CN1252255C - 辅酶nadh的光化学再生方法 - Google Patents
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Abstract
本发明公开了一种辅酶NADH的光化学再生方法。属于辅酶再生技术。以可见光或紫外光为光源，实现辅酶NADH的光化学再生方法，其特征在于：以含碳的TiO2作为光催化剂，以水、抗坏血酸、甲酸钠、EDTA或巯基乙醇为电子供体，光体系下在铑金属络合物[Cp*Rh(bpy)(H2O)]存在和无酶参与的情况下使NADH再生。本发明的优点在于：含碳的TiO2能吸收可见光，制备过程简单，成本较低，NADH的光化学再生在可见光照射下进行，能量利用率高，而且避免了紫外光对酶的影响；不需要有毒电子媒介物甲基紫晶的参与；不需要酶的参与，避免了再生酶与合成酶最适条件不同而使体系难以控制的弊病。
Description
技术领域
本发明是涉及一种NADH的光化学再生方法，属于辅酶再生技术。
背景技术
在酶的六大类型中，30％～35％为氧化还原酶。大部分氧化还原酶催化作用的发挥需要烟酰胺腺嘌呤二核苷酸辅酶(NAD(P)+、NAD(P)H)作为氧化剂或还原剂直接参与反应。然而，NAD(P)+和NAD(P)H的价格昂贵，通常比酶促反应所得产物要贵得多。因此，从技术经济性的角度来看，对辅酶进行再生并循环使用是很有必要的。此外，辅酶再生能够简化产物的分离，并有利于酶促反应向正反应方向移动。
目前已有的再生方法包括酶法、电化学法、光化学法等。酶法再生的优点在于反应速率快，选择性高，再生体系与合成体系兼容性好。但所用酶往往比较昂贵，且体系涉及两种或两种以上酶。酶的最适应用条件往往不一致，给过程优化带来困难。电化学法的再生能量来自于洁净的电能，与酶法相比成本低。电化学法中氧化还原电势的控制和反应过程的监测都较为容易。但是它与酶促合成体系的兼容性差，选择性低(尤其是对于还原态辅酶的再生)，因此电化学法比较适合于氧化态辅酶的再生。光化学法再生辅酶利用的是廉价且洁净的光能，通常需要光敏剂、电子媒介物和电子供体。光化学再生法目前还未获得理想的效果，其再生效率很低，但具有广阔的潜在应用前景。
现有的NADH光化学再生法常采用金属络合物，如Ru(II)-三(二嘧啶)(Ru(bpy)3 2+)、Zn(II)-N-(四甲基吡啶)卟啉(Zn-TMPy4+)或半导体(TiO2、CdS)为光敏剂，在电子媒介物甲基紫晶(MV2+)和酶(铁氧化还原酶FDR或硫辛酰胺脱氢酶LipDH)存在下将NAD+还原为NADH。这些方法都存在着严重的不足：光敏剂Ru(bpy)3 2+和Zn-TMPy4+在光体系下极不稳定，易发生光降解；TiO2虽然在光体系下很稳定，而且无毒、价格便宜，但TiO2是宽禁带半导体，只能被波长较短的紫外光(λ＜387nm)激发，而这部分光只占到太阳光的8％，能量利用率低；CdS虽然能够被可见光激发，但它在光体系下易发生光腐蚀；电子媒介物MV2+是有毒物质，容易污染产物，且MV+具有很强的还原性，不仅能将NAD+还原，在有些情况下还能将酶促合成的产物还原。将无机半导体颗粒CdS直接与氢化酶的活性中心相连接，使电子直接在酶与半导体间传递虽然可以避免使用甲基紫晶，但氢化酶价格昂贵且不稳定，而且将CdS与其活性中心相连的反应过程复杂。此外，上述方法都需要有酶的参与，从而再生的成本较高。
发明内容
本发明的目的在于提供一种辅酶NADH的光化学再生方法。该方法过程简单，再生成本较低。
本发明是通过下述技术方案实现的。以可见光或紫外光为光源，实现辅酶NADH的光化学再生方法，其特征在于：以1mol辅酶NAD+为基准，加入1～5mol的水、抗坏血酸、甲酸钠、EDTA或巯基乙醇电子供体，加入0.4～2.5mol的铑金属络合物[Cp*Rh(bpy)(H2O)]，加入含碳量为18.3～49.2％的100～600mol的TiO2光催化剂，在温度25～37℃，pH值在6.5～7.5条件下，反应得到NADH。
