CN111363732B - 来源于土曲霉菌nih2624的转氨酶突变体及其应用 - Google Patents
来源于土曲霉菌nih2624的转氨酶突变体及其应用 Download PDFInfo
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- CN111363732B CN111363732B CN202010172593.5A CN202010172593A CN111363732B CN 111363732 B CN111363732 B CN 111363732B CN 202010172593 A CN202010172593 A CN 202010172593A CN 111363732 B CN111363732 B CN 111363732B
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Abstract
本发明公开涉及来源于土曲霉菌Aspergillus terreus NIH2624转氨酶突变体的多肽、编码所述多肽的多核苷酸以及使用所述多肽在(R)‑3‑氨基‑1‑苄基哌啶制备上的应用。本发明通过计算机模拟为指导定向改造土曲霉菌Aspergillus terreus NIH2624转氨酶，改造氨基酸位点选自Y60、R79、W184、E213、G216、F217、N218、L235、G237、V238、T239、C273、T274及T275中的一个或多个。经改造获得的突变体应用于(R)‑3‑氨基‑1‑苄基哌啶的生产，具有优良的立体选择性和催化活力，且其催化的反应温和，在相同的生产条件下，其活力相对于野生型转氨酶提高了86.96倍，产物手性纯度及收率均有明显提高，具有较好的应用前景。
Description
技术领域：
本发明属于生物制药领域，具体涉及一种来源于土曲霉菌NIH2624的转氨酶突变体及其应用。
背景技术：
2017年底，国际糖尿病联盟(IDF)公布了第八版的全球糖尿病概览。结果显示，全球糖尿病成人患者(20-79岁)从2000年的1.51亿，到2017年已达到4.25亿，增加近2倍。预计到2045年，糖尿病患者可能达到6.29亿。糖尿病主要是由于机体胰岛素分泌不足或胰岛素作用障碍引起的以高血糖为特征的代谢疾病。其中胰岛素分泌不足被定义为Ⅰ型、胰岛素作用障碍被定义为Ⅱ型糖尿病，Ⅱ型糖尿病患者占糖尿病患者的90％以上。目前治疗Ⅱ型糖尿病的药物主要分为磺酰脲类、双胍类、α-葡萄糖苷酶抑制剂、胰岛素增敏剂、噻唑烷二酮类、二肽基肽酶IV(DPP-IV)抑制剂等几大类，其中目前公认最为高效安全的是(DPP-IV)抑制剂，该类药物能够选择性与二肽基肽酶IV 可逆性结合从而抑制二肽基肽酶IV的活性，进而延缓胰高血糖素样肽 -1(GLP-1)和抑胃肽(GIP)降解，从而调节糖尿病Ⅱ型患者的血糖水平。
目前(DPP-IV)抑制剂类降血糖药物阿格列汀、曲格列汀及利拉列汀的重要药物中间体(R)-3-氨基-1-苄基哌啶，在总生产成本中占有较大比例。现在主要生产工艺是采用化学还原的方法制备手性胺，即在Pd/C的催化作用下，前手性酮与甲酸以及无机氨/有机伯胺进行反应生成外消旋胺。此类反应中金属催化剂是非常关键的因素，且对金属催化剂要求苛刻，反应需要在高压条件下完成，操作设备要求高，同时产生大量含重金属离子的废水难以处理，对环境造成较大污染。另外，金属催化合成所需构型的手性胺含量通常为50％，远远不能满足手性胺含量≥99.5％的需求，因此需要后续进行拆分精制，在浪费一半的产品、降低生产效率的同时，还产生大量废有机溶剂，加大了环保处理的压力。
发明内容：
为解决上述问题，本发明以来源于土曲霉菌 Aspergillus terreus NIH2624的转氨酶基因XP_001209325.1为研究对象，通过计算机模拟软件对XP_001209325.1理性预测并进行定点突变，以提高转氨酶的选择性及活性。
本发明的主要目的是提高来源于土曲霉菌Aspergillus terreus NIH2624 的转氨酶对非天然底物1-苄基-3-哌啶酮催化活力，得到酶活显著提高的转氨酶突变体，解决现有(R)-3-氨基-1-苄基哌啶合成技术中存在的缺陷。
本发明的技术方案如下：
步骤1：候选转氨酶与反应底物1-苄基-3-哌啶酮的分子对接评估(三级结构未鉴定的候选转氨酶以同源建模的形成完成对接)。
步骤2：针对分子对接结果优选评估分数较高的转氨酶进行活性测试。
步骤3：选择催化活性与立体选择性最高的Aspergillus terreus NIH2624 的转氨酶作为本发明的野生型转氨酶。
