CN112877386B - 一种基于酶法合成烟酰胺单核苷酸的方法 - Google Patents
一种基于酶法合成烟酰胺单核苷酸的方法 Download PDFInfo
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- CN112877386B CN112877386B CN202110120111.6A CN202110120111A CN112877386B CN 112877386 B CN112877386 B CN 112877386B CN 202110120111 A CN202110120111 A CN 202110120111A CN 112877386 B CN112877386 B CN 112877386B
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Abstract
本发明提供了一种基于酶法合成烟酰胺单核苷酸(NMN)的方法，其以D‑核糖、烟酰胺、ATP为底物，在核糖激酶、磷酸核糖变位酶及烟酰胺核糖激酶的偶联催化作用下，一锅法合成得到烟酰胺单核苷酸。本发明制备NMN的方法开辟了一条酶法合成NMN的新途径。本发明中的三种酶可以循环利用、成本低、节能环保，适用于规模化工业生产。
Description
技术领域
本发明属于生物制药和生物催化技术领域，涉及一种基于酶法合成烟酰胺单核苷酸的新方法。
背景技术
烟酰胺单核苷酸(β-Nicotinamide mononucleotide，简称β-NMN或者NMN)是烟酰胺磷酸核糖转移酶反应的产物，是NAD+的关键前体，其在被烟酰胺核苷酸腺苷转移酶腺苷化后变成NAD+。人体细胞外NMN需要去磷酸转化为烟酰胺核糖(NR)才能进入肝细胞内部，随后NR在烟酰胺核苷激酶1的作用下生成NMN。NMN在人体内通过转化为NAD+来发挥其生理功能，如激活NAD+底物依赖性酶Sirt1(组蛋白脱乙酰酶，又称沉默调节蛋白)、调节细胞存活和死亡、维持氧化还原状态等。近期研究发现，通过补充NMN可以明显提高体内的NAD+水平，对心脑血管疾病、神经退行性病及老化退行性疾病等有较好的治疗和修复作用，还可以调节内分泌，起到保护和修复胰岛功能，增加胰岛素的分泌，对糖尿病和肥胖等代谢性疾病具有预防作用。
目前NMN的合成方法包括化学法和酶法。人们试图通过化学法来生产NMN，该方法通常是以烟酰胺核糖(NR)为原料，用三氯氧磷等进行磷酸化得到(Chem.Commun.1999,729-730，CN 107613990 A)，但所获产品杂质多、收率低、成本高，且涉及大量有毒有害试剂的使用，造成严重的环境污染，限制了其在食品级NMN的生产应用。现以酶法生产NMN成为了当前NMN生产的主流方式。例如，CN108026130A和CN110195089A公开了以烟酰胺、ATP和核糖为原料，在烟酰胺磷酸核糖转移酶、核糖磷酸焦磷酸激酶以及核糖激酶的催化作用下发生反应，制备烟酰胺单核苷酸；CN108949865A公开了以D-核糖-5-磷酸、ATP和烟酰胺为原料，通过固定化含有磷酸核糖焦磷酸合成酶和烟酰胺磷酸核糖转移酶的全细胞催化合成烟酰胺单核苷酸。CN106755209A公开了以烟酰胺核糖、ATP为底物，在烟酰胺核糖激酶的催化下生成烟酰胺单核苷酸；CN108026535A公开了以烟酰胺、ATP和AMP为原料，在烟酰胺磷酸核糖转移酶、核糖磷酸焦磷酸激酶以及核苷酶的催化作用下发生反应，制备烟酰胺单核苷酸。PCT/CN2016/092457发明公开了一种制备烟酰胺单核苷酸的方法，以烟酰胺、ATP和木糖为原料，在烟酰胺磷酸核糖转移酶、核糖磷酸焦磷酸激酶、核糖-5-磷酸异构酶、核酮糖-3-磷酸异构酶、木酮糖激酶以及木糖异构酶的催化作用下发生反应，合成烟酰胺单核苷酸。
