CN113801858A - 一种脱氢酶突变体l283v/l286v及其制备方法和应用 - Google Patents
一种脱氢酶突变体l283v/l286v及其制备方法和应用 Download PDFInfo
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Abstract
一种脱氢酶突变体L283V/L286V及其制备方法和应用，涉及生物医药技术领域；所述突变体L283V/L286V的氨基酸序列为如SEQ ID NO：1所示；为将氨基酸序列为如SEQ ID NO：3所示的脱氢酶的第283位和第286位的的亮氨酸同时突变为缬氨酸。脱氢酶突变体L283V/L286V在全细胞体系催化麦斯明还原反应生成S‑去甲烟碱中展示出高度选择性，且同时具有较的高脱氢酶和亚胺还原酶活性，酶还原时间短，转化率高。反应获得的产物S‑去甲烟碱具有极高的光学纯度，降低后续纯化工作难度。
Description
技术领域
本发明涉及生物医药技术领域，尤其涉及一种脱氢酶突变体L283V/L286V及其制备方法和应用。
背景技术
尼古丁（nicotine），俗称烟碱，是茄科植物烟草中所持有的生物碱，占烟草中全部生物碱的95%以上，其化学名为1-甲基（2-吡啶基）吡咯烷，分子式为C10H14N2，是具有较强碱性的有机二元弱碱，能和多种无机酸或有机酸生成结晶的单盐或双盐，在烟叶中多以有机盐形式存在。
烟碱的用途十分广泛，烟碱系列农药属植物杀虫剂，因其具有蒸薰、胃毒、触杀功能及迅速降解无残留等特点广泛用于粮食、油料、蔬菜、水果、牧草等农作物的杀虫剂，是生产绿色食品的理想高效杀虫剂和生物性农药。它还广泛应用于医药工业上，是研制治疗心血管、皮肤病、蛇毒等疾病药物的特种原料。在临床研究中，尼古丁还对预防并治疗老年痴呆症（Alzheimer’s disease, AD）和帕金森氏综合症（Parkinson’s disease, PD）起积极作用。一些实验和临床结果显示，尼古丁可以影响AD中神经纤维缠结改变的程度，通过皮下和静脉给药可以改善AD病人的认知障碍，尤其是信息加工能力和短期记忆能力。
目前市场上使用的天然尼古丁（S-尼古丁）主要还是依赖植物提取的方式，其中包括微波法、超声波提取法、超临界萃取法等。但是，植物提取的方法效率低，而且因为受到原材料、气候和生长周期等多方面的影响，提取尼古丁的过程中还会含有烟草特有的尼古丁相关杂质，这类杂质的长期使用会对健康产生潜在危害，通过植物提取获得的尼古丁还需要一系列纯化步骤才能获得高纯度的S-尼古丁。因此，高效合成高纯度S-尼古丁的制备方法被广泛关注。
S-去甲烟碱（S-nornicotine）是合成S-尼古丁的重要前体物质，S-去甲烟碱只需要一步甲基化反应即可生成S-尼古丁，而高光学纯度的S-去甲烟碱是合成高纯度S-尼古丁的保证。
酶是由活细胞产生的、对其底物具有高度特异性和高度催化效能的一种生物催化剂；酶的高效性和专一性，使其在合成高纯度化合物中得到广泛应用。随着分子生物学、蛋白质工程等相关技术逐渐成熟，酶催化机理的解读手段以及酶的设计技术越来越丰富，利用生物酶法完成高光学纯度S-去甲烟碱的合成是一种值得研究的方向。蛋白质工程理性设计是以定点突变技术为主要手段，根据对蛋白质结构与功能关系地了解，有目的地设计改造蛋白质，使其性能达到预期效果。目前，已有大量研究成功利用理性设计技术对天然酶的催化活性、底物特异性、热稳定性以及酶的别构效应等进行改造。随着计算生物学的发展，计算机辅助设计指导酶改造的方法日趋成熟。利用计算机模拟的方法，建立酶的三维结构模型，分析酶与底物的构象关系，快速定位与催化反应相关的区域，缩小突变库容量，高效获得相关靶点，为蛋白质工程改造提供指导。但是目前未发现通过蛋白质工程对酶进行设计和改造，实现利用生物酶法完成高光学纯度S-去甲烟碱的合成的相关研究。
发明内容
为了克服现有技术的不足，本发明的目的之一在于设计并提供一种脱氢酶突变体L283V/L286V，用于制备S-去甲烟碱，其底物多样性丰富且产物光学纯度高，且酶解时间短，转化率高。
本发明的目的之二在于提供上述脱氢酶突变体L283V/L286V的制备方法，通过定点突变技术制备脱氢酶突变体L283V/L286V，稳定性高。