步骤4：利用模拟软件虚拟突变Aspergillus terreus NIH2624转氨酶活性中心周围单个氨基酸。
步骤5:Aspergillus terreus NIH2624转氨酶活性中心氨基酸区域虚拟组合突变。
步骤6：Aspergillus terreus NIH2624转氨酶活性中心氨基酸区域虚拟组合突变体活性测试
步骤7：1-苄基-3-哌啶酮的催化条件优化。
本发明所述的分区域，指将15个单突变位点分为Ⅰ区(60位氨基酸)、Ⅱ区(79位氨基酸)、Ⅲ区(184位氨基酸)、Ⅳ区(213、216、217、218 位氨基酸)、Ⅴ区(235、237、238、239、240位氨基酸)、Ⅵ区(273、274、 275位氨基酸)。
本发明所述的区域组合，指将6个区域进行2-6个区域中1-6个突变位点组合得到转氨酶组合突变体。
本发明还提供了一类转氨酶组合突变体，所述的转氨酶组合突变体来源于上述的区域组合所述。
本发明还提供了所述转氨酶突变体的编码多核苷酸序列。
本发明还提供了所述转氨酶突变体的编码多核苷酸序列的表达载体与工程菌。
本发明还提供了所述转氨酶突变体在催化1-苄基-3-哌啶酮制备(R)-3- 氨基-1-苄基哌啶的应用。
本发明还提供了一种催化1-苄基-3-哌啶酮制备(R)-3-氨基-1-苄基哌啶的方法，包括：
(1)制备表达转氨酶突变体的基因工程菌，所述的转氨酶突变体来源于上述的区域组合所述一种或多种；
(2)培养所述基因工程菌，制备酶液；
步骤(2)中，所述的酶液最优为粗酶液进行纯化获得的纯酶，当然基因工程菌的静息细胞悬液或者破胞粗酶液也适用本发明所述的(R)-3-氨基-1-苄基哌啶制备方法。
(3)将所述酶液加入含有底物1-苄基-3-哌啶酮、异丙氨及PLP的混合体系中，进行转氨反应，制得(R)-3-氨基-1-苄基哌啶。
在上述(R)-3-氨基-1-苄基哌啶的酶催化制备方法中，作为优选，所述酶催化反应的温度为35℃～37℃，该温度区间内具有反应条件温和转化率高的优点。
在上述(R)-3-氨基-1-苄基哌啶的酶催化制备方法中，作为优选，所述酶催化反应在pH值为8.0～9.0。该pH区间能更有效的保证酶的活性，提高酶催化反应的效果。
在上述(R)-3-氨基-1-苄基哌啶的酶催化制备方法中，作为优选，所述酶催化反应还在磷酸盐缓冲液存在下进行。通过该缓冲液的存在，能够反应体系处于一定较平稳的pH值体系，避免体系中的pH值变化过大，更有效的保证反应的稳定进行。
在上述(R)-3-氨基-1-苄基哌啶的酶催化制备方法中，作为优选，所述酶催化反应在最适合酶用量为8mg/ml。
综上所述，本发明与现有技术相比，具有以下优点：
通过采用本发明的具有如SEQ ID NO：2所示序列具有至少约85％同一性的氨基酸序列的相应转氨酶，在相应的催化体系下，能够有效的使底物1-苄基-3-哌啶酮转化成高手性的产物(R)-3-氨基-1-苄基哌啶，且具有收率高的优点，解决现有(R)-3-氨基-1-苄基哌啶合成技术中存在的缺陷。
附图说明：
图1：1-苄基-3-哌啶酮与Aspergillus terreus NIH2624转氨酶相互作用图；
图2：1-苄基-3-哌啶酮与Aspergillus terreus NIH2624转氨酶相互作用图二维平面图；
图3：转氨酶酶液SDS-PAGE电泳检测图。
具体实施方式：
下面结合具体的实施例，对本发明实施例中的技术方案进行清楚、完整地描述。应理解，所描述的实施例是本发明一部分实施例，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
为了使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解，下面结合具体实施例，进一步阐述本发明。
实施例1候选转氨酶分子对接评估
以pymol、AutoDock等计算机模拟软件结合使用，完成对本发明中未进行三级结构解析的蛋白进行同源蛋白建模并评估，得到本发明所需的转氨酶。反应底物1-苄基-3-哌啶酮与候选转氨酶进行分子对接，将RMSD阈值设为 0.5埃以确保对接构象尽可能具有多样性，选择打分函数最高的对接方式。综合评价本发明的12个候选转氨酶，选择其中包括Aspergillus terreus NIH2624转氨酶在内的6个候选转氨酶进行后续的转氨酶进行活性测试。
实施例2 6个候选转氨酶催化活性评价
将6个候选转氨酶mRNA序列进行密码子优化并加上相关调控序列后(片段两侧加BglII和XhoI内切酶基因片段)送往安徽通用生物有限公司进行合成。
重组质粒转染：
1.从-80℃取大肠杆菌感受态细胞BL21，室温下置于冰盒解冻。
2.向感受态细胞中加入1ul质粒，置于4℃冰箱或冰盒30min.
