CN116790686A

CN116790686A - 一种生物酶法催化合成(r)-2-(1-氨乙基)-4-氟苯酚的方法

Info

Publication number: CN116790686A
Application number: CN202210663645.8A
Authority: CN
Inventors: 吕雪峰; 周冠; 徐卉芳; 罗泉; 赵航; 冯岩; 徐扬军; 姚庆佳
Original assignee: Sphinx Scientific Laboratory Tianjin Co ltd; Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Current assignee: Sphinx Scientific Laboratory Tianjin Co ltd; Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Priority date: 2022-06-14
Filing date: 2022-06-14
Publication date: 2023-09-22

Abstract

本发明公开了一种生物酶法催化合成(R)‑2‑(1‑氨乙基)‑4‑氟苯酚的方法。所述方法包括：对候选转氨酶序列进行异源表达，筛选具有目标活性的转氨酶催化剂；利用含目标转氨酶的生物材料(包括纯酶、细胞破碎液、重组菌全细胞等)为催化剂，以5‑氟‑2‑羟基苯乙酮为底物，在水相缓冲体系里进行目标转氨反应，利用酸碱处理、有机溶剂萃取以及浓缩干燥，得到(R)‑2‑(1‑氨乙基)‑4‑氟苯酚。本发明所述的生物酶催化工艺方法，转氨酶来源明确、氨基酸序列清晰，催化剂制备过程简单、成本低廉，反应过程简单、温和、环保、立体选择性高，产品分离纯化方法简便、回收率高，有利于规模化应用。

Description

一种生物酶法催化合成(R)-2-(1-氨乙基)-4-氟苯酚的方法

技术领域

本发明属于生物催化和生物制药领域，具体涉及一种生物酶法催化合成 (R)-2-(1-氨乙基)-4-氟苯酚的方法。

背景技术

TRK(Tropomyosin related kinase，原肌球蛋白相关激酶)是存在于人体多种组织中的一类酪氨酸激酶，在调控细胞的增殖、分化、凋亡等过程中发挥着重要作用。由于基因重排等因素引起的TRK异常表达，有时会引起肿瘤等疾病的发生。开发TRK抑制剂药物用于治疗TRK异常表达引起的各类肿瘤疾病，具有巨大的医学价值和市场前景。

在开发新一代小分子TRK抑制剂药物的过程中，(R)-2-(1-氨乙基)-4-氟苯酚是多种候选药物实体的重要中间体。因此，开发简捷、经济、绿色的(R)-2-(1-氨乙基)-4-氟苯酚合成路线，对于相关领域的肿瘤新药研发具有重要意义。

目前，(R)-2-(1-氨乙基)-4-氟苯酚的合成可通过化学法和生物法实现。化学合成法是以5-氟-2-羟基苯甲醛(化合物1)为起始底物，其首先与手性辅剂2-甲基丙烷-2-亚磺酰胺缩合，形成中间化合物N-(5-氟-2-羟基苯亚甲基)-2-甲基丙烷-2- 亚磺酰胺(化合物2)；在-65℃低温及氮气保护下，该亚胺中间体与甲基溴化镁反应，引入手性中心，生成中间体N-(1-(5-氟-2-羟基苯基)乙基)-2-甲基丙烷-2-丙磺酰胺(化合物3)；最后，在HCl及二恶烷存在条件下，脱去保护基团，生成 (R)-2-(1-氨乙基)-4-氟苯酚(化合物4)，具体步骤如下：

上述合成方法中，由于反应条件要求高(如-65℃低温)且第二步反应立体选择性较低，很难进行工业化放大生产。相比于化学法，生物酶法具有效率高、反应条件温和、立体特异性强且环境友好等特点，能够有效弥补化学法的不足。然而，目前关于生物酶法合成(R)-2-(1-氨乙基)-4-氟苯酚的报道极少，仅有的报道采用未知生物来源的商品化转氨酶干粉来合成该化合物。在该方法中，由于所用酶剂的生物来源不明，氨基酸序列未知，纯酶制剂生产成本高，购买价格昂贵，且反应需在-0.03Mpa～-0.06Mpa的真空条件下进行，很大程度上限制了该方法的规模化应用。

因此，继续开发简捷、高效、绿色、经济的(R)-2-(1-氨乙基)-4-氟苯酚生物合成方法具有十分重要的意义。

发明内容

本发明的目的在于提供一种生物酶法催化合成(R)-2-(1-氨乙基)-4-氟苯酚的方法，所述方法简单高效、成本低、构型纯，适合于工业化放大生产。

本发明公开了一种利用所述转氨酶或其重组表达菌的全细胞或破碎液作为催化剂，一步不对称催化转化底物5-氟-2-羟基苯乙酮(化合物5)生产(R)-2-(1- 氨乙基)-4-氟苯酚(化合物4)的方法，如下所示：

为实现上述发明目的，本发明采用以下技术方案予以实现：

一种生物酶法催化合成(R)-2-(1-氨乙基)-4-氟苯酚的方法，包括以下步骤：

(1)对候选转氨酶编码DNA序列进行密码子优化，合成转氨酶基因片段，构建转氨酶表达质粒并转入目标感受态细胞，得到转氨酶重组表达菌株；

(2)培养所述转氨酶重组表达菌株，诱导转氨酶的表达；

(3)利用步骤(2)中表达的转氨酶，筛选具有目标活性的转氨酶；

(4)利用含有步骤(3)中的目标活性转氨酶的生物材料为催化剂，以5- 氟-2-羟基苯乙酮作为底物，在水相缓冲体系里进行目标转氨反应，得到(R)-2-(1- 氨乙基)-4-氟苯酚粗品；

(5)将所述(R)-2-(1-氨乙基)-4-氟苯酚粗品经酸碱处理、有机溶剂萃取、浓缩干燥，得到(R)-2-(1-氨乙基)-4-氟苯酚。

进一步的，所述转氨反应的温度为20℃～60℃，反应时间为1小时～72小时。

优选的，所述转氨反应的温度为35℃～40℃，反应时间为20小时～48小时。

进一步的，所述水相缓冲体系的pH为6.0～11.0。

优选的，所述水相缓冲体系的pH为8.0～9.0。

进一步的，所述水相缓冲体系包括磷酸钾缓冲液、三乙醇胺缓冲液、甘氨酸 -NaOH缓冲液，其浓度为0.1M。

优选的，所述水相缓冲体系为0.1M、pH 8.0的磷酸钾缓冲液。

进一步的，所述转氨反应的氨基供体包括(R)-苯乙胺、(R)-1-(4-溴苯基)乙胺、(R)-1-苯丙胺、(R)-1,2,3,4-四氢-1-萘胺、(R)-1-Boc-3-氨基吡咯烷、异丙胺、仲丁胺、2-戊胺、2-氨基庚烷。

优选的，所述转氨反应中的氨基供体为(R)-苯乙胺。

进一步的，所述转氨反应中的辅酶为磷酸吡哆醛，其使用浓度为0.4mM～2.0 mM。

进一步的，所述转氨反应中助溶剂包括二甲亚砜、乙腈、甲基叔丁基醚、N,N- 二甲基甲酰胺、甲醇、乙醇、异丙醇，其浓度分数为10％～50％。

优选的，所述转氨反应中助溶剂为二甲亚砜和乙醇，浓度为10％。

进一步的，所述步骤(4)中的生物材料可为转氨酶纯酶、固定化酶、细胞破碎液或重组菌全细胞。

进一步的，所述具有目标活性的转氨酶为ω-转氨酶，其氨基酸序列如SEQ IDNo.8～SEQ ID No.16所示。

优选的，所述转氨酶的氨基酸序列如SEQ ID No.9、SEQ ID No.12或SEQ ID No.13所示。

进一步的，所述重组表达菌株包括大肠杆菌、枯草芽孢杆菌、谷氨酸棒状杆菌、酵母。

优选的，所述重组表达菌株为大肠杆菌。

本发明还提供了一种具有催化合成(R)-2-(1-氨乙基)-4-氟苯酚活性的转氨酶，所述转氨酶的氨基酸序列分别如SEQ ID No.8～SEQ ID No.16所示，其核苷酸序列分别如SEQ ID No.26～SEQ ID No.34所示。

本发明与现有技术相比，具有以下优点和有益效果：

本发明克服了现有(R)-2-(1-氨乙基)-4-氟苯酚化学合成法中反应步骤多、反应条件要求高、立体选择性较低的缺陷，以及生物合成法中生物酶背景信息不明、纯酶催化剂使用成本高的问题。本发明所述的生物酶催化法中，转氨酶来源明确、氨基酸序列清晰，催化剂制备过程简单、成本低廉，反应过程简单、温和、环保、立体选择性高，可以以>70％的转化率和高达100％的手性纯度(e.e.)来合成 (R)-2-(1-氨乙基)-4-氟苯酚，较目前方法具有更高的实用性。本发明所述的全细胞催化剂在使用过程中，可不经过细胞破碎、蛋白纯化等过程，直接以全细胞形式催化反应，很大程度上提高了该方法的规模化应用潜力。因此，本发明所述方法具有很高的工业应用潜力。

附图说明

图1为部分转氨酶(1～4号和7～13号)在重组菌中的诱导表达情况；其中1 为诱导前表达情况，2为诱导后表达情况，三角形指示了目标转氨酶在SDS-PAGE 中的位置。

图2为实施例11中反应产物的HPLC分析结果图。

图3为实施例11中反应产物的立体构型分析结果图。

图4为实施例11中反应产物的质谱分析结果图。

具体实施方式

结合以下具体实例对本发明的技术方案作进一步详细的说明。

下述实施例中，如无特殊说明，所使用的实验方法均为常规方法，所用材料、试剂等均可从生物或化学试剂公司购买。

实施例1：转氨酶大肠杆菌重组表达菌的构建

本发明对18种不同微生物来源的转氨酶进行了异源表达，转氨酶标号依次为1～18。1号转氨酶来源于脱氮副球菌Paracoccus denitrificans，其氨基酸序列如SEQ IDNo.1所示；2号转氨酶来源于简纳西氏菌Jannaschia sp.，其氨基酸序列如SEQ ID No.2所示；3号转氨酶来源于真菌Alternaria alternata，其氨基酸序列如SEQ ID No.3所示；4号转氨酶来源于细菌Mesorhizobium japonicum，其氨基酸序列如SEQ ID No.4所示；5号转氨酶来源于荧光假单胞菌 Pseudomonas fluorescens，其氨基酸序列如SEQ ID No.5所示；6号转氨酶来源于真菌Neosartorya fischeri，其氨基酸序列如SEQ ID No.6所示；7号转氨酶来源于海王生丝单胞菌Hyphomonas neptunium，其氨基酸序列如SEQ ID No.7所示；8号转氨酶来源于细菌Luminiphilus syltensis，其氨基酸序列如SEQ ID No.8 所示；9号转氨酶来源于米曲霉Aspergillus oryzae，其氨基酸序列如SEQ ID No. 9所示；10号转氨酶来源于烟曲霉Aspergillus fumigatus，其氨基酸序列如SEQ ID No.10所示；11号转氨酶来源于土曲霉Aspergillus terreus，其氨基酸序列如SEQ ID No.11所示；12～16号转氨酶均来源于节杆菌Arthrobacter sp.，其氨基酸序列如SEQ ID No.12～SEQ ID No.16所示；17号转氨酶来源于产黄青霉Penicillium chrysogenum，其氨基酸序列如SEQ ID No.17所示；18号转氨酶来源于禾谷镰孢菌Fusarium graminearum，其氨基酸序列如SEQ ID No.18所示。用于异源表达1～18号转氨酶的核苷酸序列依次如SEQ ID No.19～SEQ ID No.36所示。

