Gly Glu
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Val Leu Pro Val Pro Arg Leu Glu Trp Gly Cys Gly Met Ala His Lys
275 280 285
Thr Ile Lys Gly Gly Leu Cys Pro Gly Gly Arg Leu Arg Met Glu Arg
290 295 300
Leu Ile Asp Leu Val Phe Tyr Lys Arg Val Asp Pro Ser Lys Leu Val
305 310 315 320
Thr His Val Phe Arg Gly Phe Asp Asn Ile Glu Lys Ala Phe Met Leu
325 330 335
Met Lys Asp Lys Pro Lys Asp Leu Ile Lys Pro Val Val Ile Leu Ala
340 345 350
<210> 8
<211> 1059
<212> DNA
<213> Thermoanaerobacter brockii野生型
<400> 8
atgaaaggtt ttgcaatgct cagtatcggt aaagttggct ggattgagaa ggaaaagcct 60
gctcctggcc catttgatgc tattgtaaga cctctagctg tggccccttg cacttcggac 120
attcataccg tttttgaagg cgccattggc gaaagacata acatgatact cggtcacgaa 180
gctgtaggtg aagtagttga agtaggtagt gaggtaaaag attttaaacc tggtgatcgc 240
gttgttgtgc cagctattac ccctgattgg cggacctctg aagtacaaag aggatatcac 300
cagcactccg gtggaatgct ggcaggctgg aaattttcga atgtaaaaga tggtgttttt 360
ggtgaatttt ttcatgtgaa tgatgctgat atgaatttag cacatctgcc taaagaaatt 420
ccattggaag ctgcagttat gattcccgat atgatgacca ctggttttca cggagctgaa 480
ctggcagata tagaattagg tgcgacggta gcagttttgg gtattggccc agtaggtctt 540
atggcagtcg ctggtgccaa attgcgtgga gccggaagaa ttattgccgt aggcagtaga 600
ccagtttgtg tagatgctgc aaaatactat ggagctactg atattgtaaa ctataaagat 660
ggtcctatcg aaagtcagat tatgaatcta actgaaggca aaggtgtcga tgctgccatc 720
atcgctggag gaaatgctga cattatggct acagcagtta agattgttaa acctggtggc 780
accatcgcta atgtaaatta ttttggcgaa ggagaggttt tgcctgttcc tcgtcttgaa 840
tggggttgcg gcatggctca taaaactata aaaggcgggc tatgccccgg tggacgtcta 900
agaatggaaa gactgattga ccttgttttt tataagcgtg tcgatccttc taagctcgtc 960
actcacgttt tccggggatt tgacaatatt gaaaaagcct ttatgttgat gaaagacaaa 1020
ccaaaagacc taatcaaacc tgttgtaata ttagcataa 1059
<210> 9
<211> 1059
<212> DNA
<213> I140S突变体
<400> 9
atgaaaggtt ttgcaatgct cagtatcggt aaagttggct ggattgagaa ggaaaagcct 60
gctcctggcc catttgatgc tattgtaaga cctctagctg tggccccttg cacttcggac 120
attcataccg tttttgaagg cgccattggc gaaagacata acatgatact cggtcacgaa 180
gctgtaggtg aagtagttga agtaggtagt gaggtaaaag attttaaacc tggtgatcgc 240
gttgttgtgc cagctattac ccctgattgg cggacctctg aagtacaaag aggatatcac 300
cagcactccg gtggaatgct ggcaggctgg aaattttcga atgtaaaaga tggtgttttt 360
ggtgaatttt ttcatgtgaa tgatgctgat atgaatttag cacatctgcc taaagaaagt 420
ccattggaag ctgcagttat gattcccgat atgatgacca ctggttttca cggagctgaa 480
ctggcagata tagaattagg tgcgacggta gcagttttgg gtattggccc agtaggtctt 540
atggcagtcg ctggtgccaa attgcgtgga gccggaagaa ttattgccgt aggcagtaga 600
ccagtttgtg tagatgctgc aaaatactat ggagctactg atattgtaaa ctataaagat 660
ggtcctatcg aaagtcagat tatgaatcta actgaaggca aaggtgtcga tgctgccatc 720
atcgctggag gaaatgctga cattatggct acagcagtta agattgttaa acctggtggc 780
accatcgcta atgtaaatta ttttggcgaa ggagaggttt tgcctgttcc tcgtcttgaa 840
tggggttgcg gcatggctca taaaactata aaaggcgggc tatgccccgg tggacgtcta 900
agaatggaaa gactgattga ccttgttttt tataagcgtg tcgatccttc taagctcgtc 960
actcacgttt tccggggatt tgacaatatt gaaaaagcct ttatgttgat gaaagacaaa 1020
ccaaaagacc taatcaaacc tgttgtaata ttagcataa 1059
<210> 10
<211> 1059
<212> DNA
<213> I140S-D205E突变体
<400> 10
atgaaaggtt ttgcaatgct cagtatcggt aaagttggct ggattgagaa ggaaaagcct 60
gctcctggcc catttgatgc tattgtaaga cctctagctg tggccccttg cacttcggac 120
attcataccg tttttgaagg cgccattggc gaaagacata acatgatact cggtcacgaa 180
gctgtaggtg aagtagttga agtaggtagt gaggtaaaag attttaaacc tggtgatcgc 240
gttgttgtgc cagctattac ccctgattgg cggacctctg aagtacaaag aggatatcac 300
cagcactccg gtggaatgct ggcaggctgg aaattttcga atgtaaaaga tggtgttttt 360
ggtgaatttt ttcatgtgaa tgatgctgat atgaatttag cacatctgcc taaagaaagt 420
ccattggaag ctgcagttat gattcccgat atgatgacca ctggttttca cggagctgaa 480
ctggcagata tagaattagg tgcgacggta gcagttttgg gtattggccc agtaggtctt 540
atggcagtcg ctggtgccaa attgcgtgga gccggaagaa ttattgccgt aggcagtaga 600
ccagtttgtg tagaagctgc aaaatactat ggagctactg atattgtaaa ctataaagat 660
ggtcctatcg aaagtcagat tatgaatcta actgaaggca aaggtgtcga tgctgccatc 720
atcgctggag gaaatgctga cattatggct acagcagtta agattgttaa acctggtggc 780
accatcgcta atgtaaatta ttttggcgaa ggagaggttt tgcctgttcc tcgtcttgaa 840
tggggttgcg gcatggctca taaaactata aaaggcgggc tatgccccgg tggacgtcta 900
