NADH氧化酶的应用研究进展

目前辅酶再生是酶工程研究领域的一大热点,在氧化态辅酶再生方面,NADH氧化酶显示出巨大的应用前景.研究发现,许多微生物中都存在着NADH氧化酶.本文综述了近年来NADH氧化酶在应用研究方面的进展.     

工业微生物还原型辅酶Ⅱ的代谢调控研究进展

还原型辅酶Ⅱ（NADPH）主要参与细胞合成代谢,是微生物代谢网络中含量最丰富的氧化还原辅酶之一。辅酶工程作为代谢工程的重要分支,通过改变微生物胞内辅酶再生途径,进而改变细胞内代谢产物构成。本文在归纳NADPH产生途径和调控的基础上,分析和评述了工业微生物基于辅酶工程的NADPH代谢调控研究进展,包括过量表达NADPH代谢相关酶、敲除NADPH代谢相关基因及引入特定代谢途径等策略,指出今后的研究重点在于深入理解NADPH调控与中心碳代谢网络的相互作用,为利用代谢工程进行细胞工厂改造提供 基础。     

氧化还原酶和减少其使用的二酮衍生物

正对结构式二酮衍生物选择性酶促还原(Ⅰ)(其中包括无环结构,一个或多个杂原子)包括在还原型辅酶NADH或NADPH为的存在下,氧化还原酶/脱氢酶减少二酮衍生物,其中二酮衍生物的反应开始时使用浓度≥10g/L;和连续再生氧化辅酶NAD和NADP还原酶/脱氢酶的形成。R1氢或1~4个碳的烷基;R2:氢,1~8个碳的烷基或羟基保护基团,如酯;R3:氢、甲基或卤化物;而A:苯环或六碳环上有     

分段通气对Klebsiella pneumoniae生产1,3-丙二醇关键酶和辅酶的影响

通过考察1,3-丙二醇合成中关键酶、辅酶的变化,研究了分段通入空气对Klebsiella pneumoniae厌氧发酵生产1,3-丙二醇的影响. 与对照组相比,在发酵前期(12 h)通入空气4 h后,甘油脱氢酶酶活提高1.5倍,1,3-丙二醇氧化还原酶酶活提高18%,1,3-丙二醇浓度提高16%;发酵后期(28和48 h)通入空气后,甘油脱氢酶酶活不变,1,3-丙二醇氧化还原酶酶活下降,1,3-丙二醇浓度降低. 发酵前期,通气对辅酶NADH和NAD的浓度无影响;发酵后期,菌体生长停滞,辅酶的浓度也随之下降.     

无电荷超滤膜酶膜反应器中的辅酶截留

构建了无电荷超滤膜酶膜反应器,考察了影响辅酶截留率的多种因素.实验结果表明,连续超滤膜反应器中加入聚乙烯亚胺(PEI,50 kDa),NAD 能够有效地被超滤膜截留.在Tris-HCl缓冲液中,PEI/NAD 摩尔比为5:1时,达到较高的辅酶截留率(R=0.823).溶液离子强度的增加减少辅酶的截留率,但可增加超滤膜超滤流速.在含盐溶液中,添加0.5%或1.O%的牛血清白蛋白可增加NAD 截留率.本装置对NADH的截留率高于NAD 的截留率,因此PEI适用于辅酶再生体系.同时还考察了1,3-丙二醇酶催化体系中PEI对甘油脱氢酶和1,3-丙二醇氧化还原酶酶活力的影响.     