以上述18种转氨酶的氨基酸序列为出发模板，根据大肠杆菌密码子使用偏好，对这18种转氨酶的编码DNA序列进行密码子优化，合成用于异源表达的转氨酶基因片段，通过NdeI和XhoI酶切位点将目标基因克隆至pET-28b(+)载体上，获得转氨酶重组表达质粒。取1μl重组质粒，将其在42℃条件下热激90 秒，转化至100μl大肠杆菌BL21(DE3)感受态细胞中，经过37℃复苏1小时，取出100μl细胞悬液涂布于含有50μg/ml卡那霉素的LB固体培养基上。在37℃下，经过静置过夜培养，获得转氨酶重组表达菌的单克隆菌落。

实施例2：转氨酶的诱导表达及纯化

挑取实施例1中重组表达菌的单克隆菌落，接种于10ml含有50μg/ml卡那霉素的LB液体培养基中，在37℃下摇床过夜培养。取过夜培养物按1％接种量接种至500ml含有50μg/ml卡那霉素抗的LB液体培养基中，在37℃下摇床培养至对数生长期，加入0.1mM～0.3mMIPTG(异丙基-β-D-硫代半乳糖苷)，于16℃～20℃下诱导目标转氨酶表达。诱导20小时后，离心收集菌体，以 SDS-PAGE(变性聚丙烯酰胺凝胶电泳)检查转氨酶表达情况。部分结果如图1 所示，转氨酶在重组菌中被成功诱导表达。

将成功诱导表达的菌体离心收集，细胞沉淀重悬于20mM Tris-HCl缓冲液 (pH8.0)中，利用超声破碎法破碎细胞，离心分离后取上清组分，获得细胞破碎液。所得破碎液进一步通过镍柱亲和层析获得含有目标蛋白的洗脱液，再经过 10kDa规格的超滤膜浓缩后，获得纯化的转氨酶。

上述得到的转氨酶纯酶、细胞破碎液或含有转氨酶的重组菌细胞，用作下述反应的催化剂。

实施例3：具有目标催化活性转氨酶的筛选

转氨反应在0.2ml的反应体系中进行，该体系为0.1M的三乙醇胺缓冲溶液 (pH8.5)，含有5-氟-2-羟基苯乙酮1g/l、10％二甲亚砜(DMSO)、0.4mM磷酸吡哆醛(PLP)、50mM(R)-苯乙胺、2.0mg/ml 1～18号转氨酶纯酶(或其细胞破碎液)，在37℃条件下振荡(220rpm)反应24小时，利用HPLC检测产物生成，计算产物产率。HPLC分析设备及条件为：安捷伦1260型HPLC分析系统，装配有C18色谱柱(4.6mm×250mm×5μm)；流动相为0～15min阶段为 5％乙腈95％水(含0.1％磷酸)～95％乙腈5％水(含0.1％磷酸)，15～15.1min 阶段为95％乙腈5％水(含0.1％磷酸)～5％乙腈95％水(含0.1％磷酸)，15.1～20 min阶段为5％乙腈95％水(含0.1％磷酸)；流速1.5ml/min；检测波长210nm；柱温30℃。目标产物出峰时间为4.4min，底物出峰时间为10.9min。

上述分析中，8～16号转氨酶展现目标催化活性，产物产率如下表1所示。13 号转氨酶催化转氨反应效率最高，产物产率为41.0％；其次是9号转氨酶，产物产率为39.7％；再次为12号转氨酶，产物产率为33.9％；8、10、11、14、15、 16号转氨酶催化的产物产率在10.1％～23.1％之间。1～7号和17、18号转氨酶没有展现目标催化活性。

表1：不同转氨酶催化目标转氨反应的筛选结果

实施例4：低温处理对细胞催化活性的影响

将实施例2获得的表达有转氨酶的重组菌细胞(全细胞催化剂)在4℃或 -80℃下过夜保存，取出细胞恢复至室温后用于目标转氨反应。

转氨反应在0.4ml的反应体系中进行，该体系为0.1M的三乙醇胺缓冲溶液 (pH8.5)，含有5-氟-2-羟基苯乙酮1g/l、10％DMSO、0.4mM PLP、50mM(R)- 苯乙胺、转氨酶(12号、13号)全细胞催化剂50OD₆₀₀，在37℃条件下振荡(220 rpm)反应7、24、48小时，利用HPLC检测产物生成，计算产物产率。

结果如表2所示，与4℃保存的细胞相比，-80℃冷冻对细胞催化活性影响较小，其产物产率在稳定期(24～48小时)内的差别小于10％，说明全细胞催化剂可以在低温下(如-80℃)保存，且不影响作用效果。

表2：不同形式低温处理的细胞的催化效率比较

实施例5：反应温度的选择

转氨反应在0.4ml的反应体系中进行，该体系为0.1M的三乙醇胺缓冲溶液 (pH8.5)，含有5-氟-2-羟基苯乙酮1g/l、10％DMSO、0.4mM PLP、50mM(R)- 苯乙胺、转氨酶(12号)全细胞催化剂50OD₆₀₀，在30、35、40、45、50℃条件下振荡(220rpm)反应1小时，利用HPLC检测产物生成，计算产物产率。

结果如表3所示，反应温度为40℃时产物产率最高，为38.8％；反应温度为35℃时产物产率略低，为38.6％；其它温度下产物产率在22.9％～33.4％之间。因此，利用转氨酶全细胞催化剂进行催化反应时，温度优选35℃～40℃。

表3：不同反应温度下目标转氨反应的产物产率

实施例6：反应pH的选择

转氨反应在0.4ml的反应体系中进行，该体系为0.1M的不同pH缓冲液(磷酸钾缓冲液pH 6.0、7.0、8.0；三乙醇胺缓冲液pH 7.5、8.5；甘氨酸-NaOH缓冲液pH 9.0、10.0、11.0)，含有5-氟-2-羟基苯乙酮1g/l、10％DMSO、0.4mM PLP、 50mM(R)-苯乙胺、转氨酶(12号)全细胞催化剂50OD₆₀₀，在35℃条件下振荡(220rpm)反应1小时，利用HPLC检测产物生成，计算产物产率。

结果如表4所示，在反应pH为8.0时产物产率最高，为36.7％；反应pH为 8.5和9.0时，产物产率略低，分别为34.5％、33.9％；其它pH条件下产物产率在9.4％～29.6％之间。因此，利用转氨酶全细胞催化剂进行催化反应时，优选pH 8.0～9.0。

表4：不同pH条件下目标转氨反应的产物产率

实施例7：氨基供体的选择

转氨反应在0.4ml的反应体系中进行，该体系为0.1M的磷酸钾缓冲液(pH 8.0)，含有5-氟-2-羟基苯乙酮1g/l、10％DMSO、0.4mM PLP、转氨酶(12号) 全细胞催化剂50OD₆₀₀，氨基供体分别为50mM的(R)-苯乙胺(RMBA)、(R)-1-(4- 溴苯基)乙胺(RBEA)、(R)-1-苯丙胺(RPA)、(R)-1,2,3,4-四氢-1-萘胺(RTA)、 (R)-1-Boc-3-氨基吡咯烷(RAP)、异丙胺(IPA)、仲丁胺(SBA)、2-戊胺(APT)、 2-氨基庚烷(AHT)，在35℃条件下振荡(220rpm)反应1小时，利用HPLC 检测产物生成，计算产物产率。

结果如表5所示,当氨基供体为RMBA时产物产率最高，为57.8％；当氨基供体为RBEA、RPA、AHT时产物产率略低，依次为39.9％、41.0％、39.9％；其余氨基供体条件下产物产率在1.5％～17.9％之间。因此，利用转氨酶全细胞催化剂进行催化反应时，RMBA为优选氨基供体。

表5：不同氨基供体条件下目标转氨反应的产物产率

实施例8：助溶剂的选择

转氨反应在0.4ml的反应体系中进行，该体系为0.1M的磷酸钾缓冲液(pH 8.0)，含有5-氟-2-羟基苯乙酮1g/l、0.4mM PLP、50mM(R)-苯乙胺、转氨酶 (12号)全细胞催化剂50OD₆₀₀，助溶剂分别为10％的二甲亚砜(DMSO)、乙腈(ACN)、甲基叔丁基醚(MTBE)、N,N-二甲基甲酰胺(DMF)、甲醇(MeOH)、乙醇(EtOH)、异丙醇(IPN)，在35℃条件下振荡(100rpm)反应1小时，利用HPLC检测产物生成，计算产物产率。

结果如表6所示，当助溶剂为EtOH时产物产率最高，为37.7％；当助溶剂为DMSO时产物产率略低，为37.3％；其它助溶剂条件下，产物产率在4.5％～35.4％之间。因此，利用转氨酶全细胞催化剂进行催化反应时，优选助溶剂为EtOH和 DMSO。

表6：不同助溶剂条件下目标转氨反应的产物产率

实施例9：助溶剂浓度的选择

转氨反应在0.4ml的反应体系中进行，该体系为0.1M的磷酸钾缓冲液(pH 8.0)，含有5-氟-2-羟基苯乙酮1g/l、0.4mM PLP、50mM(R)-苯乙胺、转氨酶 (12号)全细胞催化剂50OD₆₀₀，DMSO浓度为10％、20％、30％、40％、50％，在35℃条件下振荡(220rpm)反应1小时，利用HPLC检测产物生成，计算产物产率。

结果如表7所示，当DMSO浓度为10％时产物产率最高，为50.5％。随着 DMSO浓度的升高，产物产率逐渐下降。因此，利用转氨酶全细胞催化剂进行催化反应时，优选DMSO浓度为10％。

表7：不同助溶剂浓度条件下目标转氨反应的产物产率

实施例10：100ml体系下的催化反应((R)-苯乙胺为氨基供体)

转氨反应在100ml的反应体系中进行，该体系为0.1M的磷酸钾缓冲液(pH 8.0)，含有5-氟-2-羟基苯乙酮5g/l、10％DMSO、1.0mM PLP、250mM(R)- 苯乙胺、转氨酶(12号)全细胞催化剂50OD₆₀₀，在35℃条件下振荡(100rpm) 反应20小时，利用HPLC检测产物生成。

分析表明产物产率为73.7％。

上述结果表明利用转氨酶全细胞催化剂进行的转氨反应可扩大到100ml体系完成。

实施例11：100ml体系下的催化反应(仲丁胺为氨基供体)

转氨反应在100ml的反应体系中进行，该体系为0.1M的磷酸钾缓冲液(pH 8.0)，含有5-氟-2-羟基苯乙酮5g/l、10％DMSO、2.0mM PLP、1M仲丁胺、转氨酶(12号)全细胞催化剂50OD₆₀₀，在35℃条件下振荡(100rpm)反应48小时，利用HPLC检测产物生成及其手性纯度。