agaatggaaa gactgattga ccttgttttt tataagcgtg tcgatccttc taagctcgtc 960
actcacgttt tccggggatt tgacaatatt gaaaaagcct ttatgttgat gaaagacaaa 1020
ccaaaagacc taatcaaacc tgttgtaata ttagcataa 1059
<210> 11
<211> 1059
<212> DNA
<213> H125Q-I140S-N329S突变体
<400> 11
atgaaaggtt ttgcaatgct cagtatcggt aaagttggct ggattgagaa ggaaaagcct 60
gctcctggcc catttgatgc tattgtaaga cctctagctg tggccccttg cacttcggac 120
attcataccg tttttgaagg cgccattggc gaaagacata acatgatact cggtcacgaa 180
gctgtaggtg aagtagttga agtaggtagt gaggtaaaag attttaaacc tggtgatcgc 240
gttgttgtgc cagctattac ccctgattgg cggacctctg aagtacaaag aggatatcac 300
cagcactccg gtggaatgct ggcaggctgg aaattttcga atgtaaaaga tggtgttttt 360
ggtgaatttt ttcaagtgaa tgatgctgat atgaatttag cacatctgcc taaagaaagt 420
ccattggaag ctgcagttat gattcccgat atgatgacca ctggttttca cggagctgaa 480
ctggcagata tagaattagg tgcgacggta gcagttttgg gtattggccc agtaggtctt 540
atggcagtcg ctggtgccaa attgcgtgga gccggaagaa ttattgccgt aggcagtaga 600
ccagtttgtg tagatgctgc aaaatactat ggagctactg atattgtaaa ctataaagat 660
ggtcctatcg aaagtcagat tatgaatcta actgaaggca aaggtgtcga tgctgccatc 720
atcgctggag gaaatgctga cattatggct acagcagtta agattgttaa acctggtggc 780
accatcgcta atgtaaatta ttttggcgaa ggagaggttt tgcctgttcc tcgtcttgaa 840
tggggttgcg gcatggctca taaaactata aaaggcgggc tatgccccgg tggacgtcta 900
agaatggaaa gactgattga ccttgttttt tataagcgtg tcgatccttc taagctcgtc 960
actcacgttt tccggggatt tgacagtatt gaaaaagcct ttatgttgat gaaagacaaa 1020
ccaaaagacc taatcaaacc tgttgtaata ttagcataa 1059
<210> 12
<211> 1059
<212> DNA
<213> I140S-L294I突变体
<400> 12
atgaaaggtt ttgcaatgct cagtatcggt aaagttggct ggattgagaa ggaaaagcct 60
gctcctggcc catttgatgc tattgtaaga cctctagctg tggccccttg cacttcggac 120
attcataccg tttttgaagg cgccattggc gaaagacata acatgatact cggtcacgaa 180
gctgtaggtg aagtagttga agtaggtagt gaggtaaaag attttaaacc tggtgatcgc 240
gttgttgtgc cagctattac ccctgattgg cggacctctg aagtacaaag aggatatcac 300
cagcactccg gtggaatgct ggcaggctgg aaattttcga atgtaaaaga tggtgttttt 360
ggtgaatttt ttcatgtgaa tgatgctgat atgaatttag cacatctgcc taaagaaagc 420
ccattggaag ctgcagttat gattcccgat atgatgacca ctggttttca cggagctgaa 480
ctggcagata tagaattagg tgcgacggta gcagttttgg gtattggccc agtaggtctt 540
atggcagtcg ctggtgccaa attgcgtgga gccggaagaa ttattgccgt aggcagtaga 600
ccagtttgtg tagatgctgc aaaatactat ggagctactg atattgtaaa ctataaagat 660
ggtcctatcg aaagtcagat tatgaatcta actgaaggca aaggtgtcga tgctgccatc 720
atcgctggag gaaatgctga cattatggct acagcagtta agattgttaa acctggtggc 780
accatcgcta atgtaaatta ttttggcgaa ggagaggttt tgcctgttcc tcgtcttgaa 840
tggggttgcg gcatggctca taaaactata aaaggcggga tatgccccgg tggacgtcta 900
agaatggaaa gactgattga ccttgttttt tataagcgtg tcgatccttc taagctcgtc 960
actcacgttt tccggggatt tgacaatatt gaaaaagcct ttatgttgat gaaagacaaa 1020
ccaaaagacc taatcaaacc tgttgtaata ttagcataa 1059
<210> 13
<211> 1059
<212> DNA
<213> I140S-D150Q突变体
<400> 13
atgaaaggtt ttgcaatgct cagtatcggt aaagttggct ggattgagaa ggaaaagcct 60
gctcctggcc catttgatgc tattgtaaga cctctagctg tggccccttg cacttcggac 120
attcataccg tttttgaagg cgccattggc gaaagacata acatgatact cggtcacgaa 180
gctgtaggtg aagtagttga agtaggtagt gaggtaaaag attttaaacc tggtgatcgc 240
gttgttgtgc cagctattac ccctgattgg cggacctctg aagtacaaag aggatatcac 300
cagcactccg gtggaatgct ggcaggctgg aaattttcga atgtaaaaga tggtgttttt 360
ggtgaatttt ttcatgtgaa tgatgctgat atgaatttag cacatctgcc taaagaaagt 420
ccattggaag ctgcagttat gattccccaa atgatgacca ctggttttca cggagctgaa 480
ctggcagata tagaattagg tgcgacggta gcagttttgg gtattggccc agtaggtctt 540
atggcagtcg ctggtgccaa attgcgtgga gccggaagaa ttattgccgt aggcagtaga 600
ccagtttgtg tagatgctgc aaaatactat ggagctactg atattgtaaa ctataaagat 660
ggtcctatcg aaagtcagat tatgaatcta actgaaggca aaggtgtcga tgctgccatc 720
atcgctggag gaaatgctga cattatggct acagcagtta agattgttaa acctggtggc 780
accatcgcta atgtaaatta ttttggcgaa ggagaggttt tgcctgttcc tcgtcttgaa 840
tggggttgcg gcatggctca taaaactata aaaggcgggc tatgccccgg tggacgtcta 900
agaatggaaa gactgattga ccttgttttt tataagcgtg tcgatccttc taagctcgtc 960
actcacgttt tccggggatt tgacaatatt gaaaaagcct ttatgttgat gaaagacaaa 1020
ccaaaagacc taatcaaacc tgttgtaata ttagcataa 1059
<210> 14
<211> 1059
<212> DNA