上述专利文献用到的原料中，烟酰胺核糖暂无酶法直接制备的方法，需化学法合成，价格昂贵且不易购买，D-核糖-5-磷酸性质不稳定，价格也昂贵；而以廉价D-核糖为原料的专利中，需经核糖激酶、烟酰胺磷酸核糖转移酶和核糖磷酸焦磷酸激酶等多步反应合成NMN，转化率低(50％左右)、纯化困难、成本高；本发明首次揭示了磷酸核糖激酶的双层功能，即对核糖-1-磷酸具有解磷酸作用和对烟酰胺核糖具有磷酸化作用，在该酶的上述作用下，可以将核糖-1-磷酸、烟酰胺和ATP一步合成NMN，进而有了本发明的全新NMN合成路线，即以D-核糖、烟酰胺、ATP为底物，在核糖激酶、磷酸核糖变位酶及烟酰胺核糖激酶的偶联催化作用下，一锅法合成得到烟酰胺单核苷酸，该方法转化率高(大于90％)、收率高(大于80％)、成本低，适用于大规模化工业生产。
发明内容
鉴于烟酰胺核糖暂无酶法直接制备的相关报道，本发明的目的在于提供一种从廉价原料D-核糖出发，利用酶法先从D-核糖合成烟酰胺核糖，再合成烟酰胺单核苷酸，开发了一条酶法合成NMN的新途径，具体地是利用烟酰胺核糖激酶对核糖-1-磷酸的解磷酸和对烟酰胺核糖的磷酸化双层功能制备NMN。其具体反应过程如下：D-核糖在核糖激酶的作用下合成核糖-5-磷酸；核糖-5-磷酸在磷酸核糖变位酶的作用下变为核糖-1-磷酸；核糖-1-磷酸在烟酰胺核糖激酶的解磷酸作用下合成烟酰胺核糖；烟酰胺核糖在烟酰胺核糖激酶的磷酸化作用下合成烟酰胺单核苷酸。本发明提供以下技术方案。
一种基于酶法合成烟酰胺单核苷酸的方法，包括如下步骤：
以D-核糖、烟酰胺、ATP为底物，在核糖激酶、磷酸核糖变位酶及烟酰胺核糖激酶的偶联催化作用下，一锅法合成得到烟酰胺单核苷酸，其工艺路线如图1所示。
上述反应体系中，D-核糖浓度为20-120mM，优选50-100mM，进一步优选50mM；烟酰胺浓度为20-120mM，优选50-100mM，进一步优选50mM；ATP浓度为20-150mM，优选60-120mM，进一步优选60mM。
上述反应体系的反应温度为20-37℃，优选25-30℃，进一步优选25℃；反应体系的pH为4.5-7.0，优选5.0-6.0，进一步优选5.0；反应时间3-8h，优选4-6h，进一步优选4h。
上述的方法，反应体系的缓冲液为磷酸盐缓冲液或Tris-Hcl缓冲液，优选磷酸盐缓冲液，其浓度为20-200mM，优选50-100mM，进一步优选50mM。
上述的方法，向反应体系中添加Mg2+和Mn2+离子，反应中的Mg2+浓度为10-50mM，优选20-40mM，进一步优选20mM；反应中的Mn2+浓度为1-20mM，优选5-10mM,进一步优选5mM。
上述的方法，所述的核糖激酶来源为酿酒酵母、植物乳杆菌和大肠杆菌中的至少一种，优选酿酒酵母；磷酸核糖变位酶来源为酿酒酵母、海栖热袍菌及大肠杆菌中的至少一种，优选海栖热袍菌；烟酰胺核糖激酶来源为人和酿酒酵母中的至少一种，优选人类来源。
上述的方法，所述的酿酒酵母来源的核糖激酶氨基酸序列为SEQ ID NO.1；海栖热袍菌来源的磷酸核糖变位酶氨基酸序列为SEQ ID NO.2；人类来源的烟酰胺核糖激酶氨基酸序列为SEQ ID NO.3。
上述的方法，所述核糖激酶、磷酸核糖变位酶及烟酰胺核糖激酶的表达宿主为微生物。
上述的方法，所述微生物包括大肠杆菌、枯草芽孢杆菌和酿酒酵母中的至少一种，优选大肠杆菌，进一步优选大肠杆菌BL21(DE3)；微生物中酶的重组表达载体为本领域常规的各种载体，包括：各种质粒、粘粒、噬菌体和病毒载体中的至少一种，优选原核表达载体pET30a(+)。
上述的方法，所述核糖激酶、磷酸核糖变位酶及烟酰胺核糖激酶包括粗酶、纯化酶和固定化酶中的至少一种，优选固定化酶。
本发明采用上述方法，优选将上述用酶的编码基因克隆至原核表达载体pET30a(+)，构建重组质粒，通过电转化导入大肠杆菌BL21(DE3)，按常规方法诱导表达。
本发明优选上述用酶在大肠杆菌BL21(DE3)细胞中生产后，经超声破碎，获得粗酶，进一步采用固定化金属螯合亲和层析(IMAC)纯化，获得纯化酶，粗酶和纯化酶均采用真空冷冻干燥制备成蛋白粉末；进一步采用环氧基载体ECEP进行固定化处理，获得固定化酶。