3. 42℃水浴锅中热激120s，立即放于冰盒上2min。
4.每管加入900ul的恢复培养基，摇床150rpm、37℃培养45min。
5.低速离心取管底50ul转化液涂布于含有工作浓度的kan的培养皿中。
6.倒置于37℃培养箱，培养过夜。
工程菌诱导及酶液制备：
待转化子出现后，将转化子逐个挑至10ml试管中，每管中加入1mL含有工作浓度50ug/mlKan的培养基中，37℃，220rpm培养,测试其OD600值，在3 小时左右OD600达到0.6左右，加入工作浓度为0.5mm的IPTG并诱导6h左右，离心弃上清，菌体用缓冲液重悬，用超声波细胞粉碎机进行细胞破碎，其参数选择为3号变幅杆、总工作时间为600s、工作周期为超声2s间隔5s、输出功率为217W，获得含有转氨酶大肠杆菌细胞裂解液。
转氨酶大肠杆菌细胞裂解液酶触反应操作：
1.配制PBS缓冲液：配制0.1M，pH 8.0的PBS缓冲液。
2.称取底物1-苄基-3-哌啶酮，溶于乙腈中，配制成100mg/mL的乙腈溶液。
3.称取PLP，溶于PBS缓冲液中，配制成50mg/mL的水溶液。
4.向96孔板中依次加入酶10mg、PBS缓冲液、底物的乙腈溶液100μL (10％v/v)、相当于底物4摩尔当量的异丙胺(4eq)、PLP溶液100μL，反应液总体积为1mL。(异丙胺的体积从PBS中扣除；如酶为液体，则其体积也从缓冲液中扣除)
5.将96孔板放入35℃摇床振荡24小时。
6.取样100μL，以乙腈稀释10倍，0.45μm滤膜过滤，LC/MS检测/手性 GC检测。
经检测野生型Aspergillus terreus NIH2624转氨酶催化1-苄基-3-哌啶酮转化成高手性的产物(R)-3-氨基-1-苄基哌啶的能力基本符合预期，这与实施例1中的对接评价结果基本一致。因此选择 Aspergillus terreus NIH2624转氨酶为本发明的改造对象。
实施例3虚拟突变Aspergillus terreus NIH2624转氨酶活性中心周围单个氨基酸
本发明对Aspergillus terreus NIH2624转氨酶进行虚拟突变，利用分子模拟软件对Aspergillus terreus NIH2624转氨酶进行理性设计，选定突变位点，能有效的节省突变位点筛选的时间，提高突变效率。
本发明以实施例1中对接结果，进行数据分析，确定活性区域内各个氨基酸的空间距离与作用关系。
根据来源于实施例1的相互作用图分析，选择进行单突变的位点分别为 Y60、R79、W184、E213、G216、F217、N218、L235、G237、V238、T239、C273、 T274、T275。
虚拟突变结果如表所示
表中突变能在-0.5以下，Effect为stabilizing，即这种突变会导致亲和力上升，相互作用关系增强。
根据模拟软件对Aspergillus terreus NIH2624转氨酶进行虚拟突变的结果预测Y60K/R/W、R79K、W184R/K、E213R/K/LM/A/Q/G/P/T/Y/N/I/V /H/C/S、G216R/V/K、F217R/K、N218R/K/H/F、L235K/R、G237K/R/I、V238R/K、 T239Q/L/M/Y/N/K/R/H、C273H/M/K/R/Y/W、T274K/R/M/W/Y/F、及T275K/R/I/G /C/V/L/Y/F/W/A/N能够有效提高Aspergillus terreusNIH2624转氨酶的酶活，其中，“/”表示“或”。
实施例4 Aspergillus terreus NIH2624转氨酶活性中心周围氨基酸区域虚拟组合突变
组合突变以氨基酸位点单突变结果中表现较好的区域进行组合即：以Ⅰ区(60位氨基酸)、Ⅱ区(79位氨基酸)、Ⅲ区(184位氨基酸)、Ⅳ区(213 位氨基酸)、Ⅴ区(238位氨基酸)、Ⅵ区(275位氨基酸)进行6区域同时虚拟突变。
(表中突变能在-0.5以下，Effect为stabilizing，即这种突变会导致亲和力上升，相互作用关系增强。该突变组合总数为2304个，表中仅显示突变影响最大的5个突变组合。)
根据表可知当以Ⅰ区(60位氨基酸)、Ⅱ区(79位氨基酸)、Ⅲ区(184 位氨基酸)、Ⅳ区(213位氨基酸)、Ⅴ区(238位氨基酸)、Ⅵ区(275位氨基酸)进行6区域同时突变的情况下突变体以 TYR60>LYS.ARG79>LYS.TRP184>LYS.GLU213>ARG.VAL238>LYS.THR275>LYS方式进行突变其突变能最小相应的亲和力增加也最多。
实施例5 Aspergillus terreus NIH2624转氨酶组合突变能最小5序列突变
本发明采取利用全质粒PCR引入定点突变的方法对候选突变位点进行突变。其原理是：设计一对包含突变位点的引物，与待突变模版质粒退火后用聚合酶“循环延伸”。待反应结束以后用Dpn I酶切延伸产物，原始待突变模板来源于大肠杆菌，是经dam甲基化修饰的，能够被Dpn I进行酶切，而通过 PCR扩增的突变序列没有经过甲基化这一过程，因此得以保留。
本发明以突变点为中心，根据突变位点所在区域进行PCR引物设计完成全质粒PCR引入定点突变，此处以Y60K为例进行说明：
(1)A60定点全质粒PCR突变
Y60K-F ACTCTGACCTGACCAAAGATGTACCGTCCGTAT
Y60K-R ACGGACGGTACATCTTTGGTCAGGTCAGAGTGC
表1 Aspergillus terreus NIH2624转氨酶Y60K全质粒PCR体系
其反应程序为：95℃预变性5min，95℃变性10s，55℃退火5s，72℃延伸2min，反应进行28个循环之后，72℃再延伸10min。
(2)DpnI酶消化PCR纯化产物
将PCR产物用DpnI进行消化，其消化的体系见表2。
表2消化PCR产物的体系
(3)质粒转化入DH5α感受态细胞
1.从-80℃取大肠杆菌感受态细胞DH5α，室温下置于冰盒解冻。
2.向感受态细胞中加入1ul质粒，置于4℃冰箱或冰盒30min.
3. 42℃水浴锅中热激120s，立即放于冰盒上2min。
4.每管加入900ul的恢复培养基，摇床150rpm、37℃培养45min。
5.低速离心取管底50ul转化液涂布于含有工作浓度的kan的培养皿中。
6.倒置于37℃培养箱，培养过夜。
7.提取得到突变质粒。
本发明将得到的突变质粒按照实施例1再次进行转染大肠杆菌感受态细胞BL21与进行活性检测。