反应产物手性纯度检测方法具体为：安捷伦1260型HPLC分析系统，装配有CHIRALPAK IA手性色谱柱(4.6mm×250mm×5μm)；流动相为0～30min， 30％乙腈70％水(含0.1％甲基磺酸)；流速1ml/min；检测波长275nm；柱温 30℃；R型产物出峰时间为23.7min，S型产物出峰时间为25.1min。

分析表明，产物产率为45.1％，手性纯度(e.e.)为100％。反应产物的HPLC 分析结果如图2所示，产物手性纯度分析结果如图3所示，产物质谱分析结果如图4所示。

实施例12：1升体系下的催化反应

转氨反应在1升的反应体系中进行，该体系为0.1M的磷酸钾缓冲液(pH 8.0)，含有5-氟-2-羟基苯乙酮2g/l、10％DMSO、2.0mM PLP、0.6M仲丁胺、转氨酶(12号)全细胞催化剂50OD₆₀₀，在35℃条件下振荡(100rpm)反应 24小时，利用HPLC检测产物生成及其手性纯度。

分析表明，产物产率为51.8％，手性纯度(e.e.)为100％。

上述结果表明利用转氨酶全细胞催化剂进行的转氨反应可扩大到1升体系进行。

实施例13：目标产物的分离纯化

实施例12中催化反应进行完毕后，将反应体系在50℃条件下旋转蒸发，溶液体积浓缩至约240ml。向溶液中加入盐酸调节pH至1.0，再加入3倍体积乙酸乙酯，充分混合体系后静置分层，收集水相溶液。向收集的溶液中加入NaOH 溶液调节pH至10.0，再加入3倍体积乙酸乙酯，充分混合体系后静置分层，收集有机相溶液并在40℃下进行旋转蒸发。待溶液完全蒸干后，加入30ml 50％乙腈溶液重悬样品，所得溶液在低温下冻干后，获得0.986克固体产品。

上述分离纯化方法的产品回收率为96％，手性纯度(e.e.)为100％。

对比例1：无目标催化活性的对照

转氨反应在0.4ml的反应体系中进行，该体系为0.1M的磷酸钾缓冲液(pH 8.0)，含有5-氟-2-羟基苯乙酮1g/l、10％DMSO、PLP 0.4mM、50mM(R)-苯乙胺、对照组大肠杆菌BL21(DE3)细胞(含有pET-28b空载体)50OD₆₀₀，在35℃条件下振荡(220rpm)反应1～48小时，利用HPLC检测产物生成。

结果表明，全过程未检测到目标产物生成。

综上，利用本发明所述的转氨酶及其全细胞催化剂，能够一步催化转化5- 氟-2-羟基苯乙酮生成(R)-2-(1-氨乙基)-4-氟苯酚。

以上实施例仅用以说明本发明的技术方案，而非对其进行限制；尽管参照前述实施例对本发明进行了详细的说明，对于本领域的普通技术人员来说，依然可以对前述实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或替换，并不使相应技术方案的本质脱离本发明所要求保护的技术方案的精神和范围。

序列表

<110> 中国科学院青岛生物能源与过程研究所斯芬克司药物研发（天津）股份有限公司

<120> 一种生物酶法催化合成(R)-2-(1-氨乙基)-4-氟苯酚的方法

<141> 2022-06-10

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Gly Asn Asn His Ala Phe Val Thr Ala Thr Lys Ala Leu Glu Leu Phe