<213> P36S-I140S突变体
<400> 14
atgaaaggtt ttgcaatgct cagtatcggt aaagttggct ggattgagaa ggaaaagcct 60
gctcctggcc catttgatgc tattgtaaga cctctagctg tggcctcttg cacttcggac 120
attcataccg tttttgaagg cgccattggc gaaagacata acatgatact cggtcacgaa 180
gctgtaggtg aagtagttga agtaggtagt gaggtaaaag attttaaacc tggtgatcgc 240
gttgttgtgc cagctattac ccctgattgg cggacctctg aagtacaaag aggatatcac 300
cagcactccg gtggaatgct ggcaggctgg aaattttcga atgtaaaaga tggtgttttt 360
ggtgaatttt ttcatgtgaa tgatgctgat atgaatttag cacatctgcc taaagaaagc 420
ccattggaag ctgcagttat gattcccgat atgatgacca ctggttttca cggagctgaa 480
ctggcagata tagaattagg tgcgacggta gcagttttgg gtattggccc agtaggtctt 540
atggcagtcg ctggtgccaa attgcgtgga gccggaagaa ttattgccgt aggcagtaga 600
ccagtttgtg tagatgctgc aaaatactat ggagctactg atattgtaaa ctataaagat 660
ggtcctatcg aaagtcagat tatgaatcta actgaaggca aaggtgtcga tgctgccatc 720
atcgctggag gaaatgctga cattatggct acagcagtta agattgttaa acctggtggc 780
accatcgcta atgtaaatta ttttggcgaa ggagaggttt tgcctgttcc tcgtcttgaa 840
tggggttgcg gcatggctca taaaactata aaaggcgggc tatgccccgg tggacgtcta 900
agaatggaaa gactgattga ccttgttttt tataagcgtg tcgatccttc taagctcgtc 960
actcacgttt tccggggatt tgacaatatt gaaaaagcct ttatgttgat gaaagacaaa 1020
ccaaaagacc taatcaaacc tgttgtaata ttagcataa 1059 。
序列表
<110> 迪沙药业集团（天津）药物研究有限公司
迪沙药业集团有限公司
<120> 一种改进的酮还原酶及其应用
<141> 2018-07-10
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 352
<212> PRT
<213> 野生型(Thermoanaerobacter brockii)
<400> 1
Met Leu Gly Pro Ala Met Leu Ser Ile Gly Leu Val Gly Thr Ile Gly
1 5 10 15
Leu Gly Leu Pro Ala Pro Gly Pro Pro Ala Ala Ile Val Ala Pro Leu
20 25 30
Ala Val Ala Pro Cys Thr Ser Ala Ile His Thr Val Pro Gly Gly Ala
35 40 45
Ile Gly Gly Ala His Ala Met Ile Leu Gly His Gly Ala Val Gly Gly
50 55 60
Val Val Gly Val Gly Ser Gly Val Leu Ala Pro Leu Pro Gly Ala Ala
65 70 75 80
Val Val Val Pro Ala Ile Thr Pro Ala Thr Ala Thr Ser Gly Val Gly
85 90 95
Ala Gly Thr His Gly His Ser Gly Gly Met Leu Ala Gly Thr Leu Pro
100 105 110
Ser Ala Val Leu Ala Gly Val Pro Gly Gly Pro Pro His Val Ala Ala
115 120 125
Ala Ala Met Ala Leu Ala His Leu Pro Leu Gly Ile Pro Leu Gly Ala
130 135 140
Ala Val Met Ile Pro Ala Met Met Thr Thr Gly Pro His Gly Ala Gly
145 150 155 160
Leu Ala Ala Ile Gly Leu Gly Ala Thr Val Ala Val Leu Gly Ile Gly
165 170 175
Pro Val Gly Leu Met Ala Val Ala Gly Ala Leu Leu Ala Gly Ala Gly
180 185 190
Ala Ile Ile Ala Val Gly Ser Ala Pro Val Cys Val Ala Ala Ala Leu
195 200 205
Thr Thr Gly Ala Thr Ala Ile Val Ala Thr Leu Ala Gly Pro Ile Gly
210 215 220
Ser Gly Ile Met Ala Leu Thr Gly Gly Leu Gly Val Ala Ala Ala Ile
225 230 235 240
Ile Ala Gly Gly Ala Ala Ala Ile Met Ala Thr Ala Val Leu Ile Val
245 250 255
Leu Pro Gly Gly Thr Ile Ala Ala Val Ala Thr Pro Gly Gly Gly Gly
260 265 270
Val Leu Pro Val Pro Ala Leu Gly Thr Gly Cys Gly Met Ala His Leu
275 280 285
Thr Ile Leu Gly Gly Leu Cys Pro Gly Gly Ala Leu Ala Met Gly Ala
290 295 300
Leu Ile Ala Leu Val Pro Thr Leu Ala Val Ala Pro Ser Leu Leu Val
305 310 315 320
Thr His Val Pro Ala Gly Pro Ala Ala Ile Gly Leu Ala Pro Met Leu
325 330 335
Met Leu Ala Leu Pro Leu Ala Leu Ile Leu Pro Val Val Ile Leu Ala
340 345 350
<210> 2
<211> 352
<212> PRT
<213> I140S突变体(Thermoanaerobacter brockii)
<400> 2
Met Leu Gly Pro Ala Met Leu Ser Ile Gly Leu Val Gly Thr Ile Gly
1 5 10 15
Leu Gly Leu Pro Ala Pro Gly Pro Pro Ala Ala Ile Val Ala Pro Leu
20 25 30
Ala Val Ala Pro Cys Thr Ser Ala Ile His Thr Val Pro Gly Gly Ala
35 40 45
Ile Gly Gly Ala His Ala Met Ile Leu Gly His Gly Ala Val Gly Gly
50 55 60
Val Val Gly Val Gly Ser Gly Val Leu Ala Pro Leu Pro Gly Ala Ala
65 70 75 80
Val Val Val Pro Ala Ile Thr Pro Ala Thr Ala Thr Ser Gly Val Gly
85 90 95
Ala Gly Thr His Gly His Ser Gly Gly Met Leu Ala Gly Thr Leu Pro
100 105 110
Ser Ala Val Leu Ala Gly Val Pro Gly Gly Pro Pro His Val Ala Ala
115 120 125
Ala Ala Met Ala Leu Ala His Leu Pro Leu Gly Ser Pro Leu Gly Ala
130 135 140
Ala Val Met Ile Pro Ala Met Met Thr Thr Gly Pro His Gly Ala Gly
145 150 155 160
Leu Ala Ala Ile Gly Leu Gly Ala Thr Val Ala Val Leu Gly Ile Gly
165 170 175
Pro Val Gly Leu Met Ala Val Ala Gly Ala Leu Leu Ala Gly Ala Gly
180 185 190