本发明以Ⅰ区(60位氨基酸)、Ⅱ区(79位氨基酸)、Ⅲ区(184位氨基酸)、Ⅳ区(213位氨基酸)、Ⅴ区(238位氨基酸)、Ⅵ区(275位氨基酸)进行6区域同时虚拟突变中突变能最小的前5个组合进行实践突变并如实施例2中方法进行活性测试，结果发现5个突变体都出现了不同程度的活性提高。突变位点基因与催化活性变化如下表所示：
根据上表所示，以Ⅰ区(60位氨基酸)、Ⅱ区(79位氨基酸)、Ⅲ区(184 位氨基酸)、Ⅳ区(213位氨基酸)、Ⅴ区(238位氨基酸)、Ⅵ区(275位氨基酸)进行6区域同时虚拟突变，突变能最小的前5个组合中以SEQ ID NO：12的综合性质最佳：酶活提高倍数达到了86.96、同时ee值也达了99.53。
其中上述SEQ ID NO：4～12多肽序列对应的多核苷酸序列依次如下，如SEQ IDNO：3、SEQ ID NO：5、SEQ ID NO：7、SEQ ID NO：9、SEQ ID NO： 11。
实施例6 1-苄基-3-哌啶酮的催化条件优化
1、最适作用pH分析
采用pH值分别为5.0、5.5、6.0、6.5、7.0、7.5、8.0、8.5、9.0、9.5的PBS缓冲液，将实施例4发酵获得的酶液进行稀释测定，在37℃下进行转氨酶活力测定，计算酶活，结果显示，本发明提供的转氨酶突变体 SEQ ID NO：16的最适作用pH值为8.0-9.0区间，与野生型Aspergillus terreus NIH2624转氨酶基本一致。
2、最适反应温度分析
分别在30℃、31℃、32℃、33℃、34℃、35℃、36℃、37℃、38℃、 39℃，pH为8.0条件下，对实施例4发酵获得的酶液进行转氨酶酶活测定，以最高酶活为100％，计算相对酶活，结果显示，野生型 Aspergillus terreus NIH2624转氨酶的最适作用温度为30℃；而本发明提供的Aspergillus terreus NIH2624转氨酶突SEQ ID NO：16的最适作用温度为35-37℃，相较野生型有了较大提高。
以上实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各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；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
序列表
<110> 重庆迪维斯生物科技有限公司
<120> 来源于土曲霉菌NIH2624的转氨酶突变体及其应用
<130> 2020.03.12
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 978
<212> DNA
<213> AspergillusterreusNIH2624
<400> 1
atggcctcca tggacaaagt ctttgccggc tacgccgccc gccaagcgat cctcgaatca 60
accgagacca ccaacccctt tgcgaagggt atcgcctggg tagaaggcga gctggtgccc 120
ctggcagagg cacgcattcc actgctcgac cagggcttca tgcacagcga tctcacctac 180
gacgtgccct ccgtctggga cggccgcttc ttccggctag acgaccacat cacgcggctc 240
gaagccagct gcaccaagct ccggctgcga ctgccactcc cgcgcgacca ggtcaagcag 300
attctcgtcg agatggtggc caagagcggc atccgcgacg cctttgtcga gctgatcgtg 360
acgcgcgggc tgaagggcgt gcgggggaca cgccccgagg acatcgtcaa caatctgtac 420
atgtttgtgc agccgtacgt gtgggtgatg gagccggata tgcagcgtgt cggcggcagc 480
gcggtcgtcg cccgcaccgt gcgccgggtg cccccgggtg ccatcgaccc aaccgtcaag 540
aacctgcaat ggggcgatct cgtgcgcggc atgttcgagg ctgcggatcg cggtgcaact 600
tatccgttct tgacggacgg agatgcccat ctcaccgaag gctctgggtt caatattgtg 660
ctcgtcaagg acggcgtgct gtacacacca gaccgtggtg tgctgcaggg cgtgacacga 720
aagagtgtta tcaatgcggc ggaagccttc gggattgaag tccgcgttga gtttgtgccg 780
gttgagctgg cgtaccgttg tgatgagatc tttatgtgta ccaccgctgg cggcatcatg 840
cctatcacta cgctggatgg gatgcccgtg aatggaggac agatcggtcc tattacgaag 900
aagatttggg atggatattg ggctatgcat tatgatgcgg cttacagctt cgagattgat 960
tataacgaga ggaactga 978
<210> 2
<211> 325
<212> PRT
<213> AspergillusterreusNIH2624
<400> 2
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> 3
<211> 978
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 3
atggcatcta tggataaagt gttcgcaggt tatgcggcgc gtcaggcaat cctggaatct 60
acggaaacca ccaacccatt cgctaaaggc attgcctggg tagaaggtga actggttccg 120
ctggcggaag cgcgtattcc tctgctggac cagggtttca tgcactctga cctgaccaag 180
gatgtaccgt ccgtatggga tggccgcttt ttccgtctgg atgatcatat taccaaactg 240
gaagcttcct gtaccaaact gcgtctgcgt ctgccactgc cacgtgatca agttaaacaa 300