本发明的目的之三在于提供上述脱氢酶突变体L283V/L286V在制备高光学纯度的S-去甲烟碱中的应用。
本发明的目的之一采用如下技术方案实现：
一种脱氢酶突变体L283V/L286V，所述突变体L283V/L286V的氨基酸序列为如SEQID NO：1所示。
进一步地，编码所述突变体L283V/L286V基因的核苷酸序列为如SEQ ID NO：2所示。
进一步地，所述突变体L283V/L286V为将氨基酸序列如SEQ ID NO：3所示的MesPDH酶的第283位和第286位的亮氨酸同时突变为缬氨酸所制成。
本发明的目的之二采用如下技术方案实现：
一种脱氢酶突变体L283V/L286V的制备方法，包括以下步骤：
S1，将MesPDH酶基因连接到质粒中，得到重组质粒；
S2，设计突变引物，采用突变引物并以所述重组质粒为模板进行PCR扩增，使MesPDH酶的第283位和第286位氨基酸同时突变为缬氨酸，酶切消化去除模板DNA后，回收得到突变产物；
S3，将所述突变产物转化至宿主细胞中，培养，得到脱氢酶突变体L283V/L286V表达菌株，诱导表达，得到脱氢酶突变体L283V/L286V。
进一步地，所述MesPDH酶基因的核苷酸序列如SEQ ID NO：4所示。
进一步地，所述突变引物包括引物283/286-F和引物283/286-R；所述引物283/286-F的序列如SEQ ID NO：5所示；所述引物283/286-R的序列如SEQ ID NO：6所示。
进一步地，所述宿主细胞为大肠杆菌。
进一步地，步骤S2中，PCR扩增程序的反应条件为： 98℃预变性3min；然后循环设定为： 98℃变性10s，62℃退火15s，72℃延伸2 min；30个循环后，72℃延伸10 min。
一种脱氢酶突变体L283V/L286V在制备高光学纯度的S-去甲烟碱中的应用。
进一步地，所述脱氢酶突变体L283V/L286V用于特异性还原麦斯明制备高光学纯度的S-去甲烟碱。
相比现有技术，本发明的有益效果在于：
本发明的一种脱氢酶突变体L283V/L286V，通过定点突变技术将MesPDH酶的第283位和第286位氨基酸同时突变为缬氨酸构建，其结合全细胞生物体内的辅酶循环系统，能特异地还原麦斯明制备高光学纯度的S-去甲烟碱，酶解时间短，转化率高。脱氢酶MesPDH的第283位亮氨酸（L283）和第286位亮氨酸（L286）的位置处于活性口袋内，酶的表面位置附近，通过与麦斯明、葡萄糖、葡萄糖酸、NAD+和NADH进行分子对接，这两个氨基酸均位于麦斯明、葡萄糖和葡萄糖酸附近区域且稍微远离NAD+和NADH。本发明通过将这两个关键氨基酸突变为缬氨酸，相比于亮氨酸来说，缬氨酸的侧链基团变短了一个亚甲基，扩大活性口袋空间，增加底物多样性。同时，酶分子表面引入缬氨酸有助于增加酶表面刚性。该突变体在全细胞制备S-去甲烟碱中的脱氢酶活性得到了提高，且不影响还原酶活性与手性选择性。
本发明的一种脱氢酶突变体L283V/L286V的制备方法，对MesPDH酶进行定点突变，构建脱氢酶突变体L283V/L286V，使其特异底物的催化活性得到提高。
本发明的一种脱氢酶突变体在全细胞催化体系制备S-去甲烟碱的应用，利用该脱氢酶突变体催化制备S-去甲烟碱，减少反应中反应物成分，使反应体系更简单方便，同时产物光学纯度高。
附图说明
图1为本发明实施例1中MesPDH酶的三维结构模型图；
图2为本发明实施例2中MesPDH酶与不同底物之间的对接模拟图；
图3为本发明实施例5中各全细胞催化体系反应后麦斯明与S-去甲烟碱含量对比图；
图4为本发明实施例5中各反应体系的液相检测图；
图5为本发明实施例5中全细胞催化合成系统的反应流程图。
具体实施方式
下面，结合附图和具体实施方式，对本发明做进一步描述，需要说明的是，在不相冲突的前提下，以下描述的各实施例之间或各技术特征之间可以任意组合形成新的实施例。以下是本发明具体的实施例，在下述实施例中所采用的原材料、设备等除特殊限定外均可以通过购买方式获得。
下列实施例中为注明具体条件的实验方法，通常按照常规实验条件或制造厂商所建议的实验条件，如无特殊说明，实施例中涉及的各种反应试剂均可以通过商业渠道购买得到。本实施例中未作具体说明的分子生物学实验方法，可参照《分子克隆实验指南》。