Ala Ile Ile Ala Val Gly Ser Ala Pro Val Cys Val Ala Ala Ala Leu
195 200 205
Thr Thr Gly Ala Thr Ala Ile Val Ala Thr Leu Ala Gly Pro Ile Gly
210 215 220
Ser Gly Ile Met Ala Leu Thr Gly Gly Leu Gly Val Ala Ala Ala Ile
225 230 235 240
Ile Ala Gly Gly Ala Ala Ala Ile Met Ala Thr Ala Val Leu Ile Val
245 250 255
Leu Pro Gly Gly Thr Ile Ala Ala Val Ala Thr Pro Gly Gly Gly Gly
260 265 270
Val Leu Pro Val Pro Ala Leu Gly Thr Gly Cys Gly Met Ala His Leu
275 280 285
Thr Ile Leu Gly Gly Leu Cys Pro Gly Gly Ala Leu Ala Met Gly Ala
290 295 300
Leu Ile Ala Leu Val Pro Thr Leu Ala Val Ala Pro Ser Leu Leu Val
305 310 315 320
Thr His Val Pro Ala Gly Pro Ala Ala Ile Gly Leu Ala Pro Met Leu
325 330 335
Met Leu Ala Leu Pro Leu Ala Leu Ile Leu Pro Val Val Ile Leu Ala
340 345 350
<210> 3
<211> 352
<212> PRT
<213> I140S-D205E突变体(Thermoanaerobacter brockii)
<400> 3
Met Leu Gly Pro Ala Met Leu Ser Ile Gly Leu Val Gly Thr Ile Gly
1 5 10 15
Leu Gly Leu Pro Ala Pro Gly Pro Pro Ala Ala Ile Val Ala Pro Leu
20 25 30
Ala Val Ala Pro Cys Thr Ser Ala Ile His Thr Val Pro Gly Gly Ala
35 40 45
Ile Gly Gly Ala His Ala Met Ile Leu Gly His Gly Ala Val Gly Gly
50 55 60
Val Val Gly Val Gly Ser Gly Val Leu Ala Pro Leu Pro Gly Ala Ala
65 70 75 80
Val Val Val Pro Ala Ile Thr Pro Ala Thr Ala Thr Ser Gly Val Gly
85 90 95
Ala Gly Thr His Gly His Ser Gly Gly Met Leu Ala Gly Thr Leu Pro
100 105 110
Ser Ala Val Leu Ala Gly Val Pro Gly Gly Pro Pro His Val Ala Ala
115 120 125
Ala Ala Met Ala Leu Ala His Leu Pro Leu Gly Ser Pro Leu Gly Ala
130 135 140
Ala Val Met Ile Pro Ala Met Met Thr Thr Gly Pro His Gly Ala Gly
145 150 155 160
Leu Ala Ala Ile Gly Leu Gly Ala Thr Val Ala Val Leu Gly Ile Gly
165 170 175
Pro Val Gly Leu Met Ala Val Ala Gly Ala Leu Leu Ala Gly Ala Gly
180 185 190
Ala Ile Ile Ala Val Gly Ser Ala Pro Val Cys Val Gly Ala Ala Leu
195 200 205
Thr Thr Gly Ala Thr Ala Ile Val Ala Thr Leu Ala Gly Pro Ile Gly
210 215 220
Ser Gly Ile Met Ala Leu Thr Gly Gly Leu Gly Val Ala Ala Ala Ile
225 230 235 240
Ile Ala Gly Gly Ala Ala Ala Ile Met Ala Thr Ala Val Leu Ile Val
245 250 255
Leu Pro Gly Gly Thr Ile Ala Ala Val Ala Thr Pro Gly Gly Gly Gly
260 265 270
Val Leu Pro Val Pro Ala Leu Gly Thr Gly Cys Gly Met Ala His Leu
275 280 285
Thr Ile Leu Gly Gly Leu Cys Pro Gly Gly Ala Leu Ala Met Gly Ala
290 295 300
Leu Ile Ala Leu Val Pro Thr Leu Ala Val Ala Pro Ser Leu Leu Val
305 310 315 320
Thr His Val Pro Ala Gly Pro Ala Ala Ile Gly Leu Ala Pro Met Leu
325 330 335
Met Leu Ala Leu Pro Leu Ala Leu Ile Leu Pro Val Val Ile Leu Ala
340 345 350
<210> 4
<211> 352
<212> PRT
<213> H125Q-I140S-N329S突变体(Thermoanaerobacter brockii)
<400> 4
Met Leu Gly Pro Ala Met Leu Ser Ile Gly Leu Val Gly Thr Ile Gly
1 5 10 15
Leu Gly Leu Pro Ala Pro Gly Pro Pro Ala Ala Ile Val Ala Pro Leu
20 25 30
Ala Val Ala Pro Cys Thr Ser Ala Ile His Thr Val Pro Gly Gly Ala
35 40 45
Ile Gly Gly Ala His Ala Met Ile Leu Gly His Gly Ala Val Gly Gly
50 55 60
Val Val Gly Val Gly Ser Gly Val Leu Ala Pro Leu Pro Gly Ala Ala
65 70 75 80
Val Val Val Pro Ala Ile Thr Pro Ala Thr Ala Thr Ser Gly Val Gly
85 90 95
Ala Gly Thr His Gly His Ser Gly Gly Met Leu Ala Gly Thr Leu Pro
100 105 110
Ser Ala Val Leu Ala Gly Val Pro Gly Gly Pro Pro Gly Val Ala Ala
115 120 125
Ala Ala Met Ala Leu Ala His Leu Pro Leu Gly Ser Pro Leu Gly Ala
130 135 140
Ala Val Met Ile Pro Ala Met Met Thr Thr Gly Pro His Gly Ala Gly
145 150 155 160
Leu Ala Ala Ile Gly Leu Gly Ala Thr Val Ala Val Leu Gly Ile Gly
165 170 175
Pro Val Gly Leu Met Ala Val Ala Gly Ala Leu Leu Ala Gly Ala Gly
180 185 190
Ala Ile Ile Ala Val Gly Ser Ala Pro Val Cys Val Ala Ala Ala Leu
195 200 205
Thr Thr Gly Ala Thr Ala Ile Val Ala Thr Leu Ala Gly Pro Ile Gly
210 215 220
Ser Gly Ile Met Ala Leu Thr Gly Gly Leu Gly Val Ala Ala Ala Ile
225 230 235 240
Ile Ala Gly Gly Ala Ala Ala Ile Met Ala Thr Ala Val Leu Ile Val
245 250 255
Leu Pro Gly Gly Thr Ile Ala Ala Val Ala Thr Pro Gly Gly Gly Gly
260 265 270
Val Leu Pro Val Pro Ala Leu Gly Thr Gly Cys Gly Met Ala His Leu
275 280 285
Thr Ile Leu Gly Gly Leu Cys Pro Gly Gly Ala Leu Ala Met Gly Ala
290 295 300
Leu Ile Ala Leu Val Pro Thr Leu Ala Val Ala Pro Ser Leu Leu Val
305 310 315 320
