CN116970666A - 一种生物酶法制备的三磷酸腺苷二钠及其应用 - Google Patents
一种生物酶法制备的三磷酸腺苷二钠及其应用 Download PDFInfo
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- CN116970666A CN116970666A CN202310957258.XA CN202310957258A CN116970666A CN 116970666 A CN116970666 A CN 116970666A CN 202310957258 A CN202310957258 A CN 202310957258A CN 116970666 A CN116970666 A CN 116970666A
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/32—Nucleotides having a condensed ring system containing a six-membered ring having two N-atoms in the same ring, e.g. purine nucleotides, nicotineamide-adenine dinucleotide
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
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- A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
- A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
- A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
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- C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
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- C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
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- C12Y—ENZYMES
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- C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/04—Phosphotransferases with a phosphate group as acceptor (2.7.4)
- C12Y207/04001—Polyphosphate kinase (2.7.4.1)
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Abstract
本发明涉及一种生物酶法制备三磷酸腺苷二钠的方法，其特征在于，包括如下步骤：(1)制备固定化酶；(2)在含有腺苷、六偏磷酸钠、磷酸二氢钠、硫酸镁的反应体系中，加入固定化酶，调节pH值为7.0‑7.5，37‑45℃下搅拌反应6‑10h；(3)反应液过滤回收固定化酶，过滤液经过离子交换树脂初步纯化获得粗ATP；(4)粗ATP溶解于水中，NaOH调节pH为2.5‑3.5，加入乙醇重结晶获得三磷酸腺苷二钠。本发明首次以C18键合改性硅胶为载体制备含有腺苷激酶(EC2.7.1.20，AK)、多聚磷酸激酶(EC2.7.4.1，Ppk)、多聚磷酸‑腺苷酸磷酸转移酶(EC2.7.4.‑，Pap)的固定化酶，制备得到的固定化酶可多次重复使用，数次重复使用后酶催化的活力下降较少，相比于现有技术取得了更优的技术效果。