实施例1
MesPDH酶三级结构模型的建立：
1、分析来源于中生根瘤菌（Mesorhizobium sp. L48C026A00）的6-磷酸葡萄糖酸脱氢酶MesPDH，利用同源建模工具MODELLER对MesPDH进行同源建模，获得单体MesPDH酶模型Ma，评估模型，拉氏构象图评价所有氨基酸均落在合理区域，其中落在最佳区域的氨基酸比例高达97.3%，该模型合理，模型Ma如图1a所示。
2、利用在线同源建模工具Swiss-model对MesPDH进行同源建模，获得二聚体MesPDH酶模型Mb，评估模型，拉氏构象图评价所有氨基酸均落在合理区域，其中落在最佳区域的氨基酸比例高达94.6%，模型合理，模型Mb如图1b所示。
3、利用在线建模工具RoseTTAFold对MesPDH进行模型预测，获得单体MesPDH酶模型Mc，评估模型，拉氏构象图评价所有氨基酸均落在合理区域，其中落在最佳区域的氨基酸比例高达96.2%，模型合理，模型Mc如图1c所示。
实施例2
分子对接模拟预测各底物与酶的结合构象并选择位点：
1、分子对接模拟
利用分子模拟软件Discovery Studio对3个建模模型活性口袋中心进行定位，利用分子对接软件AutodockVina，将MesPDH酶分别与麦斯明、葡萄糖、葡萄糖酸、NAD+、NADH进行对接，借助蛋白构象分析工具PyMOL，对每个对接复合物进行分析。
2、对接结果分析
如图2所示，MesPDH酶模型Ma、Mb和Mc分别与四个反应物分子对接后发现，麦斯明（myosmine）、葡萄糖和葡萄糖酸的位置均落于相似位置，NAD+和NADH则位于另一个口袋位置，从模型Mb推测MesPDH酶极大可能以二聚体形式发挥酶的活性，而麦斯明、葡萄糖和葡萄糖酸在单体的同一个口袋中进行还原竞争，NAD+和NADH则在另一个单体的同一个口袋中同样进行竞争。二聚体模型Mb中，两个单体的这两个底物口袋位置基本一致，表明各底物在该反应区域进行多个还原反应。
3、氨基酸位点的选择
综合上述三种不同的模型以及分子对接结果，为增强脱氢酶活性，同时不影响该酶的亚胺还原活性，突变位点选择在靠近葡萄糖且靠近酶分子表面位置的氨基酸L283和L286，通过将亮氨酸突变为缬氨酸，缩短这两个氨基酸的侧链基团，增加底物活性口袋大小，同时不改变活性口袋中疏水性氨基酸的组成。
实施例3
重组野生酶菌株BL21（DE3）/pET-32a-MesPDH的获得：
全基因合成并通过大肠杆菌密码子偏好性优化得到MesPDH酶基因，其核苷酸序列如SEQ ID NO：4，将其连接到pET-32a质粒上，获得的重组质粒命名为pET-32a-MesPDH，将该质粒转化至大肠杆菌BL21（DE3）中，重组菌株命名为BL21（DE3）/pET-32a-MesPDH。该重组野生酶菌株表达的野生型MesPDH酶的氨基酸序列为如SEQ ID NO：3所示。
实施例4
MesPDH突变菌株的获得：
1、全质粒PCR构建突变体载体
（1）重组质粒pET-32a-MesPDH的质粒小量提取；
（2）设计突变引物，引物有15 bp重叠区，15 bp延伸区，将突变位点设计于重叠区。以质粒pET-32a-MesPDH为模板进行PCR全质粒扩增，PCR体系如表1所示：
所述突变引物包括引物283/286-F（如SEQ ID NO.5所示）和引物283/286-R（如SEQID NO.6所示），所述引物283/286-F和引物283/286-R是对第283位和第286位氨基酸同时突变为缬氨酸进行设计的PCR上游引物和下游引物，具体引物信息如表2所示。
通过引物283/286-F和引物283/286-R并利用PCR扩增制备突变体L283V/L286V，突变体L283V/L286V的氨基酸序列为SEQ ID NO：1；编码所述突变体L283V/L286V基因的核苷酸序列为如SEQ ID NO：2所示。
PCR扩增程序： 98℃预变性3 min；循环设定： 98℃变性10 s， 62℃退火15 s， 72℃延伸2 min，30个循环；最后，72℃延伸10 min；反应结束后，利用试剂盒进行PCR产物回收。
（3）酶切消化去除模板DNA，将PCR产物进行酶切，酶切体系如表3所示：