atcctggttg aaatggttgc caaaagcggt atccgcgatg ccttcgtcga actgatcgta 360
acccgtggcc tgaaaggtgt ccgtggtacg cgtccggaag acattgttaa caacctgtac 420
atgtttgttc agccgtacgt ttgggttatg gaaccggaca tgcaacgtgt gggtggctct 480
gcggttgtcg ctcgtaccgt gcgtcgtgta cctccgggtg ctatcgatcc gaccgttaaa 540
aacctgcaga aaggcgacct ggttcgtggt atgttcgagg ccgctgatcg cggtgcaact 600
tatccgttcc tgactgacgg tgacgctcac ctgacccgtg gttctggctt caacatcgtt 660
ctggtaaaag atggcgttct gtatacccca gaccgcggcg ttctgcaggg taagactcgt 720
aaatccgtta tcaacgctgc ggaagctttt ggcatcgaag tacgtgtaga attcgtgccg 780
gttgaactgg cttatcgctg tgatgaaatc ttcatgtgca ctaaagcagg tggtatcatg 840
ccgatcacta ctctggatgg catgccggtc aacggtggcc agatcggtcc aatcaccaag 900
aaaatctggg acggctactg ggcgatgcac tacgacgctg cgtacagctt cgaaatcgac 960
tacaacgaac gcaactga 978
<210> 4
<211> 325
<212> PRT
<213> 人工序列(Artificial sequence)
<400> 4
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 Lys Asp Val Pro Ser
50 55 60
Val Trp Asp Gly Arg Phe Phe Arg Leu Asp Asp His Ile Thr Lys 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 Lys 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 Arg 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 Lys 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 Lys 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> 5
<211> 978
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 5
atggcttcta tggataaagt gttcgcgggc tacgcagccc gtcaagctat cctggaatcc 60
accgaaacca ccaacccttt cgcgaaaggt atcgcatggg tagaaggcga actggttcca 120
ctggcagaag cgcgtatccc gctgctggac cagggtttca tgcactccga cctgaccaaa 180
gacgttccga gcgtctggga cggtcgcttt ttccgtctgg atgaccatat caccaaactg 240
gaagcgtctt gcaccaaact gcgtctgcgt ctgcctctgc cgcgtgatca ggtaaagcag 300
atcctggtgg aaatggtggc taaaagcggt atccgcgacg cttttgtcga actgattgta 360
acccgcggtc tgaaaggtgt acgcggcacg cgtccggagg atattgtaaa caacctgtat 420
atgttcgttc agccatacgt ttgggtgatg gaaccggaca tgcaacgtgt aggtggctct 480
gctgttgtgg ctcgtactgt tcgtcgtgta ccaccgggcg ctattgatcc gaccgtgaag 540
aatctgcagc gtggtgacct ggtgcgtggc atgttcgagg ctgcagaccg tggtgcaacc 600
tacccgtttc tgactgatgg cgatgcgcat ctgaccaaag gttctggttt caacatcgtg 660
ctggtgaaag atggcgttct gtacaccccg gaccgtggtg ttctgcaggg caaaacccgt 720
aaatctgtta tcaacgctgc ggaagctttc ggcattgaag ttcgcgttga atttgtgccg 780
gtggagctgg cataccgttg cgatgaaatc ttcatgtgta ccaaggcagg tggtatcatg 840
ccgattacca ctctggacgg catgcctgtg aacggtggtc aaatcggtcc gattaccaaa 900
aaaatctggg atggttactg ggcaatgcac tacgatgcgg cctactcctt tgaaattgac 960
tataacgaac gcaactga 978
<210> 6
<211> 325
<212> PRT
<213> 人工序列(Artificial sequence)
<400> 6
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 Lys Asp Val Pro Ser
50 55 60