Thr His Val Pro Ala Gly Pro Ala Ser Ile Gly Leu Ala Pro Met Leu
325 330 335
Met Leu Ala Leu Pro Leu Ala Leu Ile Leu Pro Val Val Ile Leu Ala
340 345 350
<210> 5
<211> 352
<212> PRT
<213> I140S-L294I突变体(Thermoanaerobacter brockii)
<400> 5
Met Leu Gly Pro Ala Met Leu Ser Ile Gly Leu Val Gly Thr Ile Gly
1 5 10 15
Leu Gly Leu Pro Ala Pro Gly Pro Pro Ala Ala Ile Val Ala Pro Leu
20 25 30
Ala Val Ala Pro Cys Thr Ser Ala Ile His Thr Val Pro Gly Gly Ala
35 40 45
Ile Gly Gly Ala His Ala Met Ile Leu Gly His Gly Ala Val Gly Gly
50 55 60
Val Val Gly Val Gly Ser Gly Val Leu Ala Pro Leu Pro Gly Ala Ala
65 70 75 80
Val Val Val Pro Ala Ile Thr Pro Ala Thr Ala Thr Ser Gly Val Gly
85 90 95
Ala Gly Thr His Gly His Ser Gly Gly Met Leu Ala Gly Thr Leu Pro
100 105 110
Ser Ala Val Leu Ala Gly Val Pro Gly Gly Pro Pro His Val Ala Ala
115 120 125
Ala Ala Met Ala Leu Ala His Leu Pro Leu Gly Ser Pro Leu Gly Ala
130 135 140
Ala Val Met Ile Pro Ala Met Met Thr Thr Gly Pro His Gly Ala Gly
145 150 155 160
Leu Ala Ala Ile Gly Leu Gly Ala Thr Val Ala Val Leu Gly Ile Gly
165 170 175
Pro Val Gly Leu Met Ala Val Ala Gly Ala Leu Leu Ala Gly Ala Gly
180 185 190
Ala Ile Ile Ala Val Gly Ser Ala Pro Val Cys Val Ala Ala Ala Leu
195 200 205
Thr Thr Gly Ala Thr Ala Ile Val Ala Thr Leu Ala Gly Pro Ile Gly
210 215 220
Ser Gly Ile Met Ala Leu Thr Gly Gly Leu Gly Val Ala Ala Ala Ile
225 230 235 240
Ile Ala Gly Gly Ala Ala Ala Ile Met Ala Thr Ala Val Leu Ile Val
245 250 255
Leu Pro Gly Gly Thr Ile Ala Ala Val Ala Thr Pro Gly Gly Gly Gly
260 265 270
Val Leu Pro Val Pro Ala Leu Gly Thr Gly Cys Gly Met Ala His Leu
275 280 285
Thr Ile Leu Gly Gly Ile Cys Pro Gly Gly Ala Leu Ala Met Gly Ala
290 295 300
Leu Ile Ala Leu Val Pro Thr Leu Ala Val Ala Pro Ser Leu Leu Val
305 310 315 320
Thr His Val Pro Ala Gly Pro Ala Ala Ile Gly Leu Ala Pro Met Leu
325 330 335
Met Leu Ala Leu Pro Leu Ala Leu Ile Leu Pro Val Val Ile Leu Ala
340 345 350
<210> 6
<211> 352
<212> PRT
<213> I140S-D150Q突变体(Thermoanaerobacter brockii)
<400> 6
Met Leu Gly Pro Ala Met Leu Ser Ile Gly Leu Val Gly Thr Ile Gly
1 5 10 15
Leu Gly Leu Pro Ala Pro Gly Pro Pro Ala Ala Ile Val Ala Pro Leu
20 25 30
Ala Val Ala Pro Cys Thr Ser Ala Ile His Thr Val Pro Gly Gly Ala
35 40 45
Ile Gly Gly Ala His Ala Met Ile Leu Gly His Gly Ala Val Gly Gly
50 55 60
Val Val Gly Val Gly Ser Gly Val Leu Ala Pro Leu Pro Gly Ala Ala
65 70 75 80
Val Val Val Pro Ala Ile Thr Pro Ala Thr Ala Thr Ser Gly Val Gly
85 90 95
Ala Gly Thr His Gly His Ser Gly Gly Met Leu Ala Gly Thr Leu Pro
100 105 110
Ser Ala Val Leu Ala Gly Val Pro Gly Gly Pro Pro His Val Ala Ala
115 120 125
Ala Ala Met Ala Leu Ala His Leu Pro Leu Gly Ser Pro Leu Gly Ala
130 135 140
Ala Val Met Ile Pro Gly Met Met Thr Thr Gly Pro His Gly Ala Gly
145 150 155 160
Leu Ala Ala Ile Gly Leu Gly Ala Thr Val Ala Val Leu Gly Ile Gly
165 170 175
Pro Val Gly Leu Met Ala Val Ala Gly Ala Leu Leu Ala Gly Ala Gly
180 185 190
Ala Ile Ile Ala Val Gly Ser Ala Pro Val Cys Val Ala Ala Ala Leu
195 200 205
Thr Thr Gly Ala Thr Ala Ile Val Ala Thr Leu Ala Gly Pro Ile Gly
210 215 220
Ser Gly Ile Met Ala Leu Thr Gly Gly Leu Gly Val Ala Ala Ala Ile
225 230 235 240
Ile Ala Gly Gly Ala Ala Ala Ile Met Ala Thr Ala Val Leu Ile Val
245 250 255
Leu Pro Gly Gly Thr Ile Ala Ala Val Ala Thr Pro Gly Gly Gly Gly
260 265 270
Val Leu Pro Val Pro Ala Leu Gly Thr Gly Cys Gly Met Ala His Leu
275 280 285
Thr Ile Leu Gly Gly Leu Cys Pro Gly Gly Ala Leu Ala Met Gly Ala
290 295 300
Leu Ile Ala Leu Val Pro Thr Leu Ala Val Ala Pro Ser Leu Leu Val
305 310 315 320
Thr His Val Pro Ala Gly Pro Ala Ala Ile Gly Leu Ala Pro Met Leu
325 330 335
Met Leu Ala Leu Pro Leu Ala Leu Ile Leu Pro Val Val Ile Leu Ala
340 345 350
<210> 7
<211> 352
<212> PRT
<213> P36S-I140S突变体(Thermoanaerobacter brockii)
<400> 7
Met Leu Gly Pro Ala Met Leu Ser Ile Gly Leu Val Gly Thr Ile Gly
1 5 10 15
Leu Gly Leu Pro Ala Pro Gly Pro Pro Ala Ala Ile Val Ala Pro Leu
20 25 30
Ala Val Ala Ser Cys Thr Ser Ala Ile His Thr Val Pro Gly Gly Ala
35 40 45
Ile Gly Gly Ala His Ala Met Ile Leu Gly His Gly Ala Val Gly Gly
50 55 60
Val Val Gly Val Gly Ser Gly Val Leu Ala Pro Leu Pro Gly Ala Ala
65 70 75 80
Val Val Val Pro Ala Ile Thr Pro Ala Thr Ala Thr Ser Gly Val Gly