上述酶切体系置于37℃金属浴中消化1 h，反应结束后利用试剂盒进行酶切产物回收，得到突变产物。
2、测序验证突变体菌株构建成功
将上述突变产物转化至大肠杆菌BL21（DE3）感受态细胞中，37℃倒置过夜培养，挑取拟阳性转化子进行测序验证，成功获得突变体表达菌株。
实施例5
脱氢酶突变体L283V/L286V的全细胞催化反应：
1、MesPDH重组菌株与突变体重组菌株的诱导与全细胞酶液的准备
（1）分别取BL21（DE3）/pET-32a-MesPDH和BL21（DE3）/pET-32a-L283V/L286V菌株，在含Amp（100 μg/mL）的LB平板上划线活化。经37℃倒置培养过夜后，挑取单菌落接种至5mL 含Amp的LB液体培养基中，37℃，200 r/min振荡培养12-16 h。按照1%接种量将过夜培养的种子液接种于20 mL新鲜的含Amp的LB液体培养基中，37℃，200 r/min振荡培养2-3 h至OD600为0.6-0.8时，加入终浓度为0.5 mM的IPTG，将培养液放至20℃，200 r/min振荡培养16h进行蛋白的诱导表达。
（2）诱导结束后，4℃，6000 rpm离心5 min收集菌体，菌体用磷酸盐缓冲液（pH7.0）洗涤一次后，根据菌体湿重加入适量的磷酸盐缓冲液（pH 7.0）重悬菌体，获得全细胞浓度为100 mg/mL的细胞悬浊液，所有操作均在冰上或4℃进行。
2、全细胞反应
（1）以上述实验获得的野生型MesPDH酶和突变型L283V/L286V酶的全细胞悬浊液作为酶液，分组对麦斯明进行催化反应测试，具体反应体系如表4、表5所示。
（2）上述反应体系在30℃下震荡反应24小时，转速200rpm。反应结束后，往反应液中加入2倍体积的无水乙醇终止反应，随后将该反应混合液在65℃下，100rpm旋转蒸发去除溶剂。最后加入2mL无水乙醇重溶旋蒸物，经0.45 μm有机滤头过滤后进行HPLC检测。
3、S-去甲烟碱的检测方法
采用HPLC对表4、表5的反应体系的反应结果进行S-去甲烟碱的定性定量分析，色谱条件如下所述：
高效液相色谱仪：Agilent 1100 Series
色谱柱：ChiralpakAD-Hcolumn（250 mm×4.6 mm×5 μm）
检测器：DAD检测器，检测波长254nm
流动相比例与洗脱条件：流速1 mL/min；柱温30℃；进样量10 μL；等度洗脱流动相体系为正己烷：乙醇：乙二胺=74.9：25.0：0.1。
4、结果分析
如图3所示，各全细胞催化体系反应后麦斯明与S-去甲烟碱含量对比图显示，当使用野生型MesPDH酶进行表4体系的全细胞催化反应时，反应24小时后检测反应体系中的组分含量，麦斯明残留80%，表明反应效率低，仍有大量麦斯明没参与反应；而进行表5体系的全细胞催化反应时，反应24小时检测反应体系中的组分含量，麦斯明残留25%，麦斯明残留量减少，表明在体系中补充了葡萄糖脱氢酶（GDH商业用酶）后，反应效率得到提高。结果显示野生型MesPDH酶的葡萄糖脱氢酶活性较低，需要额外补充葡萄糖脱氢酶，才能提高反应效率。
此外，利用突变型L283V/L286V酶进行表4体系的全细胞催化时，反应24小时后检测反应体系中的组分含量，麦斯明残留不足20%，大部分转化为S-去甲烟碱；进行表5体系的全细胞催化反应时，麦斯明几乎没有残留，反应能彻底转化为S-去甲烟碱，表明突变型L283V/L286V酶改良了其脱氢酶活性，同时不影响还原酶活性，在不需要额外添加葡萄糖脱氢酶的情况下可完成高度转化。
液相检测结果如图4所示，R-去甲烟碱出峰时间约7.98min，S-去甲烟碱出峰时间约9.32min，麦斯明出峰时间约13.42min，所有反应所生成的去甲烟碱均为S-去甲烟碱，没有R-去甲烟碱的生成，产物的光学纯度极高。
整个全细胞合成S-去甲烟碱的过程如图5所示，突变体L283V/L286V在整个全细胞催化中同时发挥亚胺还原酶作用和两步脱氢酶作用，结合外源加入的葡萄糖和全细胞自身的NAD供给，能高效地将麦斯明转化为S-去甲烟碱。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
核苷酸和氨基酸序列表
<110> 广东金骏康生物技术有限公司
<120> 一种脱氢酶突变体L283V/L286V及其制备方法和应用