上述反应体系中选用粗酶冻干粉时，三种酶用量:50mM D-核糖:核糖激酶粗酶冻干粉200-500mg:烟酰胺核糖激酶粗酶冻干粉200-500mg:磷酸核糖变位酶粗酶冻干粉400-800mg；
优选：50mM D-核糖:核糖激酶粗酶冻干粉300mg:烟酰胺核糖激酶粗酶冻干粉300mg:磷酸核糖变位酶粗酶冻干粉600mg。
上述反应体系中选用纯化酶冻干粉时，三种酶用量:50mM D-核糖:核糖激酶纯化酶冻干粉50-300mg、烟酰胺核糖激酶纯化酶冻干粉50-300mg、磷酸核糖变位酶纯化酶冻干粉100-400mg；
优选：50mM D-核糖:核糖激酶纯化酶冻干粉100mg、烟酰胺核糖激酶纯化酶冻干粉100mg、磷酸核糖变位酶纯化酶冻干粉200mg。
上述反应体系中选用固定化酶时，三种酶用量:50mM D-核糖:核糖激酶固定化酶8-32g，烟酰胺核糖激酶固定化酶8-32g，磷酸核糖变位酶固定化酶12-48g；
优选:50mM D-核糖:核糖激酶固定化酶16g，烟酰胺核糖激酶固定化酶16g，磷酸核糖变位酶固定化酶24g。
上述三种酶的冻干粉和固定化酶是在宿主细胞中表达，经超声破碎，获得粗酶，进一步采用固定化金属螯合亲和层析(IMAC)纯化，获得纯化酶，粗酶和纯化酶均采用真空冷冻干燥制备成蛋白粉末；进一步采用环氧基载体ECEP进行固定化处理，获得固定化酶。
上述固定化酶和粗酶的用量参考纯化酶的用量。
本发明酶基因表达工程菌株的构建进一步优选包括以下步骤：
将本发明涉及的核糖激酶、磷酸核糖变位酶及烟酰胺核糖激酶氨基酸序列(SEQID NO.1-3)，送至基因合成公司进行大肠杆菌密码子优化并人工合成编码基因，分别克隆至原核表达载体pET30a(+)的NdeI和XhoI酶切位点之间，且在上述基因的C端均添加6个组氨酸编码序列，便于后续的蛋白纯化。包含上述3种酶基因的重组表达载体，通过电转化导入大肠杆菌BL21(DE3)中，获得对应的酶基因表达工程菌株。工程菌株的发酵采用常规发酵培养基TB，先在37℃、200rpm条件下培养OD600达0.6-0.8，再采用终浓度0.5mM IPTG或0.1g/L乳糖在25℃条件下，诱导表达10h，收集菌体并破胞处理，检测目的蛋白的表达情况。
本发明固定化酶的制备进一步优选包括以下步骤：
利用低温高速离心机离心(10000rpm、4℃、10min)收集诱导10h后的核糖激酶、磷酸核糖变位酶及烟酰胺核糖激酶发酵菌体，用pH 8.0、0.1mol/L磷酸盐缓冲液重复洗涤菌体两次，将浓缩20倍的菌体重悬于50ml磷酸盐缓冲液中，在冰浴中超声破碎(超声条件为：工作2s，间隔5s)直至澄清。将上述破碎液置于低温高速离心机中离心(10000rpm、4℃、20min)，收集上清分别得到核糖激酶、磷酸核糖变位酶及烟酰胺核糖激酶的粗酶溶液，再利用真空冷冻干燥制备成粗酶蛋白粉末。进一步将上述粗酶蛋白溶液进样到已活化并结合Ni+的IDA树脂上，先依次用1-2倍柱体积的10和30mM的低浓度咪唑溶液梯度洗脱除杂，再用1倍柱体积的300mM的高浓度咪唑洗脱目的蛋白，蛋白洗脱液采用常规透析或超滤的方法除咪唑，从而获得上述酶的纯化酶，进一步采用真空冷冻干燥制备成纯化酶蛋白粉末。取适量的上述纯化酶蛋白粉末，加入pH 8.0、0.1mol/L磷酸盐缓冲液中，然后加入50g经活化处理后的环氧基载体ECEP，于30℃、120rpm条件下低速搅拌固定化48h，最后将所得固定化酶用去离子水洗涤2-3遍，真空滤干后得到上述酶的固定化酶。
本发明利用上述的反应条件进行催化反应，具有以下增益效果：本发明利用烟酰胺核糖激酶的双层功能催化合成NMN，间接解决了烟酰胺核糖的酶法制备难题；采用固定化多酶体系一锅法催化反应，比其他现有NMN合成方法，具有反应条件温和、操作简单、转化率高等优点，其反应转化率大于90％、总收率大于80％，NMN结晶粉纯度大于99.5％，产品纯度高、质量可靠。