85 90 95
Ala Gly Thr His Gly His Ser Gly Gly Met Leu Ala Gly Thr Leu Pro
100 105 110
Ser Ala Val Leu Ala Gly Val Pro Gly Gly Pro Pro His Val Ala Ala
115 120 125
Ala Ala Met Ala Leu Ala His Leu Pro Leu Gly Ser Pro Leu Gly Ala
130 135 140
Ala Val Met Ile Pro Ala Met Met Thr Thr Gly Pro His Gly Ala Gly
145 150 155 160
Leu Ala Ala Ile Gly Leu Gly Ala Thr Val Ala Val Leu Gly Ile Gly
165 170 175
Pro Val Gly Leu Met Ala Val Ala Gly Ala Leu Leu Ala Gly Ala Gly
180 185 190
Ala Ile Ile Ala Val Gly Ser Ala Pro Val Cys Val Ala Ala Ala Leu
195 200 205
Thr Thr Gly Ala Thr Ala Ile Val Ala Thr Leu Ala Gly Pro Ile Gly
210 215 220
Ser Gly Ile Met Ala Leu Thr Gly Gly Leu Gly Val Ala Ala Ala Ile
225 230 235 240
Ile Ala Gly Gly Ala Ala Ala Ile Met Ala Thr Ala Val Leu Ile Val
245 250 255
Leu Pro Gly Gly Thr Ile Ala Ala Val Ala Thr Pro Gly Gly Gly Gly
260 265 270
Val Leu Pro Val Pro Ala Leu Gly Thr Gly Cys Gly Met Ala His Leu
275 280 285
Thr Ile Leu Gly Gly Leu Cys Pro Gly Gly Ala Leu Ala Met Gly Ala
290 295 300
Leu Ile Ala Leu Val Pro Thr Leu Ala Val Ala Pro Ser Leu Leu Val
305 310 315 320
Thr His Val Pro Ala Gly Pro Ala Ala Ile Gly Leu Ala Pro Met Leu
325 330 335
Met Leu Ala Leu Pro Leu Ala Leu Ile Leu Pro Val Val Ile Leu Ala
340 345 350
<210> 8
<211> 1059
<212> DNA
<213> 野生型(Thermoanaerobacter brockii)
<400> 8
atgaaaggtt ttgcaatgct cagtatcggt aaagttggct ggattgagaa ggaaaagcct 60
gctcctggcc catttgatgc tattgtaaga cctctagctg tggccccttg cacttcggac 120
attcataccg tttttgaagg cgccattggc gaaagacata acatgatact cggtcacgaa 180
gctgtaggtg aagtagttga agtaggtagt gaggtaaaag attttaaacc tggtgatcgc 240
gttgttgtgc cagctattac ccctgattgg cggacctctg aagtacaaag aggatatcac 300
cagcactccg gtggaatgct ggcaggctgg aaattttcga atgtaaaaga tggtgttttt 360
ggtgaatttt ttcatgtgaa tgatgctgat atgaatttag cacatctgcc taaagaaatt 420
ccattggaag ctgcagttat gattcccgat atgatgacca ctggttttca cggagctgaa 480
ctggcagata tagaattagg tgcgacggta gcagttttgg gtattggccc agtaggtctt 540
atggcagtcg ctggtgccaa attgcgtgga gccggaagaa ttattgccgt aggcagtaga 600
ccagtttgtg tagatgctgc aaaatactat ggagctactg atattgtaaa ctataaagat 660
ggtcctatcg aaagtcagat tatgaatcta actgaaggca aaggtgtcga tgctgccatc 720
atcgctggag gaaatgctga cattatggct acagcagtta agattgttaa acctggtggc 780
accatcgcta atgtaaatta ttttggcgaa ggagaggttt tgcctgttcc tcgtcttgaa 840
tggggttgcg gcatggctca taaaactata aaaggcgggc tatgccccgg tggacgtcta 900
agaatggaaa gactgattga ccttgttttt tataagcgtg tcgatccttc taagctcgtc 960
actcacgttt tccggggatt tgacaatatt gaaaaagcct ttatgttgat gaaagacaaa 1020
ccaaaagacc taatcaaacc tgttgtaata ttagcataa 1059
<210> 9
<211> 1059
<212> DNA
<213> I140S突变体(Thermoanaerobacter brockii)
<400> 9
atgaaaggtt ttgcaatgct cagtatcggt aaagttggct ggattgagaa ggaaaagcct 60
gctcctggcc catttgatgc tattgtaaga cctctagctg tggccccttg cacttcggac 120
attcataccg tttttgaagg cgccattggc gaaagacata acatgatact cggtcacgaa 180
gctgtaggtg aagtagttga agtaggtagt gaggtaaaag attttaaacc tggtgatcgc 240
gttgttgtgc cagctattac ccctgattgg cggacctctg aagtacaaag aggatatcac 300
cagcactccg gtggaatgct ggcaggctgg aaattttcga atgtaaaaga tggtgttttt 360
ggtgaatttt ttcatgtgaa tgatgctgat atgaatttag cacatctgcc taaagaaagt 420
ccattggaag ctgcagttat gattcccgat atgatgacca ctggttttca cggagctgaa 480
ctggcagata tagaattagg tgcgacggta gcagttttgg gtattggccc agtaggtctt 540
atggcagtcg ctggtgccaa attgcgtgga gccggaagaa ttattgccgt aggcagtaga 600
ccagtttgtg tagatgctgc aaaatactat ggagctactg atattgtaaa ctataaagat 660
ggtcctatcg aaagtcagat tatgaatcta actgaaggca aaggtgtcga tgctgccatc 720
atcgctggag gaaatgctga cattatggct acagcagtta agattgttaa acctggtggc 780
accatcgcta atgtaaatta ttttggcgaa ggagaggttt tgcctgttcc tcgtcttgaa 840
tggggttgcg gcatggctca taaaactata aaaggcgggc tatgccccgg tggacgtcta 900
agaatggaaa gactgattga ccttgttttt tataagcgtg tcgatccttc taagctcgtc 960
actcacgttt tccggggatt tgacaatatt gaaaaagcct ttatgttgat gaaagacaaa 1020
ccaaaagacc taatcaaacc tgttgtaata ttagcataa 1059
<210> 10
<211> 1059
<212> DNA
<213> I140S-D205E突变体(Thermoanaerobacter brockii)
<400> 10
atgaaaggtt ttgcaatgct cagtatcggt aaagttggct ggattgagaa ggaaaagcct 60
gctcctggcc catttgatgc tattgtaaga cctctagctg tggccccttg cacttcggac 120
