CN1597940A - 辅酶nadh的光化学再生方法 - Google Patents

辅酶nadh的光化学再生方法 Download PDF

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CN1597940A
CN1597940A CN 200410020227 CN200410020227A CN1597940A CN 1597940 A CN1597940 A CN 1597940A CN 200410020227 CN200410020227 CN 200410020227 CN 200410020227 A CN200410020227 A CN 200410020227A CN 1597940 A CN1597940 A CN 1597940A
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nadh
nad
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CN1252255C (zh
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姜忠义
吴洪
吕陈秋
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Tianjin University
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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

辅酶NADH的光化学再生方法
                         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-TMPy 4+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%
1       6.0    31          0.2              0.2              39.9
2       6.5    31          0.2              0.2              25.8
3       7.0    31          0.2              0.2              17.8
4       7.5    31          0.2              0.2              14.3
5       8.0    31          0.2              0.2              11.2
                      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%
1       6.5    25          0.2              0.2              11.2
2       6.5    28          0.2              0.2              17.0
3       6.5    31          0.2              0.2              25.8
4       6.5    34         0.2               0.2             39.0
5       6.5    37         0.2               0.2             57.4
                 表3 NAD+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%
1       6.5    31         0.1               0.2             19.8
2       6.5    31         0.2               0.2             25.8
3       6.5    31         0.3               0.2             30.5
4       6.5    31         0.4               0.2             33.3
5       6.5    31         0.5               0.2             34.2
            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%
1       6.5    31         0.2               0.1             15.7
2       6.5    31         0.2               0.2             25.8
3       6.5    31         0.2               0.3             34.1
4       6.5    31         0.2               0.4             38.9
5       6.5    31         0.2               0.5             40.9
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的铑金属络合物,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。
CN 200410020227 2004-08-03 2004-08-03 辅酶nadh的光化学再生方法 Expired - Fee Related CN1252255C (zh)

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Cited By (4)

* Cited by examiner, † Cited by third party
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CN102469770A (zh) * 2009-06-29 2012-05-23 拜尔生物科学公司 通过选择表观遗传学修饰种群改进作物产量
ITMI20131135A1 (it) * 2013-07-05 2015-01-06 Catalisi Innovativa Per Il Riciclo Del Carbonio E Fotocatalizzatore per la riduzione nel visibile di nad+ a nadh in un processo ibrido chemo-enzimatico di riduzione di co2 a metanolo
CN110327926A (zh) * 2019-06-18 2019-10-15 中国石油大学(华东) 一种铁离子掺杂二氧化钛纳米材料的制备方法
CN114192173A (zh) * 2021-11-05 2022-03-18 五邑大学 一种用于nadh再生的光催化剂及其制备方法和应用

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102469770A (zh) * 2009-06-29 2012-05-23 拜尔生物科学公司 通过选择表观遗传学修饰种群改进作物产量
CN102469770B (zh) * 2009-06-29 2013-12-11 拜尔作物科学公司 通过选择表观遗传学修饰种群改进作物产量
ITMI20131135A1 (it) * 2013-07-05 2015-01-06 Catalisi Innovativa Per Il Riciclo Del Carbonio E Fotocatalizzatore per la riduzione nel visibile di nad+ a nadh in un processo ibrido chemo-enzimatico di riduzione di co2 a metanolo
CN110327926A (zh) * 2019-06-18 2019-10-15 中国石油大学(华东) 一种铁离子掺杂二氧化钛纳米材料的制备方法
CN110327926B (zh) * 2019-06-18 2023-07-14 中国石油大学(华东) 一种铁离子掺杂二氧化钛纳米材料的制备方法
CN114192173A (zh) * 2021-11-05 2022-03-18 五邑大学 一种用于nadh再生的光催化剂及其制备方法和应用

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