本发明的方法是创造性地利用了烟酰胺核糖激酶的双层功能，以D-核糖为原料，先合成烟酰胺核糖，再合成NMN，无需直接使用价格昂贵的烟酰胺核糖为原料，开创了一条新的NMN酶法合成途径。而且本发明中的三种酶可以循环利用、成本低、节能环保。由于该制备NMN的方法是采用一锅法反应，故操作简单、反应收率高、杂质少，显著降低了成本，适用于规模化工业生产。
附图说明
图1为本发明一锅法合成得到烟酰胺单核苷酸NMN工艺路线图；
图2为本发明核糖激酶BL21(DE3)表达蛋白电泳图；
图3为本发明磷酸核糖变位酶BL21(DE3)表达蛋白电泳图；
图4为本发明烟酰胺核糖激酶BL21(DE3)表达蛋白电泳图；
图5为本发明在25mM底物浓度下，采用固定化酶一锅法生产NMN的结果分析图；
图6为本发明在50mM底物浓度下，采用固定化酶一锅法生产NMN的结果分析图；
图7为本发明在50mM底物浓度下，采用固定化酶一锅法生产NMN的结晶粉结果分析图；
图8为本发明在100mM底物浓度下，采用固定化酶一锅法生产NMN的结果分析图。
具体实施方式
为了进一步阐述本发明，下面结合实施例对本发明提供的技术方案进行详细地描述，所描述的实施例仅为本发明的部分实施例，而非全部实施例。因此，本领域普通技术人员在基于本发明的实施例和没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明下述实施例涉及的核糖激酶、磷酸核糖变位酶及烟酰胺核糖激酶是利用原核表达载体pET30a(+)构建表达且在大肠杆菌BL21(DE3)细胞中生产。
本发明下述实施例涉及的上述用酶在大肠杆菌BL21(DE3)细胞中生产后，经超声破碎，获得粗酶，进一步采用固定化金属螯合亲和层析(IMAC)纯化，获得纯化酶，粗酶和纯化酶均采用真空冷冻干燥制备成蛋白粉末；进一步采用环氧基载体ECEP进行固定化处理，获得固定化酶。
实施例1酶基因表达工程菌株的构建
将本发明涉及的核糖激酶、磷酸核糖变位酶及烟酰胺核糖激酶氨基酸序列，送至基因合成公司进行大肠杆菌密码子优化并人工合成编码基因，分别克隆至原核表达载体pET30a(+)的NdeI和XhoI酶切位点之间，且在上述基因的C端均添加6个组氨酸编码序列，便于后续的蛋白纯化。包含上述3种酶基因的重组表达载体，通过电转化导入大肠杆菌BL21(DE3)中，获得对应的酶基因表达工程菌株。工程菌株的发酵采用常规发酵培养基TB，先在37℃、200rpm条件下培养OD600达0.6-0.8，再采用终浓度0.5mM IPTG或0.1g/L乳糖在25℃条件下，诱导表达10h，收集菌体并破胞处理，检测目的蛋白的表达情况(见图2、图3、图4)。
实施例2固定化酶的制备
利用低温高速离心机离心(10000rpm、4℃、10min)收集诱导10h后的核糖激酶、磷酸核糖变位酶及烟酰胺核糖激酶发酵菌体，用pH 8.0、0.1mol/L磷酸盐缓冲液重复洗涤菌体两次，将浓缩20倍的菌体重悬于50ml磷酸盐缓冲液中，在冰浴中超声破碎(超声条件为：工作2s，间隔5s)直至澄清。将上述破碎液置于低温高速离心机中离心(10000rpm、4℃、20min)，收集上清分别得到核糖激酶、磷酸核糖变位酶及烟酰胺核糖激酶的粗酶溶液，再利用真空冷冻干燥制备成粗酶蛋白粉末。进一步将上述粗酶蛋白溶液进样到已活化并结合Ni+的IDA树脂上，先依次用1-2倍柱体积的10和30mM的低浓度咪唑溶液梯度洗脱除杂，再用1倍柱体积的300mM的高浓度咪唑洗脱目的蛋白，蛋白洗脱液采用常规透析或超滤的方法除咪唑，从而获得上述酶的纯化酶，进一步采用真空冷冻干燥制备成纯化酶蛋白粉末。取适量的上述纯化酶蛋白粉末，加入pH 8.0、0.1mol/L磷酸盐缓冲液中，然后加入50g经活化处理后的环氧基载体ECEP，于30℃、120rpm条件下低速搅拌固定化48h，最后将所得固定化酶用去离子水洗涤2-3遍，真空滤干后得到上述酶的固定化酶。
实施例3以D-核糖为底物，采用粗酶一锅法生产NMN