本发明提出的光化学再生NADH的优点在于：含碳的TiO2能吸收可见光，与掺杂贵金属(Mo、Co、Pt)以降低TiO2激发能的方法相比，掺碳的方法更为简单，成本较低，是一种新型材料，此前从未用于NADH的再生；NADH的光化学再生在可见光照射下进行，能量利用率高，而且避免了紫外光对酶的影响；不需要有毒电子媒介物甲基紫晶的参与；不需要酶的参与，不仅降低了成本，而且避免了再生酶与合成酶最适条件不同而使体系难以控制的弊病。
附图说明
图1为NADH纯品的质谱图。
图2为NAD+纯品的质谱图。
图3为反应产物混合物的质谱图，图3中丰度在710.2处为产物NADH的特征峰。
具体实施方式
实施例一
分别称取80mg含碳量分别为0、18.3％、27.5％、39.7％和49.2％的五种TiO2催化剂，在T＝37℃、pH＝6.5下进行光照实验，[Cp*Rh(bpy)(H2O)]2+起始浓度为0.2mM，NAD+起始浓度0.2mM，反应总体积为50mL，反应进行11小时。
(1)在λ≤365nm(紫外光)光照下反应。
(2)在λ≥400nm(可见光)光照下反应。
产物NADH由质谱进行定性测量，NADH的特征峰为733.2、732.3和710.2；由于NADH在340nm处有强吸收，因此NADH的浓度由紫外-可见分光光度计测量。每次取样0.6mL，稀释至3mL后在紫外-可见分光光度计上测量340nm处的吸光度。
实施例二
称取80mg含碳量49.2％的TiO2样品，在λ≥400nm(可见光)光照下反应，反应体积为50mL。通过测量反应10h后NAD+的转化率，考察了pH值、反应温度、NAD+浓度、[Cp*Rh(bpy)(H2O)]2+浓度等反应条件对反应转化率的影响。反应条件及NAD+转化率列于下表：
表1反应体系pH值对NAD+转化率的影响
| 序号 | pH | T/℃ | NAD+浓度/mM | 铑络合物浓度/mM | NAD+转化率％ |
| 12345 | 6.06.57.07.58.0 | 3131313131 | 0.20.20.20.20.2 | 0.20.20.20.20.2 | 39.925.817.814.311.2 |
表2温度对NAD+转化率的影响
| 序号 | pH | T/℃ | NAD+浓度/mM | 铑络合物浓度/mM | NAD+转化率％ |
| 123 | 6.56.56.5 | 252831 | 0.20.20.2 | 0.20.20.2 | 11.217.025.8 |
| 45 | 6.56.5 | 3437 | 0.20.2 | 0.20.2 | 39.057.4 |
表3NAD+浓度对NAD+转化率的影响
| 序号 | pH | T/℃ | NAD+浓度/mM | 铑络合物浓度/mM | NAD+转化率％ |
| 12345 | 6.56.56.56.56.5 | 3131313131 | 0.10.20.30.40.5 | 0.20.20.20.20.2 | 19.825.830.533.334.2 |
表4[Cp*Rh(bpy)(H2O)]2+浓度对NAD+转化率的影响
| 序号 | pH | T/℃ | NAD+浓度/mM | 铑络合物浓度/mM | NAD+转化率％ |
| 12345 | 6.56.56.56.56.5 | 3131313131 | 0.20.20.20.20.2 | 0.10.20.30.40.5 | 15.725.834.138.940.9 |
本发明采用含碳TiO2和铑金属络合物为催化剂，以可见光或紫外光为光源，实现了辅酶NADH的再生，实验中，NAD+的转化率可达到57.4％。
Claims (1)
1.一种辅酶NADH的光化学再生方法，该方法是以可见光或紫外光为光源，实现辅酶NADH的光化学再生，其特征在于：以1mol辅酶NAD+为基准，加入1～5mol的水、抗坏血酸、甲酸钠、EDTA或巯基乙醇电子供体，加入0.4～2.5mol的铑合金络合物，加入含碳量为18.3～49.2％的100mol的TiO2光催化剂，在温度25～37℃，pH值在6.5～7.5条件下，反应得到NADH。
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