attcataccg tttttgaagg cgccattggc gaaagacata acatgatact cggtcacgaa 180
gctgtaggtg aagtagttga agtaggtagt gaggtaaaag attttaaacc tggtgatcgc 240
gttgttgtgc cagctattac ccctgattgg cggacctctg aagtacaaag aggatatcac 300
cagcactccg gtggaatgct ggcaggctgg aaattttcga atgtaaaaga tggtgttttt 360
ggtgaatttt ttcatgtgaa tgatgctgat atgaatttag cacatctgcc taaagaaagt 420
ccattggaag ctgcagttat gattcccgat atgatgacca ctggttttca cggagctgaa 480
ctggcagata tagaattagg tgcgacggta gcagttttgg gtattggccc agtaggtctt 540
atggcagtcg ctggtgccaa attgcgtgga gccggaagaa ttattgccgt aggcagtaga 600
ccagtttgtg tagaagctgc aaaatactat ggagctactg atattgtaaa ctataaagat 660
ggtcctatcg aaagtcagat tatgaatcta actgaaggca aaggtgtcga tgctgccatc 720
atcgctggag gaaatgctga cattatggct acagcagtta agattgttaa acctggtggc 780
accatcgcta atgtaaatta ttttggcgaa ggagaggttt tgcctgttcc tcgtcttgaa 840
tggggttgcg gcatggctca taaaactata aaaggcgggc tatgccccgg tggacgtcta 900
agaatggaaa gactgattga ccttgttttt tataagcgtg tcgatccttc taagctcgtc 960
actcacgttt tccggggatt tgacaatatt gaaaaagcct ttatgttgat gaaagacaaa 1020
ccaaaagacc taatcaaacc tgttgtaata ttagcataa 1059
<210> 11
<211> 1059
<212> DNA
<213> H125Q-I140S-N329S突变体(Thermoanaerobacter brockii)
<400> 11
atgaaaggtt ttgcaatgct cagtatcggt aaagttggct ggattgagaa ggaaaagcct 60
gctcctggcc catttgatgc tattgtaaga cctctagctg tggccccttg cacttcggac 120
attcataccg tttttgaagg cgccattggc gaaagacata acatgatact cggtcacgaa 180
gctgtaggtg aagtagttga agtaggtagt gaggtaaaag attttaaacc tggtgatcgc 240
gttgttgtgc cagctattac ccctgattgg cggacctctg aagtacaaag aggatatcac 300
cagcactccg gtggaatgct ggcaggctgg aaattttcga atgtaaaaga tggtgttttt 360
ggtgaatttt ttcaagtgaa tgatgctgat atgaatttag cacatctgcc taaagaaagt 420
ccattggaag ctgcagttat gattcccgat atgatgacca ctggttttca cggagctgaa 480
ctggcagata tagaattagg tgcgacggta gcagttttgg gtattggccc agtaggtctt 540
atggcagtcg ctggtgccaa attgcgtgga gccggaagaa ttattgccgt aggcagtaga 600
ccagtttgtg tagatgctgc aaaatactat ggagctactg atattgtaaa ctataaagat 660
ggtcctatcg aaagtcagat tatgaatcta actgaaggca aaggtgtcga tgctgccatc 720
atcgctggag gaaatgctga cattatggct acagcagtta agattgttaa acctggtggc 780
accatcgcta atgtaaatta ttttggcgaa ggagaggttt tgcctgttcc tcgtcttgaa 840
tggggttgcg gcatggctca taaaactata aaaggcgggc tatgccccgg tggacgtcta 900
agaatggaaa gactgattga ccttgttttt tataagcgtg tcgatccttc taagctcgtc 960
actcacgttt tccggggatt tgacagtatt gaaaaagcct ttatgttgat gaaagacaaa 1020
ccaaaagacc taatcaaacc tgttgtaata ttagcataa 1059
<210> 12
<211> 1059
<212> DNA
<213> I140S-L294I突变体(Thermoanaerobacter brockii)
<400> 12
atgaaaggtt ttgcaatgct cagtatcggt aaagttggct ggattgagaa ggaaaagcct 60
gctcctggcc catttgatgc tattgtaaga cctctagctg tggccccttg cacttcggac 120
attcataccg tttttgaagg cgccattggc gaaagacata acatgatact cggtcacgaa 180
gctgtaggtg aagtagttga agtaggtagt gaggtaaaag attttaaacc tggtgatcgc 240
gttgttgtgc cagctattac ccctgattgg cggacctctg aagtacaaag aggatatcac 300
cagcactccg gtggaatgct ggcaggctgg aaattttcga atgtaaaaga tggtgttttt 360
ggtgaatttt ttcatgtgaa tgatgctgat atgaatttag cacatctgcc taaagaaagc 420
ccattggaag ctgcagttat gattcccgat atgatgacca ctggttttca cggagctgaa 480
ctggcagata tagaattagg tgcgacggta gcagttttgg gtattggccc agtaggtctt 540
atggcagtcg ctggtgccaa attgcgtgga gccggaagaa ttattgccgt aggcagtaga 600
ccagtttgtg tagatgctgc aaaatactat ggagctactg atattgtaaa ctataaagat 660
ggtcctatcg aaagtcagat tatgaatcta actgaaggca aaggtgtcga tgctgccatc 720
atcgctggag gaaatgctga cattatggct acagcagtta agattgttaa acctggtggc 780
accatcgcta atgtaaatta ttttggcgaa ggagaggttt tgcctgttcc tcgtcttgaa 840
tggggttgcg gcatggctca taaaactata aaaggcggga tatgccccgg tggacgtcta 900
agaatggaaa gactgattga ccttgttttt tataagcgtg tcgatccttc taagctcgtc 960
actcacgttt tccggggatt tgacaatatt gaaaaagcct ttatgttgat gaaagacaaa 1020
ccaaaagacc taatcaaacc tgttgtaata ttagcataa 1059
<210> 13
<211> 1059
<212> DNA
<213> I140S-D150Q突变体(Thermoanaerobacter brockii)
<400> 13
atgaaaggtt ttgcaatgct cagtatcggt aaagttggct ggattgagaa ggaaaagcct 60
gctcctggcc catttgatgc tattgtaaga cctctagctg tggccccttg cacttcggac 120
attcataccg tttttgaagg cgccattggc gaaagacata acatgatact cggtcacgaa 180
gctgtaggtg aagtagttga agtaggtagt gaggtaaaag attttaaacc tggtgatcgc 240