Description
技术领域
本发明属于生物合成领域，具体涉及一种生物酶法制备的三磷酸腺苷二钠及其应用。
背景技术
三磷酸腺苷(ATP)是生物体内的能量转换器和贮存器，在人体能量代谢中起着重要的作用，作为代谢的中间体、辅酶参与生物体内糖类、蛋白质、核酸和脂肪等的代谢。当体内吸收、分泌、肌肉收缩及进行生化合成反应等需要能量时，三磷酸腺苷即分解成二磷酸腺苷及磷酸基，同时释放出能量。
三磷酸腺苷二钠(ATP二钠)是临床上广泛应用的ATP的二钠盐，三磷酸腺苷二钠能够穿透血-脑脊液屏障，能提高神经细胞膜性结构的稳定性和重建能力、促进神经突起的再生长。三磷酸腺苷二钠片剂产品适用于进行性肌萎缩、脑出血后遗症、心功能不全、心肌疾患及肝炎等的辅助治疗。三磷酸腺苷二钠-氯化镁注射液临床上适用于急性黄疸型肝炎、慢性活动性肝炎、缺血性脑血管后遗症、脑损伤、脑性小儿麻痹、心肌炎等病症的辅助治疗。
ATP或ATP二钠的合成主要有化学合成法、生物酶催化法、光合磷酸化和氧化磷酸化法以及微生物酶系发酵等方法。化学合成法通常步骤比较冗长或者产率低、成本比较高，因此很少在工业化生产中实际使用。光合磷酸化和氧化磷酸化法主要是在植物或动物体内进行的生化反应。生物酶催化法和微生物酶系发酵的方法是目前ATP的工业生产中最为常用的两种方法。目前关于微生物酶系发酵方法，工业生产ATP普遍采用的方法是以腺苷或腺苷酸(AMP)为底物，利用酵母菌酶系，通过糖酵解途径，进行底物水平磷酸化合成ATP。此方法虽然生产效果良好，成本不高，但是经酵母细胞酶系催化合成ATP的反应过程复杂，参与催化反应的酶系众多，反应过程不易控制，产品批次间质量差异较大；同时，酵母质量常因供应的厂家不同、批次不同、甚至季节不同而有很大差异。且酵母酶系活力下降快，使用寿命短，一般是一次性使用，大量废弃酵母对大气、水源、土壤环境污染相对较大；另外，反应过程中添加大量酵母细胞酶液，引入了大量的蛋白、色素等杂质给ATP后期纯化带来很多困难。
生物酶法催化生产ATP技术更加高效稳定、容易控制且节能环保。在原料成本方面，除底物和一定量镁盐等无机盐外，酵母发酵法需消耗大量酵母泥、葡萄糖及磷酸盐，而生物酶法则需要添加一定量的磷酸供体和相应的催化酶。因此，长期以来，磷酸供体和催化用酶的选择是酶法催化工艺无法规模化应用于生产的限制条件。根据文献报道，乙酸激酶、氨激酶、丙酮酸激酶、精氨酸激酶及肌酸激酶等多种酶均可用于制备ATP。但是，上述酶所利用的磷酸供体或者价值昂贵，如丙酮酸激酶、肌酸激酶所需磷酸供体磷酸烯醇式丙酮酸和磷酸肌酸，或者生成的副产物有一定的生物毒性和污染性，如乙酸激酶、氨激酶催化反应的产物分别为乙酸和氨气，因此都较难在工业生产中大量使用。
由于酶促反应需要使用到的酶价格昂贵，因此现有技术中想要达到工业化的生产就必须降低酶的成本，因此，现有技术中通常使用使用可以回收重复利用的固定化酶。中国专利CN105647996B报道了以多聚磷酸激酶(Ppk)、腺苷酸激酶(Adk)、多聚磷酸-腺苷酸磷酸转移酶(Pap)作为ATP生产酶并将酶固定化，以AMP作为反应底物通过两步酶促反应制备ATP。中国专利CN106191170B报道了以腺苷激酶(Ak)、多聚磷酸激酶(包括Ppk1和Ppk2)、腺苷酸激酶(Adk)，多聚磷酸-腺苷酸磷酸转移酶(Pap)中的两种、三种或四种作为酶促反应制备ATP的酶。中国专利CN110777180A报道了以腺苷激酶(Ark)、腺苷酸激酶(Adk)、多聚磷酸-腺苷酸磷酸转移酶(Pap)作为酶促反应的酶，以腺苷为底物制备获得ATP。
发明内容
本发明要解决的技术问题是现有技术中通过生物酶法制备ATP及其盐ATP二钠时，酶的固定化技术缺乏而导致酶促反应成本高的问题。为了解决上述问题，本法明提供了一种低成本的生物酶法制备的三磷酸腺苷二钠，本发明实现了对全新的酶的固定化技术的发现和验证。
具体的，本发明涉及一种生物酶法制备三磷酸腺苷二钠的方法，其特征在于，其采用固定化酶技术，所采用的ATP生产酶包括腺苷激酶(EC2.7.1.20，AK)、多聚磷酸激酶(EC2.7.4.1，Ppk)、多聚磷酸-腺苷酸磷酸转移酶(EC2.7.4.-，Pap)。