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325 330 335

Leu Arg Ala Gly Leu Glu Lys Ile Ala Ala Ser Ala Ser Phe Ala Gly

340 345 350

Arg Leu Lys Gly Lys Gly Met Met Ser Gly Ile Glu Met Glu Ser Gly

355 360 365

Asp Val Ala Ala Glu Ile Cys Thr Glu Cys Phe Gln Asn Gly Leu Ile

370 375 380

Ile Glu Thr Ser Gly Ser Met Asp Glu Val Val Lys Val Leu Ala Pro

385 390 395 400

Leu Thr Ile Thr Asp Ala Glu Leu Asp Ala Gly Leu Lys Ile Leu Ala

405 410 415

Asp Ala Val Arg Ala Val Ser Ala Arg Arg Gln Lys Ser Ala Ala

420 425 430

<210> 8

<211> 293

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 8

Met Ser Asp Glu Pro Ile Ile Tyr Ile Asn Gly Asp Tyr Leu Pro Leu

1 5 10 15

Ser Gln Ala Arg Val Ser Pro Val Asp Gln Gly Phe Leu Leu Gly Asp

20 25 30

Gly Val Phe Asp Val Val Ser Ala Trp Lys Gly Asn Ile Phe Lys Leu

35 40 45

Asp Ala His Leu Asp Arg Phe Phe Asp Ser Ile Gln Ala Ala Arg Leu

50 55 60

Asn His Asp Met Ser Arg Asp Ala Trp Lys Glu Ala Ile Ile Glu Thr

65 70 75 80

Thr Arg Arg Asn Gly Leu Asp Asp Ala Ser Ile Arg Phe Ile Val Thr

85 90 95

Arg Gly Glu Pro Lys Gly Val Val Ala Asp Pro Arg Asp Phe Lys Pro

100 105 110

Thr Cys Ile Val Trp Val Ala Pro Tyr Ile Phe Leu Ala Asp Glu Glu

115 120 125

Lys Arg Arg Asn Gly Ile Arg Leu Met Ile Ser Ala Thr Arg Gly Phe

130 135 140

Pro Ala Asp Thr Leu Asp Pro Arg Tyr Lys Cys Leu Asp Arg Leu His

145 150 155 160

Ser Gln Leu Ile Arg Leu Glu Ala Leu Glu Ala Gly Tyr Asp Asp Ala

165 170 175

Leu Trp Leu Asp His Ser Gly His Val Ser Glu Ser Ala Ala Ser Asn

180 185 190

Leu Phe Ile Val Lys Asn Gly Val Leu Tyr Thr Pro Ser Ala Gly Ile

195 200 205

Leu Arg Gly Ile Thr Arg Asp Thr Ile Leu Glu Leu Ala Thr Glu Leu

210 215 220

Asp Ile Pro Trp Lys Glu Arg Gln Leu Ser Ala Phe Asp Val Tyr Ile

225 230 235 240

Ala Asp Glu Val Phe Thr Cys Ser Thr Ala Gly Gly Ala Leu Pro Val

245 250 255

Arg Glu Val Ala Gly Arg Thr Ile Arg Gly Thr Thr Pro Gly Pro Ile

260 265 270

Thr Gln Ala Ile Asp Asn Ala Tyr Trp Ala Met Arg Glu Thr Asp Arg

275 280 285

Tyr Ala Thr Pro Leu

290

<210> 9

<211> 326

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 9

Met Thr Ser Met Asn Lys Val Phe Ser Gly Tyr Tyr Glu Arg Lys Ala

1 5 10 15

Arg Leu Asp Asn Ser Asp Asn Arg Phe Ala Lys Gly Ile Ala Tyr Val

20 25 30

Gln Gly Ser Phe Val Pro Leu Ala Asp Ala Arg Val Pro Leu Leu Asp

35 40 45

Glu Gly Phe Met His Ser Asp Leu Thr Tyr Asp Val Pro Ser Val Trp

50 55 60

Asp Gly Arg Phe Phe Arg Leu Asp Asp His Leu Ser Arg Leu Glu Asp

65 70 75 80

Ser Cys Glu Lys Met Arg Leu Lys Ile Pro Leu Ser Arg Asp Glu Val

85 90 95

Lys Gln Thr Leu Arg Glu Met Val Ala Lys Ser Gly Ile Glu Asp Ala

100 105 110

Phe Val Glu Leu Ile Val Thr Arg Gly Leu Lys Gly Val Arg Gly Asn

115 120 125

Lys Pro Glu Asp Leu Phe Asp Asn His Leu Tyr Leu Ile Val Met Pro

130 135 140

Tyr Val Trp Val Met Glu Pro Ala Ile Gln His Thr Gly Gly Thr Ala

145 150 155 160

Ile Ile Ala Arg Thr Val Arg Arg Thr Pro Pro Gly Ala Phe Asp Pro

165 170 175

Thr Ile Lys Asn Leu Gln Trp Gly Asp Leu Thr Arg Gly Leu Phe Glu

180 185 190

Ala Ala Asp Arg Gly Ala Asp Tyr Pro Phe Leu Ser Asp Gly Asp Thr

195 200 205

Asn Leu Thr Glu Gly Ser Gly Phe Asn Ile Val Leu Val Lys Asp Gly

210 215 220

Ile Ile Tyr Thr Pro Asp Arg Gly Val Leu Glu Gly Ile Thr Arg Lys

225 230 235 240

Ser Val Phe Asp Ile Ala Gln Val Lys Asn Ile Glu Val Arg Val Gln

245 250 255

Val Val Pro Leu Glu His Ala Tyr His Ala Asp Glu Ile Phe Met Cys

260 265 270

Thr Thr Ala Gly Gly Ile Met Pro Ile Thr Lys Leu Asp Gly Lys Pro

275 280 285

Ile Arg Asn Gly Glu Val Gly Pro Leu Thr Thr Lys Ile Trp Asp Glu

290 295 300

Tyr Trp Ala Met His Tyr Asp Pro Lys Tyr Ser Ser Ala Ile Asp Tyr

305 310 315 320

Arg Gly His Glu Gly Asn

325

<210> 10

<211> 323

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 10

Met Ala Ser Met Asp Lys Val Phe Ser Gly Tyr Tyr Ala Arg Gln Lys

1 5 10 15

Leu Leu Glu Arg Ser Asp Asn Pro Phe Ser Lys Gly Ile Ala Tyr Val

20 25 30

Glu Gly Lys Leu Val Leu Pro Ser Asp Ala Arg Ile Pro Leu Leu Asp

35 40 45

Glu Gly Phe Met His Ser Asp Leu Thr Tyr Asp Val Ile Ser Val Trp

50 55 60

Asp Gly Arg Phe Phe Arg Leu Asp Asp His Leu Gln Arg Ile Leu Glu

65 70 75 80

Ser Cys Asp Lys Met Arg Leu Lys Phe Pro Leu Ala Leu Ser Ser Val

85 90 95

Lys Asn Ile Leu Ala Glu Met Val Ala Lys Ser Gly Ile Arg Asp Ala

100 105 110

Phe Val Glu Val Ile Val Thr Arg Gly Leu Thr Gly Val Arg Gly Ser

115 120 125

Lys Pro Glu Asp Leu Tyr Asn Asn Asn Ile Tyr Leu Leu Val Leu Pro

130 135 140

Tyr Ile Trp Val Met Ala Pro Glu Asn Gln Leu His Gly Gly Glu Ala

145 150 155 160

Ile Ile Thr Arg Thr Val Arg Arg Thr Pro Pro Gly Ala Phe Asp Pro

165 170 175

Thr Ile Lys Asn Leu Gln Trp Gly Asp Leu Thr Lys Gly Leu Phe Glu

180 185 190

Ala Met Asp Arg Gly Ala Thr Tyr Pro Phe Leu Thr Asp Gly Asp Thr

195 200 205

Asn Leu Thr Glu Gly Ser Gly Phe Asn Ile Val Leu Val Lys Asn Gly

210 215 220

Ile Ile Tyr Thr Pro Asp Arg Gly Val Leu Arg Gly Ile Thr Arg Lys

225 230 235 240

Ser Val Ile Asp Val Ala Arg Ala Asn Ser Ile Asp Ile Arg Leu Glu

245 250 255

Val Val Pro Val Glu Gln Ala Tyr His Ser Asp Glu Ile Phe Met Cys

260 265 270

Thr Thr Ala Gly Gly Ile Met Pro Ile Thr Leu Leu Asp Gly Gln Pro

275 280 285

Val Asn Asp Gly Gln Val Gly Pro Ile Thr Lys Lys Ile Trp Asp Gly

290 295 300

Tyr Trp Glu Met His Tyr Asn Pro Ala Tyr Ser Phe Pro Val Asp Tyr

305 310 315 320

Gly Ser Gly

<210> 11

<211> 325

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 11

Met Ala Ser Met Asp Lys Val Phe Ala Gly Tyr Ala Ala Arg Gln Ala

1 5 10 15

Ile Leu Glu Ser Thr Glu Thr Thr Asn Pro Phe Ala Lys Gly Ile Ala

20 25 30

Trp Val Glu Gly Glu Leu Val Pro Leu Ala Glu Ala Arg Ile Pro Leu

35 40 45

Leu Asp Gln Gly Phe Met His Ser Asp Leu Thr Tyr Asp Val Pro Ser

50 55 60

Val Trp Asp Gly Arg Phe Phe Arg Leu Asp Asp His Ile Thr Arg Leu

65 70 75 80

Glu Ala Ser Cys Thr Lys Leu Arg Leu Arg Leu Pro Leu Pro Arg Asp

85 90 95

Gln Val Lys Gln Ile Leu Val Glu Met Val Ala Lys Ser Gly Ile Arg

100 105 110

Asp Ala Phe Val Glu Leu Ile Val Thr Arg Gly Leu Lys Gly Val Arg

115 120 125

Gly Thr Arg Pro Glu Asp Ile Val Asn Asn Leu Tyr Met Phe Val Gln

130 135 140

Pro Tyr Val Trp Val Met Glu Pro Asp Met Gln Arg Val Gly Gly Ser

145 150 155 160

Ala Val Val Ala Arg Thr Val Arg Arg Val Pro Pro Gly Ala Ile Asp

165 170 175

Pro Thr Val Lys Asn Leu Gln Trp Gly Asp Leu Val Arg Gly Met Phe

180 185 190

Glu Ala Ala Asp Arg Gly Ala Thr Tyr Pro Phe Leu Thr Asp Gly Asp

195 200 205

Ala His Leu Thr Glu Gly Ser Gly Phe Asn Ile Val Leu Val Lys Asp

210 215 220

Gly Val Leu Tyr Thr Pro Asp Arg Gly Val Leu Gln Gly Val Thr Arg

225 230 235 240

Lys Ser Val Ile Asn Ala Ala Glu Ala Phe Gly Ile Glu Val Arg Val

245 250 255

Glu Phe Val Pro Val Glu Leu Ala Tyr Arg Cys Asp Glu Ile Phe Met

260 265 270

Cys Thr Thr Ala Gly Gly Ile Met Pro Ile Thr Thr Leu Asp Gly Met

275 280 285

Pro Val Asn Gly Gly Gln Ile Gly Pro Ile Thr Lys Lys Ile Trp Asp

290 295 300

Gly Tyr Trp Ala Met His Tyr Asp Ala Ala Tyr Ser Phe Glu Ile Asp

305 310 315 320

Tyr Asn Glu Arg Asn

325

<210> 12

<211> 330

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 12

Met Ala Phe Ser Ala Asp Thr Ser Glu Ile Val Tyr Thr His Asp Thr

1 5 10 15

Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn

20 25 30

Pro Leu Ala Gly Gly Ala Ala Trp Ile Glu Gly Ala Phe Val Pro Pro

35 40 45

Ser Glu Ala Arg Ile Ser Ile Phe Asp Gln Gly Tyr Leu His Ser Asp

50 55 60

Val Thr Tyr Thr Val Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu

65 70 75 80

Asp Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Met Arg Ile

85 90 95

Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu

100 105 110

Val Ala Lys Thr Glu Leu Arg Glu Ala Phe Val Ser Val Ser Ile Thr

115 120 125

Arg Gly Tyr Ser Ser Thr Pro Gly Glu Arg Asp Ile Thr Lys His Arg

130 135 140

Pro Gln Val Tyr Met Tyr Ala Val Pro Tyr Gln Trp Ile Val Pro Phe

145 150 155 160

Asp Arg Ile Arg Asp Gly Val His Ala Met Val Ala Gln Ser Val Arg

165 170 175

Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp

180 185 190

Gly Asp Leu Ile Arg Ala Val Gln Glu Thr His Asp Arg Gly Phe Glu

195 200 205

Ala Pro Leu Leu Leu Asp Gly Asp Gly Leu Leu Ala Glu Gly Ser Gly

210 215 220

Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg

225 230 235 240

Ala Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu

245 250 255

Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Leu Ala Glu Leu

260 265 270

Leu Asp Ala Asp Glu Val Leu Gly Cys Thr Thr Ala Gly Gly Val Trp

275 280 285

Pro Phe Val Ser Val Asp Gly Asn Pro Ile Ser Asp Gly Val Pro Gly

290 295 300

Pro Ile Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu

305 310 315 320