gttgttgtgc cagctattac ccctgattgg cggacctctg aagtacaaag aggatatcac 300
cagcactccg gtggaatgct ggcaggctgg aaattttcga atgtaaaaga tggtgttttt 360
ggtgaatttt ttcatgtgaa tgatgctgat atgaatttag cacatctgcc taaagaaagt 420
ccattggaag ctgcagttat gattccccaa atgatgacca ctggttttca cggagctgaa 480
ctggcagata tagaattagg tgcgacggta gcagttttgg gtattggccc agtaggtctt 540
atggcagtcg ctggtgccaa attgcgtgga gccggaagaa ttattgccgt aggcagtaga 600
ccagtttgtg tagatgctgc aaaatactat ggagctactg atattgtaaa ctataaagat 660
ggtcctatcg aaagtcagat tatgaatcta actgaaggca aaggtgtcga tgctgccatc 720
atcgctggag gaaatgctga cattatggct acagcagtta agattgttaa acctggtggc 780
accatcgcta atgtaaatta ttttggcgaa ggagaggttt tgcctgttcc tcgtcttgaa 840
tggggttgcg gcatggctca taaaactata aaaggcgggc tatgccccgg tggacgtcta 900
agaatggaaa gactgattga ccttgttttt tataagcgtg tcgatccttc taagctcgtc 960
actcacgttt tccggggatt tgacaatatt gaaaaagcct ttatgttgat gaaagacaaa 1020
ccaaaagacc taatcaaacc tgttgtaata ttagcataa 1059
<210> 14
<211> 1059
<212> DNA
<213> P36S-I140S突变体(Thermoanaerobacter brockii)
<400> 14
atgaaaggtt ttgcaatgct cagtatcggt aaagttggct ggattgagaa ggaaaagcct 60
gctcctggcc catttgatgc tattgtaaga cctctagctg tggcctcttg cacttcggac 120
attcataccg tttttgaagg cgccattggc gaaagacata acatgatact cggtcacgaa 180
gctgtaggtg aagtagttga agtaggtagt gaggtaaaag attttaaacc tggtgatcgc 240
gttgttgtgc cagctattac ccctgattgg cggacctctg aagtacaaag aggatatcac 300
cagcactccg gtggaatgct ggcaggctgg aaattttcga atgtaaaaga tggtgttttt 360
ggtgaatttt ttcatgtgaa tgatgctgat atgaatttag cacatctgcc taaagaaagc 420
ccattggaag ctgcagttat gattcccgat atgatgacca ctggttttca cggagctgaa 480
ctggcagata tagaattagg tgcgacggta gcagttttgg gtattggccc agtaggtctt 540
atggcagtcg ctggtgccaa attgcgtgga gccggaagaa ttattgccgt aggcagtaga 600
ccagtttgtg tagatgctgc aaaatactat ggagctactg atattgtaaa ctataaagat 660
ggtcctatcg aaagtcagat tatgaatcta actgaaggca aaggtgtcga tgctgccatc 720
atcgctggag gaaatgctga cattatggct acagcagtta agattgttaa acctggtggc 780
accatcgcta atgtaaatta ttttggcgaa ggagaggttt tgcctgttcc tcgtcttgaa 840
tggggttgcg gcatggctca taaaactata aaaggcgggc tatgccccgg tggacgtcta 900
agaatggaaa gactgattga ccttgttttt tataagcgtg tcgatccttc taagctcgtc 960
actcacgttt tccggggatt tgacaatatt gaaaaagcct ttatgttgat gaaagacaaa 1020
ccaaaagacc taatcaaacc tgttgtaata ttagcataa 1059
Claims (9)
1.一种酮还原酶突变体，其特征在于，该酮还原酶突变体的氨基酸序列是SEQ ID NO:1所示氨基酸序列发生突变的氨基酸序列，发生突变的氨基酸序列突变位点分别为：第140位I突变为S，可选的突变点位至少还包括下列点位之一：第205位D突变为E、第125位H突变为Q、第329位N突变为S、第294位L突变为I、第150位D突变为Q、第36位P突变为S。
2.根据权利要求1所述酮还原酶突变体，其特征在于，具有下列之一编码的氨基酸序列：SEQ ID NO: 2、SEQ ID NO: 3、SEQ ID NO: 4、SEQ ID NO: 5、SEQ ID NO: 6或SEQ IDNO:7。
3.根据权利要求1所述的酮还原酶突变体，其特征在于，对应的编码基因具有下述之一的核苷酸序列：SEQ ID NO: 8、SEQ ID NO: 9、SEQ ID NO: 10、SEQ ID NO: 11、SEQ ID NO:12、SEQ ID NO: 13或SEQ ID NO: 14。
4.一种重组质粒，其特征在于，含有权利要求3所述的酮还原酶突变体编码基因。
5.根据权利要求4所述的重组质粒，其特征在于，所述质粒为pET-28a(+)、pET-28b(+)、pET-28c(+)、pET-5b(+)、pET-15b、pET-24a(+)、pET-24c(+)、pET-24d(+)、pET-25b(+)、pET-27b(+)、pET-28c(+)、pET-29a(+)、pET-29b(+)、pET-29c(+)、pET-30b(+)、pET-30c(+)、pET-30 Xa/LIC、pET-30 EK/LIC、pET-31b(+)、pET-32b(+)、pET-32c(+)、pET-32 EK/LIC、pET-32Xa/LIC、pET-33b(+)、pET-37b(+)、pET-39b(+)、pET-40b(+)、pET-41a(+)、pET-41b(+)、pET-42b(+)、pET-42c(+)、pET-43.1a(+)、pET-43.1b(+)、pET-43.1c(+)、pET-43.1 EK/LIC、pET-44a(+)、pET-44b(+)、pET-44c(+)、pET-44 EK/LIC、pET-45b(+)、pET-46 EK/LIC、pET-47b(+)、pET-48b(+)、pET-49b(+)、pET-51b(+)、pET-52b(+)、pQE30、pQE31、pQE32、pQE40、pBV220、pBV221、pCold-GST、pCold IV、pCold-GST或pTrcHis C。
6.一种宿主细胞，其特征在于，含有权利要求4所述的重组质粒。
7.一种宿主细胞，其特征在于，含有权利要求5所述的质粒。
8.根据权利要求6或7所述宿主细胞，其特征在于，所述宿主细胞包括原核细胞、酵母或真核细胞，所述原核细胞为大肠杆菌BL21(DE3)细胞。
9.权利要求1所述的酮还原酶突变体在将5-((4S)-2-氧代-4-苯基(1, 3-恶唑烷-3-基))-1-(4-氟苯基) 戊烷-1, 5-二酮还原为(4S)-3-[(5S)-5-(4-氟苯基)-5-羟基戊酰基]-4-苯基-1, 3-氧氮杂环戊烷-2-酮中的应用。
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| CN111996176A (zh) * | 2020-10-29 | 2020-11-27 | 中国科学院天津工业生物技术研究所 | 羰基还原酶突变体及其应用 |
| CN113072516A (zh) * | 2021-03-30 | 2021-07-06 | 浙江亚瑟医药有限公司 | 一种胆固醇吸收选择性抑制剂类药物中间体的合成方法 |
| CN114752574A (zh) * | 2021-09-13 | 2022-07-15 | 台州酶易生物技术有限公司 | 一种酶催化体系、加氢酶及制备方法和应用 |
| EP4163368A4 (en) * | 2020-06-08 | 2024-05-01 | Asymchem Life Science Tianjin Co Ltd | CETOREDUCTASE MUTANT AND ITS APPLICATION |
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