进一步地，所述的固定化酶技术是以改性硅胶为固定化载体。
进一步地，所述改性硅胶的粒度为100-400目，优选为200目。
进一步地，所述改性硅胶为C18键合改性硅胶。
进一步地，所述腺苷激酶(EC2.7.1.20，AK)、多聚磷酸激酶(EC2.7.4.1，Ppk)、多聚磷酸-腺苷酸磷酸转移酶(EC2.7.4.-，Pap)的浓度比为1-2:1-3:1-3，优选为1:1:1。
进一步地，一种生物酶法制备三磷酸腺苷二钠的方法，其特征在于，包括如下步骤：(1)制备固定化酶；(2)在含有腺苷、六偏磷酸钠、磷酸二氢钠、硫酸镁的反应体系中，加入固定化酶，调节pH值为7.0-7.5，37-45℃下搅拌反应6-10h；(3)反应液过滤回收固定化酶，过滤液经过离子交换树脂初步纯化获得粗ATP；(4)粗ATP溶解于水中，NaOH调节pH为2.5-3.5，加入乙醇重结晶获得三磷酸腺苷二钠。
进一步地，所述步骤(1)中固定化酶的制备方法为：将腺苷激酶(EC2.7.1.20，AK)、多聚磷酸激酶(EC2.7.4.1，Ppk)、多聚磷酸-腺苷酸磷酸转移酶(EC2.7.4.-，Pap)按照比例配成混合酶液，加入搅拌反应器中室温下搅拌，接着加入适量改性硅胶与上述酶液混合，在室温下搅拌8-12小时，过滤收集固体，用0.02M磷酸钾缓冲液(pH 7.5)洗涤1-3次，获得固定化酶。
进一步地，所述步骤(2)中腺苷、六偏磷酸钠、磷酸二氢钠、硫酸镁的重量比例为5-10：4-8：1-2：3-6，优选重量比例为5:4:1:3。
进一步地，所述步骤(3)中的过滤是采用超滤膜过滤，所回收的固定化酶用0.02M磷酸钾缓冲液(pH 7.5)洗涤数次后重复利用。
进一步地，所述步骤(3)中的离子交换树脂选自阴离子交换树脂，优选型号为HZ-201的阴离子交换树脂。
进一步地，所述步骤(4)中所用溶解用水的用量为粗ATP质量的5-10倍，优选8倍；所述pH优选调节为3.0。
进一步地，所述步骤(4)中加入乙醇使得乙醇含量为60-80％，优选为70％。
另外，本发明还涉及一种生物酶法制备的三磷酸腺苷二钠，具体地，其由上述的的生物酶法制备得到。
此外，本发明还涉及上述生物酶法制备的三磷酸腺苷二钠的应用，具体地，将所述生物酶法制备的三磷酸腺苷二钠应用于制备药品、保健品中。
相比于现有技术，本发明所达到的有益效果包括：
本发明首次以C18键合改性硅胶为载体制备含有腺苷激酶(EC2.7.1.20，AK)、多聚磷酸激酶(EC2.7.4.1，Ppk)、多聚磷酸-腺苷酸磷酸转移酶(EC2.7.4.-，Pap)的固定化酶，制备得到的固定化酶可多次重复使用，数次重复使用后酶催化的活力下降较少，相比于现有技术取得了更优的技术效果。另外，本发明制备ATP的反应体系相比于现有技术更加简单，不需要加入ATP、硫酸铵等，进一步节约了成本。此外，本发明将ATP制备成更加稳定的三磷酸腺苷二钠(ATP二钠)，还通过简单的重结晶方法即可获得纯度很高的三磷酸腺苷二钠。
具体实施方式
以下结合具体实施例对本发明进行更详细地说明。
实施例1固定化酶的制备
分别取含腺苷激酶(EC2.7.1.20，AK)、多聚磷酸激酶(EC2.7.4.1，Ppk)、多聚磷酸-腺苷酸磷酸转移酶(EC2.7.4.-，Pap)的菌体各500g，用10L 0.1MpH为7.5Tris盐酸缓冲液混溶悬浮后，用高压均质机破碎菌，离心收集上清。上清液加入搅拌反应器中室温下搅拌，接着加入1.5kg的C18键合改性硅胶填料(青岛邦凯)与上述酶液混合，在室温下搅拌8-12小时，过滤收集固体，用0.02M磷酸钾缓冲液(pH 7.5)洗涤3次，获得固定化酶。
实施例2三磷酸腺苷二钠的制备
反应罐中加入腺苷1200g、六偏磷酸钠960g、磷酸二氢钠240g、硫酸镁720g，水15L，加入实施例1获得的全部固定化酶，NaOH调节pH值为7.5，40℃下搅拌反应8h；(3)反应液采用超滤膜过滤回收固定化酶，过滤液经过型号为HZ-201的阴离子交换树脂初步纯化获得粗ATP约2090g；上述获得的粗ATP溶解于16.7L水中，NaOH调节pH为3.0，搅拌下加入乙醇使得乙醇含量为70％，室温下放置24小时，重结晶，过滤获得三磷酸腺苷二钠(三水合物)2213g，纯度大于97％，产率约81.4％。