Ser Ser Ser Leu Leu Thr Pro Val Gln Tyr

325 330

<210> 13

<211> 330

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 13

Met Ala Phe Ser Ala Asp Thr Pro Glu Ile Val Tyr Thr His Asp Thr

1 5 10 15

Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn

20 25 30

Pro Leu Ala Gly Gly Ala Ala Trp Ile Glu Gly Ala Phe Val Pro Pro

35 40 45

Ser Glu Ala Arg Ile Ser Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp

50 55 60

Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu

65 70 75 80

Gly Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu

85 90 95

Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu

100 105 110

Val Ala Lys Thr Glu Leu Arg Glu Ala Met Val Thr Val Thr Ile Thr

115 120 125

Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg

130 135 140

Pro Gln Val Tyr Met Ser Ala Cys Pro Tyr Gln Trp Ile Val Pro Phe

145 150 155 160

Asp Arg Ile Arg Asp Gly Val His Leu Met Val Ala Gln Ser Val Arg

165 170 175

Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp

180 185 190

Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu

195 200 205

Leu Pro Leu Leu Leu Asp Cys Asp Asn Leu Leu Ala Glu Gly Pro Gly

210 215 220

Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg

225 230 235 240

Ala Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu

245 250 255

Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Glu Leu

260 265 270

Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp

275 280 285

Pro Phe Val Ser Val Asp Gly Asn Ser Ile Ser Asp Gly Val Pro Gly

290 295 300

Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu

305 310 315 320

Pro Ser Ser Leu Leu Thr Pro Val Gln Tyr

325 330

<210> 14

<211> 330

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 14

Met Ala Phe Ser Ala Asp Thr Ala Glu Ile Val Tyr Thr His Asp Thr

1 5 10 15

Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn

20 25 30

Pro Leu Ala Gly Gly Ala Ala Trp Ile Glu Gly Ala Phe Val Pro Pro

35 40 45

Ala Glu Ala Arg Ile Ser Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp

50 55 60

Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu

65 70 75 80

Asp Gly His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu

85 90 95

Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu

100 105 110

Val Ala Lys Thr Glu Leu Arg Glu Ala Met Val Thr Val Ser Ile Thr

115 120 125

Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg

130 135 140

Pro Gln Val Tyr Met Ser Ala Cys Pro Tyr Gln Trp Ile Val Pro Phe

145 150 155 160

Asp Arg Ile Arg Asp Gly Val His Leu Met Val Ala Gln Ser Val Arg

165 170 175

Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp

180 185 190

Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu

195 200 205

Leu Pro Leu Leu Leu Asp Ala Asp Asn Leu Leu Ala Glu Gly Pro Gly

210 215 220

Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg

225 230 235 240

Ala Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu

245 250 255

Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Glu Leu

260 265 270

Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp

275 280 285

Pro Phe Val Ser Val Asp Gly Asn Ala Ile Ser Asp Gly Ala Pro Gly

290 295 300

Pro Ile Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu

305 310 315 320

Ser Ser Ser Leu Leu Thr Pro Val Gln Tyr

325 330

<210> 15

<211> 330

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 15

Met Ala Phe Ser Ala Asp Thr Ala Glu Ile Val Tyr Thr His Asp Thr

1 5 10 15

Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn

20 25 30

Pro Leu Ala Gly Gly Ala Ala Trp Ile Glu Gly Ala Phe Val Pro Pro

35 40 45

Ser Glu Ala Arg Ile Ser Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp

50 55 60

Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu

65 70 75 80

Asp Gly His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu

85 90 95

Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu

100 105 110

Val Ala Lys Thr Glu Leu Arg Glu Ala Met Val Thr Val Ser Ile Thr

115 120 125

Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg

130 135 140

Pro Gln Val Tyr Met Ser Ala Cys Pro Tyr Gln Trp Ile Val Pro Phe

145 150 155 160

Asp Arg Ile Arg Asp Gly Val His Leu Met Val Ala Gln Ser Val Arg

165 170 175

Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp

180 185 190

Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu

195 200 205

Leu Pro Leu Ala Leu Asp Ala Asp Asn Leu Leu Ala Glu Gly Pro Gly

210 215 220

Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg

225 230 235 240

Ala Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu

245 250 255

Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Glu Leu

260 265 270

Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp

275 280 285

Pro Phe Val Ser Val Asp Gly Asn Ala Ile Ser Asp Gly Ala Pro Gly

290 295 300

Pro Ile Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu

305 310 315 320

Ser Ser Ser Leu Leu Thr Pro Val Gln Tyr

325 330

<210> 16

<211> 330

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 16

Met Ala Phe Ser Ala Asp Thr Ala Glu Ile Val Tyr Thr His Asp Thr

1 5 10 15

Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn

20 25 30

Pro Leu Ala Gly Gly Ala Ala Trp Ile Glu Gly Ala Phe Val Pro Pro

35 40 45

Ala Glu Ala Arg Ile Ser Ile Phe Asp Gln Gly Phe Ala Thr Ser Asp

50 55 60

Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu

65 70 75 80

Asp Gly His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu

85 90 95

Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu

100 105 110

Val Ala Lys Thr Glu Leu Arg Glu Ala Met Val Thr Val Ser Ile Thr

115 120 125

Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg

130 135 140

Pro Gln Val Tyr Met Ser Ala Cys Pro Tyr Gln Trp Ile Val Pro Phe

145 150 155 160

Asp Arg Ile Arg Asp Gly Val His Leu Met Val Ala Gln Ser Val Arg

165 170 175

Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp

180 185 190

Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu

195 200 205

Leu Pro Leu Leu Leu Asp Ala Asp Asn Leu Leu Ala Glu Gly Pro Gly

210 215 220

Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg

225 230 235 240

Ala Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu

245 250 255

Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Glu Leu

260 265 270

Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp

275 280 285

Pro Phe Val Ser Val Asp Gly Asn Ala Ile Ser Asp Gly Ala Pro Gly

290 295 300

Pro Ile Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu

305 310 315 320

Ser Ser Ser Leu Leu Thr Pro Val Gln Tyr

325 330

<210> 17

<211> 319

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 17

Met Ala Thr Met Glu Lys Ile Phe Ala Ala Tyr His Glu Arg Gln Lys

1 5 10 15

Leu Leu Ala Ala Asn Thr His Pro Phe Ala Lys Gly Val Ala Trp Val

20 25 30

Glu Gly Glu Leu Thr Pro Leu His Glu Ala Arg Ile Pro Ile Leu Asp

35 40 45

Gln Gly Phe Met His Ser Asp Leu Thr Tyr Asp Val Pro Ser Val Trp

50 55 60

Asp Gly Arg Phe Phe Arg Leu Asp Asp His Ile Thr Arg Leu Glu Ala

65 70 75 80

Ser Cys Thr Lys Leu Arg Met Lys Leu Pro Leu Pro Arg Asp Glu Val

85 90 95

Lys Gln Ile Leu Val Asp Met Val Ala Lys Ser Gly Ile Arg Asp Ala

100 105 110

Phe Val Glu Ile Ile Val Thr Arg Gly Leu Lys Gly Val Arg Gly Ser

115 120 125

Arg Pro Glu Asp Ile Val Asn Arg Ile Tyr Met Phe Ile Gln Pro Tyr

130 135 140

Val Trp Cys Met Glu Pro Glu Val Gln Pro Val Gly Gly Ser Ala Ile

145 150 155 160

Ile Ala Arg Thr Val Arg Arg Val Pro Pro Gly Cys Ile Asp Pro Thr

165 170 175

Val Lys Asn Leu Gln Trp Gly Asp Leu Val Arg Gly Leu Phe Glu Ala

180 185 190

Ser Asp Arg Gly Ala Glu Tyr Pro Phe Leu Thr Asp Gly Asp Thr Asn

195 200 205

Leu Thr Glu Gly Ser Gly Phe Asn Ile Val Leu Val Lys Asp Asn Ile

210 215 220

Leu Tyr Thr Pro Ala Arg Gly Val Leu Glu Gly Val Thr Arg Lys Ser

225 230 235 240

Val Ile Asp Val Ala Arg Ala Ser Gly Phe Asp Ile Lys Val Glu Leu

245 250 255

Val Pro Val Gln Met Ala Tyr Asp Ala Asp Glu Ile Phe Met Cys Thr

260 265 270

Thr Ala Gly Gly Ile Met Pro Ile Thr Ser Leu Asp Gly Lys Pro Val

275 280 285

Asn Asp Gly Lys Val Gly Ser Val Thr Lys Lys Ile Trp Asp Gly Tyr

290 295 300

Trp Ala Ile His Tyr Asp Pro Ala Tyr Ser Phe Glu Ile Ala Tyr

305 310 315

<210> 18

<211> 325

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 18

Met Ser Thr Met Asp Lys Ile Phe Ala Gly His Ala Gln Arg Gln Ala

1 5 10 15

Thr Leu Val Ala Ser Asp Asn Ile Phe Ala Asn Gly Ile Ala Trp Ile

20 25 30

Gln Gly Glu Leu Val Pro Leu Asn Glu Ala Arg Ile Pro Leu Met Asp

35 40 45

Gln Gly Phe Met His Gly Asp Leu Thr Tyr Asp Val Pro Ala Val Trp

50 55 60

Asp Gly Arg Phe Phe Arg Leu Asp Asp His Leu Asp Arg Leu Glu Ala

65 70 75 80

Ser Val Lys Lys Met Arg Met Gln Phe Pro Ile Pro Arg Asp Glu Ile