<130> 2021.10.29
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Claims (10)
1.一种脱氢酶突变体L283V/L286V，其特征在于，所述突变体L283V/L286V的氨基酸序列如SEQ ID NO：1所示。
2.根据权利要求1所述的一种脱氢酶突变体L283V/L286V，其特征在于，编码所述突变体L283V/L286V的核苷酸序列如SEQ ID NO：2所示。
3.根据权利要求1所述的一种脱氢酶突变体L283V/L286V，其特征在于，所述突变体L283V/L286V为将氨基酸序列如SEQ ID NO：3所示的MesPDH酶的第283位和第286位的亮氨酸同时突变为缬氨酸所制成。
4.一种权利要求1-3任一项所述的脱氢酶突变体L283V/L286V的制备方法，其特征在于，包括以下步骤：
S1，将编码MesPDH酶的基因连接到质粒中，得到重组质粒；所述MesPDH酶的氨基酸序列如SEQ ID NO：3所示；
S2，设计突变引物，采用突变引物并以所述重组质粒为模板进行PCR扩增，使MesPDH酶的第283位和第286位氨基酸同时突变为缬氨酸，酶切消化去除模板DNA后，回收得到突变产物；
S3，将所述突变产物转化至宿主细胞中，培养，得到脱氢酶突变体L283V/L286V表达菌株，诱导表达，得到脱氢酶突变体L283V/L286V。
5.根据权利要求4所述的一种脱氢酶突变体L283V/L286V的制备方法，其特征在于，所述编码MesPDH酶的基因的核苷酸序列如SEQ ID NO：4所示。
6.根据权利要求4所述的一种脱氢酶突变体L283V/L286V的制备方法，其特征在于，所述突变引物包括引物283/286-F和引物283/286-R；所述引物283/286-F的序列如SEQ IDNO：5所示；所述引物283/286-R的序列如SEQ ID NO：6所示。
7.根据权利要求4所述的一种脱氢酶突变体L283V/L286V的制备方法，其特征在于，所述宿主细胞为大肠杆菌。
8.根据权利要求4所述的一种脱氢酶突变体L283V/L286V的制备方法，其特征在于，步骤S2中，PCR扩增程序的反应条件为： 98℃预变性3min；然后循环设定为： 98℃变性10s，62℃退火15s，72℃延伸2 min；30个循环后，72℃延伸10 min。
9.权利要求1-3任一项所述的一种脱氢酶突变体L283V/L286V在制备高光学纯度的S-去甲烟碱中的应用。
10.根据权利要求9所述的应用，其特征在于，所述脱氢酶突变体L283V/L286V用于特异性还原麦斯明制备高光学纯度的S-去甲烟碱。
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115948360A (zh) * | 2023-01-16 | 2023-04-11 | 浙江安诺和生物医药有限公司 | 一种亚胺还原酶突变体及其应用和应用方法 |
| WO2023088098A1 (zh) * | 2021-11-18 | 2023-05-25 | 广东金骏康生物技术有限公司 | 一种脱氢酶突变体l283v/l286v及其制备方法和应用 |
| CN116218803A (zh) * | 2023-01-31 | 2023-06-06 | 河北工业大学 | 亚胺还原酶及其制备方法和编码亚胺还原酶的dna |
| CN116622792A (zh) * | 2022-10-19 | 2023-08-22 | 修实生物医药(南通)有限公司 | 一种使用酶催化合成（s）-烟碱的方法 |
| CN117965479A (zh) * | 2023-12-13 | 2024-05-03 | 华南理工大学 | 一种亚胺还原酶突变体及其应用 |
Families Citing this family (1)
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5881791A (ja) * | 1981-11-12 | 1983-05-17 | Japan Tobacco Inc | Ｓ−ノルニコチンの製造法 |