在反应体系中依次加入终浓度为50mM的D-核糖，50mM的烟酰胺，60mM ATP，5mM氯化锰，20mM氯化镁，用1L的pH 6.0、50mM磷酸盐缓冲液，充分振荡溶解后调pH 5.00。加入实施例2中制备的核糖激酶粗酶冻干粉300mg、烟酰胺核糖激酶粗酶冻干粉300mg、磷酸核糖变位酶粗酶冻干粉600mg，混合均匀后，控制反应温度为25℃，300rpm搅拌反应，采用自动滴定仪，全程用1mol/l氢氧化钠控制pH 5.0，反应过程中用液相色谱HPLC检测NMN的生成浓度，反应在4小时内结束，反应得到NMN 14.8g，反应收率88.62％，反应液经膜设备微滤、高效液相色谱分离纯化、纳滤浓缩、真空冷冻干燥等后处理后得到结晶粉12.87g，总收率77.06％，结晶粉纯度大于99.5％。
实施例4以D-核糖为底物，采用纯化酶一锅法生产NMN
在反应体系中依次加入终浓度为50mM的D-核糖，50mM的烟酰胺，60mM ATP，5mM氯化锰，20mM氯化镁，用1L的pH 6.0、50mM磷酸盐缓冲液，充分振荡溶解后调pH 5.00。加入实施例2中制备的核糖激酶纯化酶冻干粉100mg、烟酰胺核糖激酶纯化酶冻干粉100mg、磷酸核糖变位酶纯化酶冻干粉200mg，混合均匀后，控制反应温度为25℃，300rpm搅拌反应，采用自动滴定仪，全程用1mol/l氢氧化钠控制pH 5.0，反应过程中用液相色谱HPLC检测NMN的生成浓度，反应在4小时内结束，反应得到NMN 15.1g，反应收率90.42％，反应液经膜设备微滤、高效液相色谱分离纯化、纳滤浓缩、真空冷冻干燥等后处理后得到结晶粉13.07g，总收率78.26％，结晶粉纯度大于99.5％。
实施例5以D-核糖为底物，采用固定化酶一锅法生产NMN
在反应体系中依次加入终浓度为25mM的D-核糖，25mM的烟酰胺，30mM ATP，2.5mM氯化锰，10mM氯化镁，用1L的pH 6.0、50mM磷酸盐缓冲液，充分振荡溶解后调pH 5.00。加入实施例2中制备的核糖激酶固定化酶8g，烟酰胺核糖激酶固定化酶8g，磷酸核糖变位酶固定化酶12g，混合均匀后，控制反应温度为25℃，300rpm搅拌反应，采用自动滴定仪，全程用1mol/l氢氧化钠控制pH 5.0，反应过程中用液相色谱检测NMN的生成浓度，反应在4小时内结束，反应得到NMN 7.7g，反应收率92.21％(见图5)，反应液经筛网过滤、高效液相色谱分离纯化、纳滤浓缩、真空冷冻干燥等后处理后得到结晶粉6.7g，总收率80.23％，结晶粉纯度大于99.5％。
实施例6以D-核糖为底物，采用固定化酶一锅法生产NMN
在反应体系中依次加入终浓度为50mM的D-核糖，50mM的烟酰胺，60mM ATP，5mM氯化锰，20mM氯化镁，用1L的pH 6.0、50mM磷酸盐缓冲液，充分振荡溶解后，用1mol/l氢氧化钠调pH 5.00。加入实施例2中制备的核糖激酶固定化酶16g、烟酰胺核糖激酶固定化酶16g，磷酸核糖变位酶固定化酶24g，混合均匀后，控制反应温度为25℃，300rpm搅拌反应，采用自动滴定仪，全程用1mol/l氢氧化钠控制pH 5.0，反应过程中用液相色谱HPLC检测NMN的生成浓度，反应在4小时内结束，反应得到NMN 15.9g，反应收率95.20％(见图6)，反应液经筛网过滤、高效液相色谱分离纯化、纳滤浓缩、真空冷冻干燥等后处理后得到结晶粉14.07g，总收率84.25％，结晶粉纯度大于99.5％(见图7)。
实施例7以D-核糖为底物，采用固定化酶一锅法生产NMN
在反应体系中依次加入终浓度为100mM的D-核糖，100mM的烟酰胺，120mM ATP，10mM氯化锰，40mM氯化镁，用1L的pH 6.0、50mM磷酸盐缓冲液，充分振荡溶解后，用1mol/l氢氧化钠调pH 5.00。加入实施例2中制备的核糖激酶固定化酶32g、烟酰胺核糖激酶固定化酶32g，磷酸核糖变位酶固定化酶48g，混合均匀后，控制反应温度为25℃，300rpm搅拌反应，采用自动滴定仪，全程用1mol/l氢氧化钠控制pH 5.0，反应过程中用液相色谱HPLC检测NMN的生成浓度，反应在4小时内结束，反应得到NMN 30.9g，反应收率92.51％(见图8)，反应液经筛网过滤、高效液相色谱分离纯化、纳滤浓缩、真空冷冻干燥等后处理后得到结晶粉28.06g，总收率84.01％，结晶粉纯度大于99.5％。酿酒酵母来源的核糖激酶氨基酸序列