85 90 95

Arg Met Thr Leu Leu Asp Met Leu Ala Lys Ser Gly Ile Lys Asp Ala

100 105 110

Phe Val Glu Leu Ile Val Thr Arg Gly Leu Lys Pro Val Arg Glu Ala

115 120 125

Lys Pro Gly Glu Val Leu Asn Asn His Leu Tyr Leu Ile Val Gln Pro

130 135 140

Tyr Val Trp Val Met Ser Pro Glu Ala Gln Tyr Val Gly Gly Asn Ala

145 150 155 160

Val Ile Ala Arg Thr Val Arg Arg Ile Pro Pro Gly Ser Met Asp Pro

165 170 175

Thr Ile Lys Asn Leu Gln Trp Ser Asp Phe Thr Arg Gly Met Phe Glu

180 185 190

Ala Tyr Asp Arg Gly Ala Gln Tyr Pro Phe Leu Thr Asp Gly Asp Thr

195 200 205

Asn Ile Thr Glu Gly Ser Gly Phe Asn Val Val Phe Val Lys Asn Asn

210 215 220

Val Ile Tyr Thr Pro Asn Arg Gly Val Leu Gln Gly Ile Thr Arg Lys

225 230 235 240

Ser Val Ile Asp Ala Ala Lys Trp Cys Gly His Glu Val Arg Val Glu

245 250 255

Tyr Val Pro Val Glu Met Ala Tyr Glu Ala Asp Glu Ile Phe Met Cys

260 265 270

Thr Thr Ala Gly Gly Ile Met Pro Ile Thr Thr Met Asp Gly Lys Pro

275 280 285

Val Lys Asp Gly Lys Val Gly Pro Val Thr Lys Ala Ile Trp Asp Arg

290 295 300

Tyr Trp Ala Met His Trp Glu Asp Glu Phe Ser Phe Lys Ile Asp Tyr

305 310 315 320

Gln Lys Leu Lys Leu

325

<210> 19

<211> 1362

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 19

atgaatcagc cgcagagctg ggaagcgcgg gcggaaacct atagcctgta tggctttacc 60

gatatgccga gcgtgcatca gcgcggcact gttgttgtta cgcatggcga aggtccatat 120

attgtggatg tgcatggccg ccgctatctg gatgcgaata gcggcttatg gaatatggtg 180

gcgggctttg atcataaagg cctgattgaa gcggcgaaag cgcagtatga tcgctttccg 240

ggctatcatg cgttctttgg ccgcatgagc gatcagaccg tgatgctgag cgaaaaactg 300

gtggaagtga gcccgtttga taatggccgc gtgttttata ccaatagcgg cagcgaagcg 360

aatgatacga tggtgaaaat gctgtggttt ctgcatgcgg cggaaggcaa accgcagaaa 420

cgcaaaattc tgacccgctg gaatgcgtat catggcgtga ccgcggtgag cgcgagcatg 480

accggtaaac cgtataatag cgtgtttggc ctgccgctgc cgggctttat tcatctgacc 540

tgcccgcatt attggcgcta tggcgaagaa ggcgaaaccg aagcgcagtt tgtggcccgc 600

ttggcgcgtg aattagaaga taccattacc cgcgaaggcg cggataccat tgcgggcttc 660

tttgcggaac cggtgatggg cgcgggcggc gttattccac cagcgaaagg ttattttcag 720

gcgattctgc cgattctgcg caaacatgat attccgatga ttagcgatga agtgatttgc 780

ggctttggcc gcaccggcaa tacctggggt tgtctgactt atgattttat gccggatgcg 840

attattagca gcaaaaatct gaccgcgggc ttctttccga tgggcgcggt tattctgggc 900

ccggatttag cgaaacgcgt gaaagcggcg gttgaagcga ttgaagaatt tccgcatggc 960

tttaccgcga gcggccatcc ggttggctgt gcgattgcat taaaagcgat tgatgtggtg 1020

atgaatgaag gcctggcgga aaatgtgcgc cgcctggcgc cacgttttga agcgggctta 1080

aaacgcattg cggatcgccc gaatattggc gaatatcgcg gcattggctt tatgtgggcg 1140

ctggaagcgg tgaaagataa accgaccaaa accccgtttg atgcgaatct gagcgtgagc 1200

gaacgcattg cgaatacctg caccgatctg ggcctgattt gccgcccgtt aggccaaagc 1260

attgtgctgt gtccgccgtt tattctgacc gaagcgcaga tggatgaaat gtttgaaaaa 1320

ctggaaaagg cgctggataa agtgtttgcg gaagtggcgt aa 1362

<210> 20

<211> 900

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 20

atgaatgatc tgagcaatgg cgcggcgtgg atgggcagcc gtattattcc aattgcggaa 60

gcggcgattc cagtgaccga ttggggtctg acccatagcg atattgcgta tgatgtggtg 120

ccggtgtggc gcggcggttt ctttcgctta gatgattatg tggcccgctt tatggcgagc 180

gtgggcgcgt tgcgtatgga tattggccgc gatggcgatg gcgtgaaaac cgcgttaacc 240

cgcatggtgg cggcgagcgg tttacgtgat agctatgtgg cgatggtggc ggcccgcggt 300

cgtaatccag ttccaggtag ccgcgatcca cgcgattgcg cgaatcattt ttatgcgtgg 360

tgcgtgccgt atgtgcatat tgtgaaaccg gaaattgcgg aacgcggcac cagcgtgtgg 420

attgcgaaac gcacccgccg cattccggcg gatagtgttg atccaaccgt gaaaaattat 480

cattggggcg attttaccgg cggcctgttt gaagcgaaag ataaaggctt tgaaaccgtg 540

ctgctgctgg atcatgcggg ccatgtgacc gaaggcccgg gttttaatgc gtttgcgctg 600

tttggcgatc gcattgtgac cagcgatcat ggcgtgctgc atggcattac ccgccgcacc 660

gttctggaaa tggcggcgga agcgggctta accgttgaaa cccgtccact gccgctggat 720

gaatttctgg aagcggatga agtgtttctg agcagcagcg gcggcggcgt tattccggtt 780

gcgcgtgttg ataatcgcgt gtttagcaat gatgcggcgg gcccggttgc gctggattta 840

agacgtcgct attttgattg gattacccgc gcggaacatc gcaccgatat tgcgtattaa 900

<210> 21

<211> 1122

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 21

atggcgagct atggctttcc gctgaccgcg agcagcctgg tggattggac cagtttaacc 60

tttagcccga ttgaagtgaa tggccatatt cagtgcacct atagcccgga agtggcggaa 120

tggggcgccc cacattttgt gaaagatccg tatctgcgcg tgcatggcct ggcgccagcg 180

ttgaattatg gccagcagat ttttgaaggc atgaaagcgt ttcgcacccc gaccggcagc 240

attcgcttat ttcgcccgaa aatgaatgcg gtgcgctttg cgcatagcgc gagctttgtg 300

gcgattccgc cggtgccaga agcgttgttt ctgcgtgcag ttcatctggc ggtgggctta 360

aatagcgaat ttgtgccgcc gtatgatagc cgcggcagcg cgttatatat tcgcccgatt 420

gcgtttgcga gcagcgcgac cgttaatctg gcgccggcgg atcattttac cttttgcgtg 480

tttgtgatgc cggtggcgcc gctgagcacc ggtgcgggtc aaggtttacg tgcgttggtt 540

gtggaagatg tggatcgcgc ggcgccgaaa ggcaccggta gcgcgaaagt tggtggtaat 600

tatgcgccga ttgtgaccac catgcaacgc gcgaaagcgg atggctatgg cctgacctta 660

catctggata gcgcgaccca taccatggtg gatgaattta gcgcgagcgg ctttattggc 720

gtgcgcgtgg atgcgggcaa aaccaccatg gtggtgccgg atagcccgac cattctgcgc 780

agcattaccg tggatagcat gtgccgcatt gcggaaagct ttggctggca ggtgcagcgc 840

cgtgcggtta gctttaccga actggcggaa ctgagcgaag cgtttgcggt gggcactgcg 900

tttattctga ccccggttcg cgcgattacc cgcccatgta cccatacctg cattgaatat 960

accgcggatt atcgcagcag cgcgagcgcg tatacccgct tattagaaac cctgcagggc 1020

attcagcagg gctggctgga tgatgcgtgg ggctggactg aagaagtgca ggatccgagc 1080

agcgatgaat ttattaccga taccgtgcag gcgcgtcgct aa 1122

<210> 22

<211> 963

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 22

atggatcaga ccaccgcgac ccaagcgagc aaaccgttgc caaccgttgg cgatcgccat 60

gtggatccgc atagctatcc ggatggcatt gcgtttctgg atggccagta tctgccgatg 120

agccaggcga aagtgagcgt gctggattgg ggctttctgc atagcgatgc gacctatgat 180

accgtgcatg tgtggaatgg ccgctttttc cgcctggatc tgcatctgga tcgcttcttt 240

ggcggcctgg aaaaactgcg catgaccatt ccgtttgatc gcgatggcgt ggcggaaatt 300

ctgcataatt gcgtggcgct gagcggccat cgcgcggcgt atgttgaaat gctgtgcacc 360

cgcggcgcga gcccgacctt tagccgtgat ccacgtcaag cgattaatcg ctttatggcg 420

tttgcggtgc cgtttggcag cgtggcgaat gcggaacagc tgcagcgtgg cttacgcgtg 480

gcgattagcg ataaagtgcg cattccgccg gcgagcgtgg atccgagcat taaaaattat 540

cattggctgg atctggtgcg cggcctgtat gatgcgtatg atagcggcgc ggaaaccgcg 600

ctgattctgg attttaatgg caatgtggcg gaaggcccgg gctttaatgt gttttgcgtg 660

aaagatggca aactgagcac cccggcgatt ggcgtgttac cgggtattac ccgtcgcacc 720

gtttttgatc tgtgcgcgga agaaggcctg gcggcggcgg cggcggatgt tagcgttgcg 780

gcgttaaaag cggcggatga agtgtttatt accagcaccg cgggcggcat tatgccggtg 840

acggaaattg atggcgcggc gattgcggat ggcaaagtgg gcccagttac cagccgttta 900

atggcgttgt attggcagaa acatgatgat ccggcgtgga gcagccaggt gaaatatccg 960

taa 963

<210> 23

<211> 1365

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 23

atgaacagca ataataaagc gtggctgaaa gaacataata ccgtgcacat gatgcatccg 60

atgcaggatc cgaaagcgct gcatgaacag cgcccgctga ttattcagag cggcaaaggc 120

gtgcatatta ccgatgtgga tggccgccgc tttattgatt gccagggcgg cttatggtgc 180

gtgaatgcgg gttatggccg tcgcgaaatt attgatgccg tgacccgcca gatggaagaa 240

ctggcgtatt atagcctgtt tccgggcagc accaatgcgc cggcgattgc gttaagccag 300

aaactgaccg aagtggcggc ggaagaaggc atggtgaaag cgagctttgg cctgggcggc 360

agcgatgcgg ttgaaaccgc attaaaaatt gcccgccagt attggaaact ggaaggccag 420

ccggataaag tgaaatttgt gagcctgtat aatggctatc atggcctgaa ttttggcggc 480

atgagcgcgt gcggcggcaa tgcgtggaaa agcagctatg aaccgctgat gccgggcttt 540

ttccaggtgg aaagcccgca tctgtatcgc aatccgttta ccaatgatcc ggaagaactg 600

gcggaaattt gcgcccagat tctggaacgc cagattgaaa tgcaggcgcc gggcaccgtt 660

gcggcgttaa ttgcggaacc gattcagggc gcgggcggcg ttattgtgcc gccagcaagc 720

tattggccgc gcttaagaca aatttgcgat aaatatgata tcctgctgat cgcggatgaa 780

gtgattaccg gcctgggccg cagtggcagc ttatttggta gccgcggttg gggcgttaaa 840

ccggatatta tgtgcctggc gaaaggcatt agcagcggct atgtgccgct gagcgcgacc 900

ctggttaata gccgcgttgc gcgtgcgtgg gaacgcgatg cgggttttac cagcgtgtat 960

atgcatggct atacctatag cggccatccg gtgagctgcg cggcggcgtt agcggcgatt 1020

gatattgtgc tgcaggaaaa tctggcggaa aatgcccgcg tggtgggcga ttattttctg 1080

gaaaaactgc tgattctgaa ggacaaacat cgcgcgattg gcgatgtgcg cggcaaaggc 1140

ttaatgctgg cggttgaact ggtgaaagaa cgcgcgacca aagaaccgtt tggcccggcg 1200

gatgcgtatc cgttagcgat tagcgaagcg tgcgtgaata atggcgtgat gattcgcacc 1260

attgtgaata aactgattat cagcccgccg ctgaccttta ccaccgaaca tgtggatgaa 1320

gtgattgaag tgctggatcg cgcgtttgtg gcgaatccgt ggtaa 1365

<210> 24

<211> 972

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 24

atggcgagca tggataaagt gtttagcggc tatcatgcgc gtcagaaact gctggaacgc 60

agcgataatc cgtttagcaa aggcattgcg tatgtggaag gcaaactggt gctgccgagc 120

gatgcgcgta ttccgttatt agatgaaggc tttatgcatg gcgatctgac ctatgatgtg 180