Val Trp Asp Gly Arg Phe Phe Arg Leu Asp Asp His Ile Thr Lys 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 Arg 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 Lys 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 Lys 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 Lys 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> 7
<211> 978
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 7
atggcttcca tggataaagt atttgcaggc tacgctgcac gtcaggcaat cctggaaagc 60
accgaaacca ccaacccttt cgccaagggc atcgcgtggg tagaaggcga actggtaccg 120
ctggcagaag ctcgcattcc gctgctggat cagggcttca tgcattctga cctgacttgg 180
gatgttccgt ctgtgtggga cggccgtttc ttccgtctgg atgaccacat cactaagctg 240
gaagcttcct gtaccaaact gcgtctgcgt ctgccgctgc cacgtgatca ggttaaacag 300
attctggtag aaatggttgc taagtctggt atccgtgacg ctttcgttga actgatcgtg 360
acccgtggcc tgaaaggtgt tcgtggcacc cgtccggagg atatcgtcaa caacctgtat 420
atgttcgtgc aaccttacgt ttgggtaatg gaaccggaca tgcagcgtgt tggcggttct 480
gcagtggtag cgcgtactgt tcgtcgtgta cctccaggcg ctatcgaccc gaccgtgaaa 540
aatctgcagc gtggtgacct ggttcgtggt atgttcgaag cggcggaccg tggtgcgact 600
tacccattcc tgactgacgg tgatgcgcac ctgacccgtg gtagcggttt caacatcgtg 660
ctggttaaag acggcgttct gtataccccg gatcgcggtg tgctgcaagg caagacccgc 720
aaatctgtta tcaacgcagc tgaagcattt ggcattgaag tacgcgtaga gtttgtccca 780
gtagaactgg cctaccgttg cgatgaaatt ttcatgtgca ctaaagccgg tggcattatg 840
ccgatcacta ctctggatgg tatgccagta aacggcggtc agatcggtcc gattaccaaa 900
aaaatctggg atggttactg ggctatgcac tacgacgcgg cgtattcctt tgagatcgat 960
tacaacgagc gtaactga 978
<210> 8
<211> 325
<212> PRT
<213> 人工序列(Artificial sequence)
<400> 8
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 Trp Asp Val Pro Ser
50 55 60
Val Trp Asp Gly Arg Phe Phe Arg Leu Asp Asp His Ile Thr Lys 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 Arg 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 Arg 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 Lys 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 Lys 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> 9
<211> 978
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 9
atggcgtcta tggataaagt tttcgctggt tatgcagccc gtcaggctat tctggaatcc 60
accgaaacca ccaacccgtt cgctaaaggt atcgcttggg ttgagggcga actggtgccg 120
ctggctgaag cccgcatccc gctgctggat caaggtttta tgcactccga cctgactcgc 180
gatgtaccgt ccgtatggga tggtcgtttc ttccgtctgg acgaccacat tactaaactg 240
gaagcgagct gcaccaaact gcgtctgcgt ctgccgctgc cgcgcgatca agtgaaacag 300
attctggtgg agatggtagc gaagtctggc atccgtgacg cttttgtgga actgatcgtg 360
acccgcggcc tgaaaggcgt tcgtggcacc cgcccggaag acatcgtgaa taacctgtac 420
atgtttgtcc agccgtacgt atgggtcatg gaaccggata tgcagcgtgt cggcggttct 480
gcagttgtgg cccgtaccgt ccgtcgcgta ccgccaggcg ctattgaccc aaccgtgaaa 540
aacctgcaga agggtgatct ggttcgtggc atgttcgaag cggccgatcg tggcgcgact 600
tatccgttcc tgactgatgg cgacgctcac ctgaccaaag gctctggctt caacatcgta 660
ctggtaaaag atggcgtact gtacactccg gaccgcggtg tgctgcaggg caaaacccgt 720
