CN112852798A - 一种核壳纳米多聚物的制备方法及其在辅酶循环再生上的应用 - Google Patents
一种核壳纳米多聚物的制备方法及其在辅酶循环再生上的应用 Download PDFInfo
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Abstract
本发明公开了一种核壳纳米多聚物的制备方法及其在辅酶循环再生上的应用，通过金属骨架化合物ZIF材料原位共固定辅酶聚合物和两种辅酶依赖性酶制备核壳结构的多聚物。本发明利用ZIF外壳限定多酶生物催化反应区域，利用双酶分别在引发剂和底物的作用下，实现辅酶的循环再生和双酶级联反应的进行。在核壳多聚物使用期间不需要再添加额外的辅酶，在5次重复使用后对底物的转化率与初始转化率的比值仍有70‑80%。核壳纳米多聚物在有辅酶参与的生物合成与转化上具有十分广阔的前景。
Description
技术领域
本发明涉及纳米材料领域，还涉及多酶共固定化和辅酶循环再生领域。
背景技术
氧化还原酶广泛应用于催化制备手性醇,羟基酸,氨基酸等方面。大多数的氧化还原酶催化反应都需要辅酶,在其所需辅酶中,NAD(P)+-NAD(P)H体系占90％左右。但这些辅酶价格昂贵,限制了氧化还原酶的应用。酶在受限结构中的空间组织为设计生物催化级联提供了一种通用方法，将一种酶的产物作为另一种酶的底物，从而实现多酶之间的相互通讯，利用这一原理可以利用在纳米尺度下实现多酶催化级联和辅酶的循环再生。
目前多酶在微尺度或纳米尺度等不同空间组织下的共固定化已经被报道，酶在DNA 支架上的空间定位，如DNA带、DNA链或DNA折叠，已被用于操作两或三酶级联。因此，对于多酶催化级联以及固定化多酶的可行性策略的研究在酶均相/非均相催化中具有重要作用。但是这种载体存在价格昂贵，操作复杂等问题限制其在实际应用中的发展。
目前关于单酶的封装和固定化的研究集中在提高封装酶的稳定性和催化活性上，并在催化代谢过程中的氧化还原反应和对底物的转化效率方面有了较深入的研究。关于多酶的共固定化以及联动体系的构建等仍需要进行大量的研究。多酶共固定化的局限性之一是无法正确地折叠大的多结构域蛋白，因此效率通常较低。尤其是多酶在固定化时或封装的同时往往会因为溶剂的作用而使活性位点损失，而合成金属骨架化合物纳米颗粒时的溶剂热、高温高压、超声辅助等条件对酶的活性位点影响较大。
以下是主要相关的文章和已经公开的专利，但明显与本申请文件不同，如表1：
表1 部分公开文章或专利与申请文件的区别
发明内容
本发明提供了一种用于核壳纳米聚合物的制备方法以及其在辅酶循环再生上的应用。本发明通过采用原位的方法利用材料的生长将不同尺寸的多种酶共固定化，利用多酶的反应催化过程中对不同状态下辅酶的转化实现辅酶的循环再生。
本发明所涉及的所有试剂均购买于试剂公司，如辅酶依赖性酶、辅酶、乙酸锌、硝酸锌、2-甲基咪唑、三乙胺、乙酸钴等。
本发明是通过以下技术方案实现的：
一种核壳纳米多聚物的制备方法，主要利用ZIF-8和ZIF-67晶体的生长特性，将两种辅酶依赖性酶、辅酶多聚物原位共固定化而得。
进一步，所述的制备方法的步骤中，在固定化辅酶时，将介孔硅表面进行羧基修饰后与辅酶的非活性位点结合实现辅酶的固定。
进一步，固定到同一核壳纳米体系中的两个辅酶依赖性酶分别为能够催化转化两种不同状态辅酶，即两个酶能分别进行脱氢和加氢从而实现辅酶的循环再生。当辅酶聚合物固定化辅酶为NAD+和NADH这一组合中的一种时，可将辅酶依赖性酶划分为乙醇脱氢酶、葡萄糖脱氢酶、3-磷酸甘油醛脱氢酶和乳酸脱氢酶、酵母酒精脱氢酶等两类酶。多聚物中固定的两种酶可分别选用这两类的某一个组合。辅酶聚合物固定化辅酶为NADPH和NADP+这一组合中的一种时，也可将辅酶依赖性酶划分为两类，一类为苹果酸酶、亚磷酸脱氢酶、异丙醇脱氢酶、葡萄糖-6-磷酸脱氢酶等。另一类为谷氨酸脱氢酶，多聚物中固定的两种酶从第一类任选一种与谷氨酸脱氢酶组合。