accaccgtgt gggatggccg cttctttcgc ctggatgatc acatgcagcg cattctggaa 240

agctgcgata aaatgcgcct gaaatttccg ctggcgccga gcaccgttaa aaatattctg 300

gcggaaatgg tggcgaaaag cggcattcgc gatgcgtttg tggaagtgat tgtgacccgc 360

ggcctgaccg gcgttcgtgg cagcaaacca gaagatctgt ataataataa tatctacctg 420

ctggtgctgc cgtatgtgtg ggtgatggcg ccggaaaatc agctgctggg cggcagtgcg 480

attattaccc gcaccgttcg tcgtaccccg ccaggcgcgt ttgatccaac cattaaaaat 540

ctgcagtggg gcgatctgac caaaggcctg tttgaagcga tggatcgcgg cgcgacctat 600

ccgtttctga ccgatggcga taccaatctg accgaaggca gcggctttaa tattgtgctg 660

gtgaaaaatg gcattatcta caccccggat cgcggcgtgc tgcgcggtat tactcgtaaa 720

agcgtgattg atgtggcccg tgcgaataat attgatattc gcctggaagt ggtgccggtg 780

gaacaggtgt atcatagcga tgaaattttt atgtgcacca ccgcgggcgg cattatgccg 840

attaccctgc tggatggcca gccggtgaat gatggccagg tgggcccaat taccaaaaag 900

atttgggatg gctattggga gatgcattat aatccggcgt atagctttcc ggtggattat 960

ggcagcggct aa 972

<210> 25

<211> 1296

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 25

atggcgtatg aacatgcgtc ggcgccagcg aatatttttg aacgccgcga aagccgcgtg 60

cgcagctatt gtcgtagctt tccggcggtg tttaaaagcg cacgcggcag cgaactgatt 120

gcggaagatg gcacccgcta tattgatttt ctgagcggct gcagcaccct gaattatggc 180

cataatcatc cggaactgaa acaggcgctg ctggattata ttggcgcgga tggcgtggcg 240

catggcctgg atatgcatac ccgcgcgaaa gcccgctttc tgaatacctt tgaacgcctg 300

attctgaaac cgcgcagcat ggatcatcgc gtgctgtttc cgggcccgac cggtgcgaat 360

gcggttgaag cagcgttgaa aaccgcgcgt aaagtgaccg gccgcaccaa tgtgattgcg 420

tttaccaatg gctttcatgg catgaccctg ggcgccctgg cggcgactgg aaatagtggt 480

aaacgtggcg gtgcgggtgt gccattaacc ggcgttaccc atgaagcgtt tgatggctat 540

tttggcgaag ataccgatac cgcggatcag ctggatcgcc gcttaagcga tccgagcagc 600

ggcctggata aaccggcggc gattattgtg gaaaccgtgc agggcgaagg cggcctgaat 660

gtggcgagcg atgcgtggtt acgtaaaatt gaaaagattg cgcgtaaaca cggcgccctg 720

tttattattg atgatattca ggcgggcatt ggccgcaccg gcggtttctt tagctttgaa 780

aaagcgggcg tgaccccgga tattattacg atggcgaaaa gcctgagcgg cctgggctta 840

ccgtttgcgt tgaccctgat tcgcccgcag catgatctgt ggaaaccggg cgaacataat 900

ggcacctttc gcggcaataa tcatgcgttt gtgaccgcga ccaaagcgct ggaactgttt 960

tgggcggatg atgcgtttga aaaagaaacc gcgcgtaaag cggcgcgtct gcgtgcaggt 1020

ttagagaaaa ttgcggcgag cgcgagcttt gcgggccgcc tgaaaggtaa aggcatgatg 1080

agcggcattg aaatggaaag cggcgatgtg gcggcggaaa tttgcaccga atgctttcag 1140

aatggcctga ttattgaaac cagcggcagc atggatgaag tggtgaaagt gctggcgccg 1200

ctgaccatta ccgatgcgga actggatgcg ggcctgaaaa ttctggcgga tgcggtgcgt 1260

gcggtgagcg cccgtcgtca gaaaagcgca gcgtaa 1296

<210> 26

<211> 882

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 26

atgagcgatg aaccgatcat ttacatcaat ggcgattatc tgccgctgag ccaggcccgc 60

gttagcccag ttgatcaagg ctttctgctg ggcgatggcg tgtttgatgt ggtgagcgcg 120

tggaaaggca atatttttaa actggatgcg catctggatc gctttttcga tagcattcag 180

gcggcccgcc tgaatcatga tatgagccgc gatgcgtgga aagaagcgat tattgaaacc 240

acccgccgca atggcctgga tgatgcgagt attcgcttta ttgtgacccg cggcgaaccg 300

aaaggcgtgg tggcggatcc acgcgatttt aaaccgacct gcattgtgtg ggtggcgccg 360

tatatttttc tggcggatga agaaaaacgc cgcaatggca ttcgcctgat gattagcgcg 420

acccgcggct ttccggcgga tactttagat ccgcgttata aatgcctgga tcgcctgcat 480

agccagctga ttcgcctgga agcgctggaa gcgggctatg atgatgcgct gtggctggat 540

catagcggcc atgtgagcga aagcgcggcg agcaatctgt ttattgtgaa aaatggcgtg 600

ctgtataccc cgagcgcggg cattttgcgc ggcattaccc gtgataccat tctggaactg 660

gcgaccgaac tggatattcc gtggaaagaa cgccagctga gcgcgtttga tgtgtatatt 720

gcggatgaag tgtttacctg cagcaccgcg ggcggcgcgt taccagttcg tgaagttgcg 780

ggtcgtacca ttcgcggtac caccccgggt ccaattaccc aagcgattga taatgcgtat 840

tgggcgatgc gcgaaaccga tcgctatgcg accccgctgt aa 882

<210> 27

<211> 981

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 27

atgaccagca tgaataaagt gtttagcggc tattatgagc gcaaagcccg cctggataat 60

agcgataatc gctttgcgaa aggcattgcg tatgtgcagg gcagctttgt gccgctggcg 120

gatgcgcgtg tgccattatt agatgaaggc tttatgcata gcgatctgac ctatgatgtg 180

ccgagcgtgt gggatggccg ctttttccgc ctggatgatc atctgagccg cctggaagat 240

agctgcgaaa agatgcgcct gaaaattccg ctgagccgcg atgaagtgaa acagaccctg 300

cgcgaaatgg tggcgaaaag cggcattgaa gatgcgtttg tggaactgat tgtgacccgc 360

ggcctgaaag gcgtgcgcgg caataaaccg gaagatctgt ttgataatca tctgtatctg 420

attgtgatgc cgtatgtgtg ggtgatggaa ccggcgattc agcataccgg cggcaccgcg 480

attattgccc gcaccgttcg tcgcaccccg ccaggtgcgt ttgatccgac cattaaaaat 540

ctgcagtggg gcgatctgac ccgcggcttg tttgaagcgg cggatcgtgg tgcggattat 600

ccgtttctga gcgatggcga taccaatctg accgaaggca gcggctttaa tattgtgctg 660

gtgaaagatg gcattatcta taccccggat cgcggcgtgc tggaaggcat tactcgtaaa 720

agcgtgtttg atattgcgca ggtgaaaaat attgaggtgc gcgtgcaggt ggtgccgctg 780

gaacatgcgt atcatgcgga tgaaattttt atgtgcacca ccgcgggcgg cattatgccg 840

attaccaaac tggatggcaa accgattcgc aatggcgaag tgggcccgct gactaccaaa 900

atttgggatg aatattgggc gatgcattat gatccgaaat atagcagcgc gattgattat 960

cgcggccatg aaggcaatta a 981

<210> 28

<211> 972

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 28

atggcgagca tggataaagt gtttagcggc tattatgcgc gtcagaaact gctggaacgc 60

agcgataatc cgtttagcaa aggcattgcg tatgtggaag gcaaactggt gctgccgagc 120

gatgcgcgta ttccgttatt ggatgaaggc tttatgcata gcgatctgac ctatgatgtg 180

attagcgtgt gggatggccg ctttttccgc ctggatgatc atctgcagcg cattctggaa 240

agctgcgata aaatgcgcct gaaatttccg ctggcgctga gcagcgtgaa aaatattctg 300

gcggaaatgg tggcgaaaag cggcattcgc gatgcgtttg tggaagtgat tgtgacccgc 360

ggcctgaccg gcgttagagg tagcaaacca gaagatctgt ataataataa catctacctg 420

ctggtgctgc cgtatatttg ggtgatggcg ccggaaaatc agctgcatgg cggcgaagcg 480

attattaccc gcaccgtgcg ccgcacccca ccaggtgcgt ttgatccaac cattaaaaat 540

ctgcagtggg gcgatctgac caaaggcctg tttgaagcga tggatcgcgg cgcgacctat 600

ccgtttctga ccgatggcga taccaatctg accgaaggca gcggctttaa tattgtgctg 660

gtgaaaaatg gcattatcta caccccggat cgcggcgtgc tgcgcggtat tactagaaaa 720

agcgtgattg atgtggcccg cgccaatagc attgatattc gcctggaagt ggtgccggtg 780

gaacaggcgt atcatagcga tgaaattttt atgtgcacca ccgcgggcgg cattatgccg 840

attaccctgc tggatggcca gccggtgaat gatggccagg tgggcccaat taccaagaaa 900

atttgggatg gctattggga gatgcattat aatccggcgt atagctttcc ggtggattat 960

ggcagcggct aa 972

<210> 29

<211> 978

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 29

atggcgagca tggataaagt gtttgcgggc tatgcggccc gccaggcgat tttagaaagc 60

accgaaacca ccaatccgtt tgcgaaaggc attgcgtggg tggaaggcga actggtgccg 120

ctggcagaag cgagaattcc attactggat cagggcttta tgcatagcga tctgacctat 180

gatgtgccga gcgtgtggga tggccgcttc tttcgcctgg atgatcatat tacccgcctg 240

gaagcgagct gcaccaaact gcgcttacgc ctgccactgc cgcgtgatca agtgaaacaa 300

attctggtgg aaatggtggc gaaaagcggc attcgcgatg cgtttgtgga actgattgtg 360

acccgcggcc tgaaaggcgt gcgcggtacc cgtccagaag atattgtgaa taatctgtat 420

atgttcgtgc agccgtatgt gtgggtgatg gaaccggata tgcagcgcgt gggcggcagc 480

gcggttgttg cgcgtactgt tcgtcgtgtt ccgccaggtg cgattgatcc gaccgtgaaa 540

aatctgcagt ggggcgatct ggtgcgcggc atgtttgaag cggcggatcg cggtgcgacc 600

tatccgtttt taaccgatgg cgatgcgcat ctgaccgaag gcagcggctt taatattgtg 660

ctggtgaaag atggcgtgct gtataccccg gatcgcggcg tgttgcaggg cgttacccgt 720

aaaagcgtga ttaatgcggc ggaagcgttt ggcattgaag tgcgcgtgga atttgtgccg 780

gtggaactgg cgtatcgctg cgatgaaatt tttatgtgca ccaccgcggg cggcattatg 840

ccgattacca ccctggatgg catgccggtg aatggcggcc aaattggccc aattaccaaa 900

aagatttggg acggctattg ggcgatgcat tatgatgcgg cgtatagctt tgaaattgat 960

tataatgagc gcaactga 978

<210> 30

<211> 993

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 30

atggcgttta gcgcggatac cagcgaaatt gtgtataccc atgataccgg cctggattat 60

attacctata gcgattatga actggacccg gcgaatccgc tggcgggcgg tgcggcgtgg 120

attgaaggtg cgtttgttcc gccgagcgaa gcgcgtatta gcatttttga tcagggctat 180

ctgcatagcg atgtgaccta taccgtgttt catgtgtgga atggcaatgc gtttcgcctg 240

gatgatcata ttgaacgcct gtttagcaat gcggaaagca tgcgcattat tccgccgctg 300

acccaggatg aagtgaaaga aattgcgctg gaactggtgg cgaaaaccga actgcgcgaa 360

gcgtttgtga gcgtgagcat tacccgcggc tatagcagca ccccgggcga aagagatatt 420

accaaacatc gcccgcaggt gtatatgtat gcggtgccgt atcagtggat tgtgccgttt 480

gatcgcattc gcgatggcgt gcatgcgatg gtggcgcaga gcgttcgtcg taccccacgt 540

agtagcattg atccgcaagt gaaaaatttt cagtggggcg atctgattcg cgcggtgcag 600

gaaacccatg atcgcggctt tgaagcgccg ctgctgctgg atggtgatgg cctgttagcg 660

gaaggtagcg gttttaatgt ggtggtgatt aaagatggcg tggtgcgcag cccgggccgc 720

gcggcgttac caggtattac cagaaaaacc gtgctggaaa ttgcggaaag cctgggccat 780

gaagcgattc tggcggatat taccctggcg gaactgctgg atgcggatga agtgctgggc 840

tgcaccaccg cgggtggtgt ttggccattt gttagcgtgg atggcaatcc gattagcgat 900

ggcgtgccgg gcccaattac ccagagtatt attcgccgct attgggaact gaatgtggaa 960