SEQ ID NO.1
MGITVIGSLNYDLDTFTDRLPNAGETFRANHFETHAGGKGLNQAAAIGKLKNPSSRYSVRMIGNVGNDTFGKQLKDTLSDCGVDITHVGTYEGINTGTATILIEEKAGGQNRILIVEGANSKTIYDPKQLCEIFPEGKEEEEYVVFQHEIPDPLSIIKWIHANRPNFQIVYNPSPFKAMPKKDWELVDLLVVNEIEGLQIVESVFDNELVEEIREKIKDDFLGEYRKICELLYEKLMNRKKRGIVVMTLGSRGVLFCSHESPEVQFLPAIQNVSVVDTTGAGDTFLGGLVTQLYQGETLSTAIKFSTLASSLTIQRKGAAESMPLYKDVQKDA
海栖热袍菌来源的磷酸核糖变位酶氨基酸序列
SEQ ID NO.2
MRVVLIVLDSVGIGEMPDAHLYGDEGSNTIVNTAKAVSGLHLPNMAKLGLGNLDDIPGVEPVKPAEGIYGKMMEKSPGKDTTTGHWEIAGVILKKPFDLFPEGFPKELIEEFERRTGRKVIGNKPASGTEIIKELGPIHEKTGALIVYTSADSVFQIAAKKEIVPLEELYRYCEIARELLNEMGYKVARVIARPFTGEWPNYVRTPERKDFSLEPEGKTLLDVLTENGIPVYGVGKIADIFAGRGVTENYKTKDNNDGIDKTISLMKEKNHDCLIFTNLVDFDTKYGHRNDPVSYAKALEEFDARLPEIMHNLNEDDVLFITADHGCDPTTPSTDHSREMVPLLGYGGRLKKDVYVGIRETFADLGQTIADIFGVPPLENGTSFKNLIWE
人类来源的烟酰胺核糖激酶氨基酸序列
SEQ ID NO.3
MKLIVGIGGMTNGGKTTLTNSLLRALPNCCVIHQDDFFKPQDQIAVGEDGFKQWDVLESLDMEAMLDTVQAWLSSPQKFARAHGVSVQPEASDTHILLLEGFLLYSYKPLVDLYSRRYFLTVPYEECKWRRSTRNYTVPDPPGLFDGHVWPMYQKYRQEMEANGVEVVYLDGMKSREELFREVLEDIQNSLLNRSQESAPSPARPARTQGPGRGCGHRTARPAASQQDSM
序列表
<110> 湖南福来格生物技术有限公司
<120> 一种基于酶法合成烟酰胺单核苷酸的方法
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Phe Ala Gly Arg Gly Val Thr Glu Asn Tyr Lys Thr Lys Asp Asn Asn
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Asp Gly Ile Asp Lys Thr Ile Ser Leu Met Lys Glu Lys Asn His Asp
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Arg Asn Asp Pro Val Ser Tyr Ala Lys Ala Leu Glu Glu Phe Asp Ala
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Arg Leu Pro Glu Ile Met His Asn Leu Asn Glu Asp Asp Val Leu Phe