实施例3固定化酶的重复利用研究
回收的固定化酶用0.02M磷酸钾缓冲液(pH 7.5)洗涤数次后回收重复利用，回收率在85-95％之间。参照实施例2的三磷酸腺苷二钠的制备方法，根据每次回收后的固定化酶的重量，反应体系中各反应物按照比例进行缩减进行三磷酸腺苷二钠的制备，固定化酶多次回收利用的酶促反应获得的三磷酸腺苷二钠的产率如下表1。
表1固定化酶的重复利用催化效率
本发明的固定化酶回收利用6次后的催化效率依然很高，相比于初始制备的固定化酶的催化效率没有明显改变，表明本发明的C18键合改性硅胶对于腺苷激酶(EC2.7.1.20，AK)、多聚磷酸激酶(EC2.7.4.1，Ppk)、多聚磷酸-腺苷酸磷酸转移酶(EC2.7.4.-，Pap)的固定效果非常好，取得了意想不到的效果。
以上虽然已经描述了本发明的一些特定形式，但在不违反本发明的原理的情况下对本发明作出的各种显而易见的改进和组合，应当也属于本发明的范围。
Claims (10)
1.一种生物酶法制备三磷酸腺苷二钠的方法，其特征在于，包括如下步骤：(1)制备固定化酶，所述固定化酶中包含腺苷激酶(EC2.7.1.20，AK)、多聚磷酸激酶(EC2.7.4.1，Ppk)、多聚磷酸-腺苷酸磷酸转移酶(EC2.7.4.-，Pap)；(2)在含有腺苷、六偏磷酸钠、磷酸二氢钠、硫酸镁的反应体系中，加入固定化酶，调节pH值为7.0-7.5，37-45℃下搅拌反应6-10h；(3)反应液过滤回收固定化酶，过滤液经过离子交换树脂初步纯化获得粗ATP；(4)粗ATP溶解于水中，NaOH调节pH为2.5-3.5，加入乙醇重结晶获得三磷酸腺苷二钠。
2.如权利要求1所述的一种生物酶法制备三磷酸腺苷二钠的方法，其特征在于，所述步骤(1)中的固定化酶是以改性硅胶为固定化载体。
3.如权利要求2所述的一种生物酶法制备三磷酸腺苷二钠的方法，其特征在于，所述改性硅胶的粒度为100-400目，所述改性硅胶为C18键合改性硅胶。
4.如权利要求1-3任一项所述的生物酶法制备三磷酸腺苷二钠的方法，其特征在于，所述步骤(1)中固定化酶的制备方法为：将腺苷激酶(EC2.7.1.20，AK)、多聚磷酸激酶(EC2.7.4.1，Ppk)、多聚磷酸-腺苷酸磷酸转移酶(EC2.7.4.-，Pap)按照比例配成混合酶液，加入搅拌反应器中室温下搅拌，接着加入适量改性硅胶与上述酶液混合，在室温下搅拌8-12小时，过滤收集固体，用0.02M磷酸钾缓冲液(pH 7.5)洗涤1-3次，获得固定化酶。
5.如权利要求4所述的生物酶法制备三磷酸腺苷二钠的方法，其特征在于，步骤(1)中的所述固定化腺苷激酶(EC2.7.1.20，AK)、多聚磷酸激酶(EC2.7.4.1，Ppk)、多聚磷酸-腺苷酸磷酸转移酶(EC2.7.4.-，Pap)的浓度比为1-2:1-3:1-3，优选为1:1:1。
6.如权利要求1-3任一项所述的生物酶法制备三磷酸腺苷二钠的方法，其特征在于，所述步骤(2)中腺苷、六偏磷酸钠、磷酸二氢钠、硫酸镁的重量比例为5-10：4-8：1-2：3-6，优选重量比例为5:4:1:3。
7.如权利要求1-3任一项所述的生物酶法制备三磷酸腺苷二钠的方法，其特征在于，所述步骤(3)中的过滤是采用超滤膜过滤，所回收的固定化酶用0.02M磷酸钾缓冲液(pH 7.5)洗涤数次后重复利用；和/或所述步骤(3)中的离子交换树脂选自阴离子交换树脂，优选型号为HZ-201的阴离子交换树脂。
8.如权利要求1-3任一项所述的生物酶法制备三磷酸腺苷二钠的方法，其特征在于，所述步骤(4)中所用溶解用水的用量为粗ATP质量的5-10倍；所述pH调节为3.0；所述步骤(4)中加入乙醇使得乙醇含量为60-80％。
9.一种生物酶法制备的三磷酸腺苷二钠，其特征在于，其由上述权利要求1-8任一所述的生物酶法制备得到。
10.一种如权利要求9所述生物酶法制备的三磷酸腺苷二钠的应用，其特征在于，将所述生物酶法制备的三磷酸腺苷二钠应用于制备药品、保健品中。
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