进一步，两种辅酶依赖性酶与辅酶聚合物的质量比为1:2-1:10。
进一步，所述的制备方法的步骤中，在15-25℃条件下磁力搅拌10-15h，以原位合成的方式进行多酶共固定，制备具有核壳结构的纳米多酶体系。
进一步，核壳多聚物的ZIF外壳，能够有效保护酶的稳定性，更重要的是ZIF材料具有亲和性将酶吸附到辅酶聚合物周围，减少了底物的扩散。
进一步，核壳多聚物依赖于所制备的纳米复合材料的限域效应，从而实现辅酶的高效率转化。
进一步，核壳多聚物在辅酶循环时，需要引发剂与底物的参与。若辅酶聚合物中固定的为NAD+，则醇脱氢酶、葡萄糖脱氢酶、3-磷酸甘油醛脱氢酶的引发剂为乙醇、葡萄糖、3-磷酸甘油醛，选用乳酸脱氢酶、酵母酒精脱氢酶作为辅酶再生酶，底物分别为丙酮酸、乙醇。反之若固定化的为NADH，则醇脱氢酶、葡萄糖脱氢酶、3-磷酸甘油醛脱氢酶作为辅酶再生酶，底物为乙醇、葡萄糖、3-磷酸甘油醛，乳酸脱氢酶或者酵母酒精脱氢酶在丙酮酸或乙醇等引发剂的作用下引发辅酶循环再生。若辅酶聚合物中固定的为NADP+，则苹果酸酶、亚磷酸脱氢酶、异丙醇脱氢酶、葡萄糖-6-磷酸脱氢酶的引发剂分别为苹果酸、亚磷酸、异丙醇和6-磷酸葡萄糖。谷氨酸脱氢酶作为辅酶还原酶，底物为谷氨酸。反之若固定化NADPH时，苹果酸酶、亚磷酸脱氢酶、异丙醇脱氢酶、葡萄糖-6-磷酸脱氢酶作为辅酶还原酶，底物分别为苹果酸、亚磷酸、异丙醇和6-磷酸葡萄糖，谷氨酸脱氢酶在引发剂谷氨酸的存在下引发辅酶循环再生。
本发明的有益效果
与游离条件下NAD+/NADH辅助因子的具有扩散作用相比，本发明研究的NAD+/NADH(NADP+/NADPH)功能化聚合物被限制在ZIF的外壳内，可以使两种酶在壳内传递有效交换本体溶液和反应位点之间的底物和产物，模拟发生在细胞环境中的生物催化，从而实现辅酶的循环再生。通过产物的转化速率来反应辅酶的再生效果，核壳多聚物相较于游离酶的转化率增加了1-3倍，表明核壳多聚物的在辅酶循环再生上的优势。
附图说明
图1位ZIF-8核壳多聚物的SEM和TEM图。
图2位ZIF-8核壳多聚物的CLSM图：FITC标记醇脱氢酶为乙醇脱氢酶，罗丹明标记乳酸脱氢酶。
具体实施方式
下面通过实施例，对本发明的具体实施方式进一步的描述。以下实施例是用来进一步说明本发明的内容，而不限制本发明的保护范围。对于本发明所涉及的相关内容及所做的修改，均属于本发明保护范围。
实施例1：
制备辅酶多聚物：称取2g羧基修饰后的介孔硅纳米粒子，在30mL去离子水中超声处理8-15 分钟后加入一定量的辅酶NAD+，用0.5M盐酸溶液调节pH至4.5-5.5，在25-30℃下磁力搅拌。在5分钟内连续加入50mg水溶性碳二亚胺EDC，在加入过程中继续滴加盐酸溶液，使 pH保持在4.5-5.5，在25-30℃下磁力搅拌25-30h。反应结束后，离心除去未反应的NAD+和其他杂质，用0.2M NaCl溶液洗涤，直至洗涤液中未检出NAD+。最后冷冻干燥得到固定化辅酶。
原位共固定多酶：将0.5-1mg乙醇脱氢酶、0.5-1mg乳酸脱氢酶、1-2mg MSN-NAD+加入5-10mL醋酸锌(40mM)溶液中，在室温搅拌下快速加5-10mL(2.8mM)2-甲基咪唑并搅拌10-12h。将溶液沉淀离心，用去离子水洗涤数次后冷冻干燥得到以ZIF-8作为外壳纳米多聚物。采用同样的方法，将0.4-0.5g的Co(CH3COO)2分散到10-15mL的水溶液中，加入0.5-1 mg的乙醇脱氢酶和乳酸脱氢酶，1-2mg的辅酶聚合物，然后在室温搅拌下快速加入10-15 mL(1-1.5g)的2-甲基咪唑并搅拌10-12h。将溶液沉淀离心，用去离子水洗涤数次后冷冻干燥得到以ZIF-67作为外壳的纳米多聚物。
辅酶循环：取100-150mM氯化钠，100-150μg/mL多聚物，2-3mM丙酮酸，20-30mM 乙醇，加入200μL PB缓冲溶液(100mM pH 7.4)。以等量的ADH、辅酶聚合物和LDH为对照。通过检测是否有乳酸的产生，监测核壳纳米体系下辅酶的循环再生效果。