| US20140171380A1 (en) * | 2011-03-08 | 2014-06-19 | The Trustees Of Columbia University In The City Of New York | Screening Assays Using Stem Cells and Stem Cell-Derived Neurons from Mouse Models of Alzheimer's Disease |
| CN108559735A (zh) * | 2018-05-10 | 2018-09-21 | 江南大学 | 一种亮氨酸脱氢酶突变体的构建及其应用 |
| CN110592042A (zh) * | 2019-10-28 | 2019-12-20 | 凯莱英医药集团（天津）股份有限公司 | 转氨酶突变体及其应用 |
| WO2021113337A1 (en) * | 2019-12-03 | 2021-06-10 | Altria Client Services Llc | Compositions and methods for producing tobacco plants and products having altered alkaloid levels |
| CN113373188A (zh) * | 2020-03-10 | 2021-09-10 | 重庆博腾制药科技股份有限公司 | 一种(s)-尼古丁的合成方法 |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10913962B2 (en) * | 2018-11-16 | 2021-02-09 | Zanoprima Lifesciences Limited | Process of making (S)-nicotine |
| CN113046335B (zh) * | 2019-12-27 | 2023-05-26 | 中国科学院天津工业生物技术研究所 | 一种仿生辅酶偏好性的葡萄糖6-磷酸脱氢酶突变体及其应用 |
| US20220154231A1 (en) * | 2020-11-18 | 2022-05-19 | Shandong Jincheng Pharmaceutical Chemical Co., Ltd | Methods for preparing nicotine and intermediates thereof |
| CN113801858B (zh) * | 2021-11-18 | 2022-02-22 | 广东金骏康生物技术有限公司 | 一种脱氢酶突变体l283v/l286v及其制备方法和应用 |
-
2021
- 2021-11-18 CN CN202111369684.9A patent/CN113801858B/zh active Active
-
2022
- 2022-11-02 WO PCT/CN2022/129268 patent/WO2023088098A1/zh not_active Ceased
- 2022-11-18 US US18/056,728 patent/US11873515B2/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5881791A (ja) * | 1981-11-12 | 1983-05-17 | Japan Tobacco Inc | Ｓ−ノルニコチンの製造法 |
| US20140171380A1 (en) * | 2011-03-08 | 2014-06-19 | The Trustees Of Columbia University In The City Of New York | Screening Assays Using Stem Cells and Stem Cell-Derived Neurons from Mouse Models of Alzheimer's Disease |
| CN108559735A (zh) * | 2018-05-10 | 2018-09-21 | 江南大学 | 一种亮氨酸脱氢酶突变体的构建及其应用 |