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Ile Thr Ala Asp His Gly Cys Asp Pro Thr Thr Pro Ser Thr Asp His
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Ser Arg Glu Met Val Pro Leu Leu Gly Tyr Gly Gly Arg Leu Lys Lys
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Asp Val Tyr Val Gly Ile Arg Glu Thr Phe Ala Asp Leu Gly Gln Thr
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Ile Ala Asp Ile Phe Gly Val Pro Pro Leu Glu Asn Gly Thr Ser Phe
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Lys Asn Leu Ile Trp Glu
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<212> PRT
<213> Homo sapiens
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Met Leu Asp Thr Val Gln Ala Trp Leu Ser Ser Pro Gln Lys Phe Ala
65 70 75 80
Arg Ala His Gly Val Ser Val Gln Pro Glu Ala Ser Asp Thr His Ile
85 90 95
Leu Leu Leu Glu Gly Phe Leu Leu Tyr Ser Tyr Lys Pro Leu Val Asp
100 105 110
Leu Tyr Ser Arg Arg Tyr Phe Leu Thr Val Pro Tyr Glu Glu Cys Lys
115 120 125
Trp Arg Arg Ser Thr Arg Asn Tyr Thr Val Pro Asp Pro Pro Gly Leu
130 135 140
Phe Asp Gly His Val Trp Pro Met Tyr Gln Lys Tyr Arg Gln Glu Met
145 150 155 160
Glu Ala Asn Gly Val Glu Val Val Tyr Leu Asp Gly Met Lys Ser Arg
165 170 175
Glu Glu Leu Phe Arg Glu Val Leu Glu Asp Ile Gln Asn Ser Leu Leu
180 185 190
Asn Arg Ser Gln Glu Ser Ala Pro Ser Pro Ala Arg Pro Ala Arg Thr
195 200 205
Gln Gly Pro Gly Arg Gly Cys Gly His Arg Thr Ala Arg Pro Ala Ala
210 215 220
Ser Gln Gln Asp Ser Met
225 230
Claims (22)
1.一种基于酶法合成烟酰胺单核苷酸的方法，其特征在于，以D-核糖、烟酰胺、ATP为底物，在核糖激酶、磷酸核糖变位酶及烟酰胺核糖激酶的偶联催化作用下，一锅法合成得到烟酰胺单核苷酸；所述的烟酰胺核糖激酶为人类来源的烟酰胺核糖激酶，其氨基酸序列为SEQID NO.3。
2.根据权利要求1所述的方法，其特征在于，反应体系中，所述的D-核糖浓度为20-120mM；烟酰胺浓度为20-120 mM；ATP浓度为20-150 mM。