aaatccgtga tcaacgcggc cgaggccttt ggtatcgaag ttcgcgttga attcgttccg 780
gttgagctgg cctaccgttg cgatgaaatc ttcatgtgca cccgtgcagg tggtattatg 840
cctatcacca cgctggacgg tatgccggtg aacggtggtc agattggtcc gatcactaag 900
aagatttggg acggctactg ggcaatgcat tacgacgcgg cttattcctt tgaaatcgat 960
tataatgaac gcaattga 978
<210> 10
<211> 325
<212> PRT
<213> 人工序列(Artificial sequence)
<400> 10
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 Arg Asp Val Pro Ser
50 55 60
Val Trp Asp Gly Arg Phe Phe Arg Leu Asp Asp His Ile Thr Lys 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 Lys 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 Lys 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 Lys 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 Arg 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> 11
<211> 978
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 11
atggctagca tggacaaggt tttcgctggt tatgccgcac gtcaggctat cctggaatcc 60
accgaaacca cgaacccgtt cgccaaaggt atcgcctggg ttgaaggcga actggtccct 120
ctggcagaag cgcgtatccc gctgctggat cagggcttta tgcactctga cctgacgcgt 180
gatgtgccgt ccgtttggga tggtcgtttt tttcgtctgg atgatcacat tactaaactg 240
gaggcatcct gtactaaact gcgtctgcgt ctgccactgc cacgtgatca ggtgaaacag 300
atcctggttg aaatggtagc caaatccggt atccgcgatg cgttcgtcga actgatcgtt 360
actcgtggcc tgaaaggcgt tcgtggtact cgtccggaag acatcgttaa caatctgtat 420
atgttcgttc agccgtacgt gtgggtaatg gaaccagaca tgcagcgtgt tggtggctct 480
gccgttgtcg ctcgtactgt ccgtcgtgta ccgccgggtg ctatcgatcc gaccgtcaaa 540
aacctgcagc gtggcgatct ggttcgtggc atgttcgaag ccgcggatcg tggcgcaact 600
taccctttcc tgactgatgg tgacgctcat ctgacccgtg gctctggttt taacatcgtg 660
ctggttaagg atggcgttct gtataccccg gatcgtggtg ttctgcaggg taaaactcgc 720
aaatccgtta ttaacgcggc ggaagctttc ggtattgagg tccgcgtaga atttgttccg 780
gtcgaactgg cttatcgctg tgacgaaatc ttcatgtgta cccgcgcagg tggcattatg 840
cctatcacta ccctggacgg tatgcctgtt aacggcggcc agatcggccc gatcaccaaa 900
aagatttggg acggttattg ggctatgcac tatgatgctg cttactcctt tgaaatcgac 960
tataacgaac gtaactga 978
<210> 12
<211> 325
<212> PRT
<213> 人工序列(Artificial sequence)
<400> 12
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 Arg Asp Val Pro Ser
50 55 60
Val Trp Asp Gly Arg Phe Phe Arg Leu Asp Asp His Ile Thr Lys 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 Arg 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 Arg 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 Lys 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 Arg 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
Claims (7)
1.一种转氨酶突变体，能够以至少约80%的立体异构体将底物1-苄基-3-哌啶酮转化为产物(R)-3-氨基-1-苄基哌啶，其特征在于，所述转氨酶突变体的氨基酸序列为SEQ ID NO：4、SEQ ID NO：6、SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12中的任一个。
2.一种转氨酶突变体的多核苷酸分子，其特征在于，所述转氨酶突变体的多核苷酸分子编码权利要求1中任一项所述的转氨酶突变体氨基酸序列。
3.一种重组质粒，其特征在于，所述重组质粒连接有权利要求2所述的转氨酶突变体的多核苷酸分子。