本实施例核壳多聚物反应体系中，基于乙醇脱氢酶在引发剂乙醇的作用下转化NAD+生成NADH，生成的NADH又参与到乳酸脱氢酶催化丙酮酸合成乳酸的反应这一实验原理。如果在最终的反应体系中能够检查到乳酸的合成，就表明了在核壳纳米多聚物中生成了 NADH，而大量的乳酸合成则证实了在多聚物体系中NAD+和NADH的转变即实现了辅酶的循环再生。两种核壳多聚物体系中合成的乳酸浓度相较于游离酶均增加了2-3倍。经过5次重复使用下，在相同条件下ZIF-8作为外壳的多聚物对乳酸的转化率为70％-80％。以ZIF-67 作为外壳的多聚物对乳酸的转化率为65-75％。这一结果归因于核壳多聚物的特殊结构。ZIF 材料所形成的具有明显尺寸选择性的过渡层，可以保护内部酶免受损伤，减少大分子背景干扰。ZIF材料具有较高的化学稳定性和结构稳定性，可以作为酶长期储存的选择。
实施例2：
称取2g基团修饰后的介孔硅纳米粒子，在30mL去离子水中超声处理8-15分钟后加入一定量的辅酶NADPH，在25-30℃下磁力搅拌。在5分钟内连续加入50mg水溶性碳二亚胺EDC，在加入过程中控制pH保持在7.4-8，在25-30℃下磁力搅拌25-30h。反应结束后，离心除去未反应的NADPH和其他杂质，用0.2M NaCl溶液洗涤，直至洗涤液中未检出NADPH。最后冷冻干燥得到固定化辅酶。分别利用ZIF-67和ZIF-8材料原位生长的方式将谷氨酸脱氢酶、葡萄糖-6-磷酸脱氢酶共固定化和辅酶聚合物封装。加入一定量的谷氨酸和6-磷酸葡萄糖引发体系内的辅酶循环和多酶级联反应。谷氨酸脱氢酶与体系内的NADPH的存在下催化谷氨酸脱氨生成α-酮戊二酸和NADP+。生成的NADP+又参与到葡萄糖-6-磷酸脱氢酶对6-磷酸葡萄糖的转化中生成NADPH和6-磷酸葡糖酸。以产生的6-磷酸葡糖酸的量来反映核壳多聚物内的辅酶循环再生效果和多酶级联效率。经过实验表明，两种ZIF材料包封的多聚物均能在不添加NADP+的条件下，能够检测到6-磷酸葡糖酸的存在，经计算有60-70％的6-磷酸葡萄糖转化为6-磷酸葡糖酸。
Claims (5)
1.一种核壳纳米多聚物的制备方法及其在辅酶循环再生上的应用包含以下步骤；
（1）辅酶多聚物的制备
利用模板法制备粒径为60-90 nm的介孔硅纳米颗粒，然后对其表面进行基团修饰，将修饰后介孔硅与辅酶（NAD+和NADH，NADP+和NADPH等两个组合中的一种）的非活性位点结合制备辅酶聚合物，备用；
（2）多酶原位共固定化
采用原位合成的方法将酶进行共固定：将乙酸锌（硝酸锌、乙酸钴等）2-甲基咪唑、三乙胺、辅酶聚合物、两种辅酶依赖性酶按照一定比例分散于pH 7-9的PBS溶液中，15-25 ℃磁力搅拌12-20 h、冷冻干燥后得。
2.根据权利要求书1所述，其特征在于，可以用ZIF-8材料或者ZIF-67材料作为多聚物外壳；用ZIF-8封装时，按质量比计，乙酸锌:2-甲基咪唑=10:1-15:1，硝酸锌：2-甲基咪唑=1:3-1:5；用ZIF-67封装时，按质量比计，乙酸钴：2-甲基咪唑：三乙胺=3:1:0-5:1:18。
3.根据权利要求书1-2所述，其特征在于，多聚物中共固定化的两种辅酶依赖性酶在催化反应过程中一个需要同一组辅酶的参与；两种辅酶依赖性酶与辅酶聚合物的质量比为1:1:2-1:5:10。
4.根据权利要求书1-3所述，其特征在于，将制备的核壳多聚物分散于PBS溶液中，添加引发剂和辅酶再生底物实现辅酶的循环再生。
5.根据权利要求书1-4所述的方法，其特征在于，ZIF外壳对酶具有亲和性，使得辅酶依赖性酶聚集到辅酶聚合物周围，减少底物的扩散，促使辅酶因子在体系内快速转化，从而实现辅酶的循环再生和进行双酶级联反应；将其应用在需要辅酶参与的生物催化中，可以避免昂贵辅酶的投入。
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