agcagcagcc tgctgacccc ggtgcaatat taa 993

<210> 31

<211> 993

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 31

atggcgttta gcgcggatac cccggaaatt gtgtataccc atgataccgg cctggattat 60

attacctata gcgattatga actggacccg gcgaatccgc tggcgggcgg tgcagcgtgg 120

attgaaggtg catttgttcc gccgagcgaa gcgcgtatta gcatttttga tcagggcttt 180

tataccagcg atgcgaccta taccaccttt catgtgtgga atggcaatgc gtttcgcctg 240

ggcgatcata ttgaacgcct gtttagcaat gcggaaagca ttcgcctgat tccgccgctg 300

acccaggatg aagtgaaaga aattgcgctg gaactggtgg cgaaaaccga actgcgcgaa 360

gcgatggtga ctgtgaccat tacccgcggc tatagcagca ccccgtttga acgcgatatt 420

accaaacatc gcccgcaggt gtatatgagc gcgtgcccgt atcagtggat tgtgccgttt 480

gatcgcattc gcgatggcgt gcatctgatg gtggcgcaga gcgtgcgtcg taccccacgt 540

agtagcattg atccacaggt gaaaaatttt cagtggggcg atctgattcg cgcgattcag 600

gaaacccatg atcgcggctt tgaactgccg ctgctgctgg attgcgataa tctgctggcg 660

gaaggcccgg gctttaatgt ggtggtgatt aaagatggcg tggtgcgcag cccgggccgc 720

gcggcgttac caggtattac cagaaaaacc gtgctggaaa ttgcggaaag cctgggccat 780

gaagcgattc tggcggatat taccccggcg gaactgtatg atgcggatga agtgctgggc 840

tgcagcaccg gcggtggtgt ttggccattt gtgagcgttg atggcaatag cattagcgat 900

ggcgtgccgg gcccggtgac tcaaagcatt attcgccgct attgggaact gaatgtggaa 960

ccgagcagcc tgctgacccc ggtgcaatat taa 993

<210> 32

<211> 993

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 32

atggcgttta gcgcggatac cgcggaaatt gtgtataccc atgataccgg cctggattat 60

attacctata gcgattatga actggacccg gcgaatccgc tggcgggcgg tgcggcgtgg 120

attgaaggtg cgtttgttcc gccagcggaa gcgcgtatta gcatttttga tcagggcttt 180

tataccagcg atgcgaccta taccaccttt catgtgtgga atggcaatgc gtttcgcctg 240

gatggccata ttgaacgcct gtttagcaat gcggaaagca ttcgcctgat tccgccgctg 300

acccaggatg aagtgaaaga aattgcgctg gaactggtgg cgaaaaccga actgcgcgaa 360

gcgatggtga cggtgagcat tacccgcggc tatagcagca ccccgtttga acgcgatatt 420

accaaacatc gcccgcaggt gtatatgagc gcgtgcccgt atcagtggat tgtgccgttt 480

gatcgcattc gcgatggcgt gcatctgatg gtggcgcaga gcgtgcgtcg caccccacgt 540

agtagcattg atccgcaggt gaaaaatttt cagtggggcg atctgattcg cgcgattcag 600

gaaacccatg atcgcggctt tgaactgccg ctgctgctgg atgcggataa tctgctggcg 660

gaaggcccgg gctttaatgt ggtggtgatt aaagatggcg tggtgcgcag cccgggccgc 720

gcggcattac caggtattac tcgtaaaacc gtgctggaaa ttgcggaaag cctgggccat 780

gaagcgattc tggcggatat taccccggcg gaactgtatg atgcggatga agtgctgggc 840

tgcagcaccg gcggtggtgt ttggccattt gttagcgttg atggcaatgc gattagcgat 900

ggcgccccgg gcccaattac ccagagcatt attcgccgct attgggaact gaatgtggaa 960

agcagcagcc tgctgacccc ggtgcaatat taa 993

<210> 33

<211> 993

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 33

atggcgttta gcgcggatac cgcggaaatt gtgtataccc atgataccgg cctggattat 60

attacctata gcgattatga actggacccg gcgaatccgc tggcgggcgg tgcggcgtgg 120

attgaaggtg cgtttgttcc gccaagcgaa gcgcgtatta gcatttttga tcagggcttt 180

tataccagcg atgcgaccta taccaccttt catgtgtgga atggcaatgc gtttcgcctg 240

gatggccata ttgaacgcct gtttagcaat gcggaaagca ttcgcctgat tccgccgctg 300

acccaggatg aagtgaaaga aattgcgctg gaactggtgg cgaaaaccga actgcgcgaa 360

gcgatggtga cggtgagcat tacccgcggc tatagcagca ccccgtttga acgcgatatt 420

accaaacatc gcccgcaggt gtatatgagc gcgtgcccgt atcagtggat tgtgccgttt 480

gatcgcattc gcgatggcgt gcatctgatg gtggcgcaga gcgtgcgtcg caccccacgt 540

agtagcattg atccgcaggt gaaaaatttt cagtggggcg atctgattcg cgcgattcag 600

gaaacccatg atcgcggctt tgaactgccg ctggcgctgg atgcggataa tctgctggcg 660

gaaggcccgg gctttaatgt ggtggtgatt aaagatggcg tggtgcgcag cccgggccgc 720

gcggcattac caggtattac tcgtaaaacc gtgctggaaa ttgcggaaag cctgggccat 780

gaagcgattc tggcggatat taccccggcg gaactgtatg atgcggatga agtgctgggc 840

tgcagcaccg gcggtggtgt ttggccattt gttagcgttg atggcaatgc gattagcgat 900

ggcgccccgg gcccaattac ccagagcatt attcgccgct attgggaact gaatgtggaa 960

agcagcagcc tgctgacccc ggtgcaatat taa 993

<210> 34

<211> 993

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 34

atggcgttta gcgcggatac cgcggaaatt gtgtataccc atgataccgg cctggattat 60

attacctata gcgattatga actggacccg gcgaatccgc tggcgggcgg tgcggcgtgg 120

attgaaggtg cgtttgttcc gccagcggaa gcgcgtatta gcatttttga tcagggcttt 180

gcgaccagcg atgcgaccta taccaccttt catgtgtgga atggcaatgc gtttcgcctg 240

gatggccata ttgaacgcct gtttagcaat gcggaaagca ttcgcctgat tccgccgctg 300

acccaggatg aagtgaaaga aattgcgctg gaactggtgg cgaaaaccga actgcgcgaa 360

gcgatggtga cggtgagcat tacccgcggc tatagcagca ccccgtttga acgcgatatt 420

accaaacatc gcccgcaggt gtatatgagc gcgtgcccgt atcagtggat tgtgccgttt 480

gatcgcattc gcgatggcgt gcatctgatg gtggcgcaga gcgtgcgtcg caccccacgt 540

agtagcattg atccgcaggt gaaaaatttt cagtggggcg atctgattcg cgcgattcag 600

gaaacccatg atcgcggctt tgaactgccg ctgctgctgg atgcggataa tctgctggcg 660

gaaggcccgg gctttaatgt ggtggtgatt aaagatggcg tggtgcgcag cccgggccgc 720

gcggcattac caggtattac tcgtaaaacc gtgctggaaa ttgcggaaag cctgggccat 780

gaagcgattc tggcggatat taccccggcg gaactgtatg atgcggatga agtgctgggc 840

tgcagcaccg gcggtggtgt ttggccattt gttagcgttg atggcaatgc gattagcgat 900

ggcgccccgg gcccaattac ccagagcatt attcgccgct attgggaact gaatgtggaa 960

agcagcagcc tgctgacccc ggtgcaatat taa 993

<210> 35

<211> 960

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 35

atggcgacca tggagaaaat ttttgcggcg tatcatgaac gccagaaact gctggcggcg 60

aatacccatc cgtttgcgaa aggcgtggcg tgggtggaag gcgaattaac cccattacat 120

gaagcgcgta ttccgattct ggatcagggc tttatgcata gcgatctgac ctatgatgtg 180

ccgagcgtgt gggatggccg cttctttcgc ctggatgatc atattacccg cctggaagcg 240

agctgcacca aactgcgcat gaaactgccg ctgccgcgcg atgaagtgaa acagattctg 300

gtggatatgg tggcgaaaag cggcattcgc gatgcgtttg tggaaattat tgtgacccgc 360

ggcctgaaag gcgtgcgcgg tagtcgtcca gaagatattg ttaatcgcat ttatatgttc 420

atccagccgt atgtgtggtg catggaaccg gaagtgcagc cggtgggcgg tagcgcaatt 480

attgcgcgta ccgtgcgccg tgtgccgccg ggttgcattg atccaaccgt taaaaatctg 540

cagtggggcg atctggtgcg cggcttattt gaagcgagcg atcgcggcgc ggaatatccg 600

tttctgaccg atggcgatac caatctgacc gaaggcagcg gctttaatat tgtgctggtg 660

aaagataata ttctgtacac cccggcgcgt ggcgtgctgg aaggtgttac ccgtaaaagc 720

gtgattgatg tggcgcgtgc cagcggcttt gatattaaag tggaactggt gccggtgcag 780

atggcgtatg atgcggatga aatttttatg tgcaccaccg cgggcggcat tatgccgatt 840

accagcctgg atggcaaacc ggtgaatgat ggcaaagtgg gcagcgtgac caaaaagatt 900

tgggatggct attgggcgat tcattatgat ccggcgtata gctttgaaat tgcgtattaa 960

<210> 36

<211> 978

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 36

atgagcacga tggataaaat ttttgcgggc catgcgcagc gccaggcgac cttagttgcg 60

agcgataata tttttgcgaa tggcattgcg tggattcagg gcgaactggt gccgctgaat 120

gaagcgcgta ttccgctgat ggatcagggc tttatgcatg gcgatctgac ctatgatgtg 180

ccggcggtgt gggatggccg ctttttccgc ctggatgatc atctggatcg cctggaagcg 240

agcgtgaaga aaatgcgcat gcagtttccg attccgcgcg atgaaattcg catgaccctg 300

ctggatatgc tggcgaaaag cggcattaaa gatgcgtttg tggaactgat tgtgacccgc 360

ggcctgaaac cggtgcgcga agcgaaacca ggcgaagtgt taaataatca tctgtatctg 420

attgtgcagc cgtatgtgtg ggtgatgagc ccggaagcgc agtatgtggg cggcaatgcg 480

gttattgccc gcaccgttcg ccgtattccg ccgggtagca tggatccgac tattaaaaat 540

ctgcagtgga gcgattttac ccgcggcatg tttgaagcgt atgatcgcgg cgcccagtat 600

ccgtttctga ccgatggcga taccaatatt accgaaggca gcggctttaa tgtggtgttt 660

gtgaaaaata atgtgatcta caccccgaac cgcggcgtgc tgcagggcat tacccgtaaa 720

agcgtgattg atgcggcgaa atggtgcggc catgaagtgc gcgttgaata tgtgccggtg 780

gaaatggcgt atgaagcgga tgaaattttt atgtgcacca ccgcgggcgg cattatgccg 840

attaccacga tggatggcaa accggtgaaa gatggcaaag tgggcccagt gaccaaagcg 900

atttgggatc gctattgggc gatgcattgg gaagatgaat ttagctttaa aatcgactac 960

cagaagctga aactgtaa 978

Claims

1.一种生物酶法催化合成(R)-2-(1-氨乙基)-4-氟苯酚的方法，其特征在于，包括以下步骤：

(2)培养所述转氨酶重组表达菌株，诱导转氨酶的表达；

(4)利用含有步骤(3)中的具有目标活性的转氨酶的生物材料为催化剂，以5-氟-2-羟基苯乙酮作为底物，在水相缓冲体系里进行目标转氨反应，得到(R)-2-(1-氨乙基)-4-氟苯酚粗品；

2.根据权利要求1所述的生物酶法催化合成(R)-2-(1-氨乙基)-4-氟苯酚的方法，其特征在于，所述转氨反应的温度为20℃～60℃，反应时间为1小时～72小时。

3.根据权利要求1所述的生物酶法催化合成(R)-2-(1-氨乙基)-4-氟苯酚的方法，其特征在于，所述水相缓冲体系的pH为6.0～11.0。

4.根据权利要求1所述的生物酶法催化合成(R)-2-(1-氨乙基)-4-氟苯酚的方法，其特征在于，所述水相缓冲体系包括磷酸钾缓冲液、三乙醇胺缓冲液、甘氨酸-NaOH缓冲液，其浓度为0.1M。

5.根据权利要求1所述的生物酶法催化合成(R)-2-(1-氨乙基)-4-氟苯酚的方法，其特征在于，所述转氨反应中的氨基供体包括(R)-苯乙胺、(R)-1-(4-溴苯基)乙胺、(R)-1-苯丙胺、(R)-1,2,3,4-四氢-1-萘胺、(R)-1-Boc-3-氨基吡咯烷、异丙胺、仲丁胺、2-戊胺、2-氨基庚烷。

6.根据权利要求1所述的生物酶法催化合成(R)-2-(1-氨乙基)-4-氟苯酚的方法，其特征在于，所述转氨反应中的辅酶为磷酸吡哆醛，其使用浓度为0.1mM～5.0mM。

7.根据权利要求1所述的生物酶法催化合成(R)-2-(1-氨乙基)-4-氟苯酚的方法，其特征在于，所述转氨反应中助溶剂包括二甲亚砜、乙腈、甲基叔丁基醚、N,N-二甲基甲酰胺、甲醇、乙醇、异丙醇，其浓度分数为5％～50％。

8.根据权利要求1所述的生物酶法催化合成(R)-2-(1-氨乙基)-4-氟苯酚的方法，其特征在于，所述具有目标活性的转氨酶为ω-转氨酶，其氨基酸序列如SEQ ID No.8～SEQ IDNo.16所示。

9.根据权利要求1所述的生物酶法催化合成(R)-2-(1-氨乙基)-4-氟苯酚的方法，其特征在于，所述重组表达菌株包括大肠杆菌、枯草芽孢杆菌、谷氨酸棒状杆菌、酵母。

10.一种具有催化合成(R)-2-(1-氨乙基)-4-氟苯酚活性的转氨酶，其特征在于，所述转氨酶的氨基酸序列分别如SEQ ID No.8～SEQ ID No.16所示，其核苷酸序列分别如SEQID No.26～SEQ ID No.34所示。