4.根据权利要求3所述的重组质粒，其特征在于，所述重组质粒为pET-28a(+)。
5.一种宿主细胞，其特征在于，所述宿主细胞含有权利要求3所述的重组质粒。
6.根据权利要求5所述的宿主细胞，其特征在于，所述宿主细胞为大肠杆菌BL21细胞。
7.一种将底物1-苄基-3-哌啶酮转化为产物(R)-3-氨基-1-苄基哌啶的方法，其特征在于:利用权利要求1至6中任一项所述的转氨酶突变体，转氨酶突变体的多核苷酸分子、重组质粒、含有重组质粒的宿主细胞转化1-苄基-3-哌啶酮成为(R)-3-氨基-1-苄基哌啶。
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996038555A2 (en) * | 1995-05-31 | 1996-12-05 | Thierry Bogaert | Unc-53 from c. elegans and its uses in testing compounds involved in the control fo cell behaviour and pharmaceutical compositions |
| CN106520716A (zh) * | 2016-10-28 | 2017-03-22 | 杭州酶易生物技术有限公司 | 一种嗜热酮还原酶突变体及其应用 |
| CN106754806A (zh) * | 2016-12-20 | 2017-05-31 | 尚科生物医药（上海）有限公司 | 一种改进的转氨酶及其在(r)‑3‑氨基丁醇制备上的应用 |
| CN106801043A (zh) * | 2016-12-28 | 2017-06-06 | 江苏阿尔法药业有限公司 | 一种重组转氨酶及其制备方法和应用 |
| CN107466322A (zh) * | 2015-04-16 | 2017-12-12 | 豪夫迈·罗氏有限公司 | 突变型转氨酶及与其相关的方法和用途 |
| CN107828751A (zh) * | 2017-11-06 | 2018-03-23 | 凯莱英生命科学技术(天津)有限公司 | 转氨酶突变体及其应用 |
| CN108866021A (zh) * | 2018-05-30 | 2018-11-23 | 浙江工业大学 | 一种转氨酶突变体及在制备西他列汀中间体中的应用 |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015078258A1 (zh) * | 2013-11-26 | 2015-06-04 | 凯莱英医药集团（天津）股份有限公司 | R型ω-转氨酶及其应用 |
| EP3075847B1 (en) * | 2013-11-26 | 2019-05-15 | Asymchem Laboratories (Tianjin) Co., Ltd | Transaminase and use thereof |
| KR101869432B1 (ko) * | 2015-04-08 | 2018-06-20 | 연세대학교 산학협력단 | 케톤에 대한 활성이 증대된 오메가 트랜스아미네이즈의 변이체 및 이것을 이용한 광학활성 아민의 제조방법 |
-
2020
- 2020-03-12 CN CN202010172593.5A patent/CN111363732B/zh active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996038555A2 (en) * | 1995-05-31 | 1996-12-05 | Thierry Bogaert | Unc-53 from c. elegans and its uses in testing compounds involved in the control fo cell behaviour and pharmaceutical compositions |
| CN107466322A (zh) * | 2015-04-16 | 2017-12-12 | 豪夫迈·罗氏有限公司 | 突变型转氨酶及与其相关的方法和用途 |
| CN106520716A (zh) * | 2016-10-28 | 2017-03-22 | 杭州酶易生物技术有限公司 | 一种嗜热酮还原酶突变体及其应用 |
| CN106754806A (zh) * | 2016-12-20 | 2017-05-31 | 尚科生物医药（上海）有限公司 | 一种改进的转氨酶及其在(r)‑3‑氨基丁醇制备上的应用 |
| CN106801043A (zh) * | 2016-12-28 | 2017-06-06 | 江苏阿尔法药业有限公司 | 一种重组转氨酶及其制备方法和应用 |
| CN107828751A (zh) * | 2017-11-06 | 2018-03-23 | 凯莱英生命科学技术(天津)有限公司 | 转氨酶突变体及其应用 |
| CN108866021A (zh) * | 2018-05-30 | 2018-11-23 | 浙江工业大学 | 一种转氨酶突变体及在制备西他列汀中间体中的应用 |
Non-Patent Citations (3)
| Title |
|---|
| Construction of stabilized (R)-selective amine transaminase from Aspergillusterreus by consensus mutagenesis.;Xie DF等;《J Biotechnol.》;全文 * |
| Crystal Structure of an (R)-Selective v-Transaminase from Aspergillus terreus;Łyskowski Andrzej等;《PLOS ONE》;全文 * |
| Engineering thermostable (R)-selective amine transaminase from Aspergillus terreus through in silico design employing B-factor and folding free energy calculations;Huang J.等;《Biochem Biophys Res Commun》;第483卷(第1期);全文 * |
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