| CN110592042A (zh) * | 2019-10-28 | 2019-12-20 | 凯莱英医药集团（天津）股份有限公司 | 转氨酶突变体及其应用 |
| WO2021113337A1 (en) * | 2019-12-03 | 2021-06-10 | Altria Client Services Llc | Compositions and methods for producing tobacco plants and products having altered alkaloid levels |
| CN113373188A (zh) * | 2020-03-10 | 2021-09-10 | 重庆博腾制药科技股份有限公司 | 一种(s)-尼古丁的合成方法 |
Non-Patent Citations (3)
| Title |
|---|
| D MERBERG ET AL.: "Regulation of hydrogenase in Rhizobium japonicum: analysis of mutants altered in regulation by carbon substrates and oxygen", 《JOURNAL OF BACTERIOLOGY》 * |
| GENBANK: "NAD(P)-dependent oxidoreductase [Mesorhizobium sp.L48C026A00，NCBI Reference Sequence:WP_023799151.1", 《GENBANK》 * |
| 马林等: "烟碱脱氢酶的纯化及性质研究", 《工业微生物》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023088098A1 (zh) * | 2021-11-18 | 2023-05-25 | 广东金骏康生物技术有限公司 | 一种脱氢酶突变体l283v/l286v及其制备方法和应用 |
| US11873515B2 (en) * | 2021-11-18 | 2024-01-16 | Golden Health (Guangdong) Biotechnology Co., Ltd. | Dehydrogenase mutant L283V/L286V, and preparation method and use thereof |
| CN116622792A (zh) * | 2022-10-19 | 2023-08-22 | 修实生物医药(南通)有限公司 | 一种使用酶催化合成（s）-烟碱的方法 |
| CN116622792B (zh) * | 2022-10-19 | 2024-03-08 | 修实生物医药(南通)有限公司 | 一种使用酶催化合成（s）-烟碱的方法 |
| CN115948360A (zh) * | 2023-01-16 | 2023-04-11 | 浙江安诺和生物医药有限公司 | 一种亚胺还原酶突变体及其应用和应用方法 |
| CN116218803A (zh) * | 2023-01-31 | 2023-06-06 | 河北工业大学 | 亚胺还原酶及其制备方法和编码亚胺还原酶的dna |
| CN116218803B (zh) * | 2023-01-31 | 2024-03-19 | 河北工业大学 | 亚胺还原酶及其制备方法和编码亚胺还原酶的dna |
| CN117965479A (zh) * | 2023-12-13 | 2024-05-03 | 华南理工大学 | 一种亚胺还原酶突变体及其应用 |
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| CN113801858B (zh) | 2022-02-22 |
| US11873515B2 (en) | 2024-01-16 |
| US20230193217A1 (en) | 2023-06-22 |
| WO2023088098A1 (zh) | 2023-05-25 |
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