3.根据权利要求2所述的方法，其特征在于，反应体系中，所述的D-核糖浓度为50-100mM；烟酰胺浓度为50-100 mM；ATP浓度为60-120 mM。
4.根据权利要求3所述的方法，其特征在于，反应体系中，所述的D-核糖浓度为50 mM；烟酰胺浓度为50 mM；ATP浓度为60 mM。
5.根据权利要求1所述的方法，其特征在于，反应体系的反应温度为20-37℃；反应体系的pH为4.5-7.0；反应时间3-8 h。
6.根据权利要求5所述的方法，其特征在于，反应体系的反应温度为25-30℃；反应体系的pH为5.0-6.0；反应时间4-6 h。
7.根据权利要求6所述的方法，其特征在于，反应体系的反应温度为25℃；反应体系的pH为5.0；反应时间4 h。
8.根据权利要求1或5或6或7所述的方法，其特征在于，反应体系的缓冲液为磷酸盐缓冲液或Tris-Hcl缓冲液，其浓度为20-200 mM。
9.根据权利要求8所述的方法，其特征在于，反应体系的缓冲液为磷酸盐缓冲液，其浓度为50-100 mM。
10.根据权利要求9所述的方法，其特征在于，反应体系的缓冲液为磷酸盐缓冲液，其浓度为50 mM。
11.根据权利要求1所述的方法，其特征在于，向反应体系中添加Mg2+和Mn2+离子，反应中的Mg2+浓度为10-50 mM；反应中的Mn2+浓度为1-20 mM。
12.根据权利要求11所述的方法，其特征在于，向反应体系中添加Mg2+和Mn2+离子，反应中的Mg2+浓度为20-40 mM；反应中的Mn2+浓度为5-10 mM。
13.根据权利要求12所述的方法，其特征在于，向反应体系中添加Mg2+和Mn2+离子，反应中的Mg2+浓度为20 mM；反应中的Mn2+浓度为5 mM。
14.根据权利要求1所述的方法，其特征在于，所述的核糖激酶来源为酿酒酵母、植物乳杆菌和大肠杆菌中的至少一种；磷酸核糖变位酶来源为酿酒酵母、海栖热袍菌及大肠杆菌中的至少一种。
15.根据权利要求14所述的方法，其特征在于，所述的核糖激酶来源为酿酒酵母；磷酸核糖变位酶来源为海栖热袍菌。
16.根据权利要求15所述的方法，其特征在于，所述的酿酒酵母来源的核糖激酶氨基酸序列为SEQ ID NO.1；海栖热袍菌来源的磷酸核糖变位酶氨基酸序列为SEQ ID NO.2。
17.根据权利要求14或15或16所述的方法，其特征在于，所述核糖激酶、磷酸核糖变位酶及烟酰胺核糖激酶的表达宿主为微生物。
18.根据权利要求17所述的方法，其特征在于，所述微生物包括大肠杆菌、枯草芽孢杆菌和酿酒酵母中的至少一种；微生物中酶的重组表达载体包括：各种质粒、粘粒、噬菌体和病毒载体中的至少一种。
19.根据权利要求18所述的方法，其特征在于，所述微生物为大肠杆菌；微生物中酶的重组表达载体为原核表达载体pET30a(+)。
20.根据权利要求19所述的方法，其特征在于，所述微生物为大肠杆菌BL21(DE3)。
21.根据权利要求14或15或16所述的方法，其特征在于，所述核糖激酶、磷酸核糖变位酶及烟酰胺核糖激酶包括粗酶、纯化酶和固定化酶中的至少一种。
22.根据权利要求21所述的方法，其特征在于，所述核糖激酶、磷酸核糖变位酶及烟酰胺核糖激酶为固定化酶。
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Denomination of invention: A Method for Enzymatic Synthesis of Nicotinamide Mononucleotide Granted publication date: 20220826 Pledgee: Changsha Bank city branch of Limited by Share Ltd. Pledgor: HUNAN FLAG BIOTECHNOLOGY Co.,Ltd. Registration number: Y2024980012003 |