重组甲酸脱氢酶对合成1,3-丙二醇的促进作用

在甘油厌氧发酵生产1,3-丙二醇的过程中，需要消耗还原当量NADH，NADH的有效供给决定了1,3-丙二醇的产量。本文从Candida boidinii基因组DNA中克隆了甲酸脱氢酶基因fdh，利用表达质粒pMAL TM-p2X-fdh转化到1,3-丙二醇生产菌 Klebsiella pneumoniae YMU2中，构建了具有NADH再生系统的重组菌Klebsiella pneumoniae F-1。在5 L发酵罐培养中，F-1合成1,3-丙二醇浓度和产率分别达到了78. 6 g·L-1 和1. 33 g·L-1·h-1，分别比YMU2提高了12. 5% 和41. 2%。根据F-1和YMU2菌株的主要代谢产物的生成情况比较了二者的代谢流分布。     

辅酶自循环的氧化还原级联体系在生物催化过程中的应用：机遇与挑战

氧化还原酶是生物催化过程中应用最为广泛的一类酶，其可以在温和条件下高选择性进行催化，在化工、医药、农业等领域发挥着重要作用。大部分氧化还原酶催化的反应需要烟酰胺辅因子参与。烟酰胺辅因子价格昂贵，大量外源添加会加重成本，无法满足工业生产的要求，因此开发高效经济的辅因子再生策略对氧化还原酶的工业应用具有重要意义。在常用的辅因子再生方法中，酶法因其高效绿色的特点得到了广泛的应用。其中，辅酶自循环的氧化还原级联体系是一种特殊的酶法再生烟酰胺辅因子策略，它能结合多酶级联催化，在不添加任何共底物的情况下，完成烟酰胺辅因子内部的自给自足循环再生。相比其他酶法再生策略，其具有副产物少、原子经济性高等突出优势。本文按照酶促反应进行的顺序将辅酶自循环的氧化还原级联体系分为四大类进行讨论，并以醇/醛脱氢酶为基础，综述了该体系应用于生物催化过程的机遇与挑战，为进一步开发更高效的辅酶自循环的氧化还原级联体系提供思路。     

辅酶NADH模型物BNAH与活化烯烃在氧气饱和混合溶剂中的反应机理研究

在氧气饱和的混合溶剂中,随着反应时间的延长,在辅酶NADH模型物BNAH与活化烯烃的反应中,氧化产物与还原产物的比例逐渐变小,但总产率越来越大。     

辅酶Q0的简便合成

以3,4,5-三甲氧基苯甲醛为原料,经Wolff-Kishner-黄鸣龙还原反应和过氧化氢氧化反应简便、高效地制备辅酶Q10的重要原料辅酶Q0.对影响反应的关键因素进行了研究,发现甲酸介质、3倍过量的水合肼、50%过氧化氢以及用磷钼酸作催化剂(用量为TMT质量的2.5%～3.0%)、反应45 min为比较合适的条件.     

乳酸生产中的微生物代谢工程

从代谢工程的角度综述了同型及异型乳酸发酵的代谢途径、乳酸菌代谢模型、乳酸脱氢酶在乳酸生产方面的应用、米根霉发酵生产乳酸的代谢工程和基因工程阻断乙醇代谢途径改造乳酸的生产过程等方面的研究进展,讨论了生物信息学及环境胁迫对乳酸代谢的影响,展望了乳酸的微生物代谢工程的发展趋势.     

纳米粒子固定化双酶耦合体系连续催化制备(R)-苯基乙二醇

将活化醇盐水解法制备的SiO2纳米粒子分别与羰基还原酶(CR)和甲酸脱氢酶(FDH)进行共价固定化,固定化CR与FDH耦合,连续催化转化b-羟基苯乙酮制备(R)-苯基乙二醇,考察了NADH的再生与循环利用性. 结果表明,纳米粒子固定化CR和FDH酶载量分别为3.32和5.55 mg/g,催化活性为游离酶的50%~60%,最适反应pH值分别为6.5和8.5,最适反应温度分别为40和45℃. 耦合体系进行12批次反应,产物(R)-苯基乙二醇累积量达35.6 g/L,纳米粒子生产能力达178 g/g. 纳米粒子固定化酶经简单离心收集后可重复利用.     

Recent Trends in Biomimetic NADH Regeneration

Nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate constitute a major cost factor in preparative biotransformations. The development of efficient methods for their regeneration with cheap reducing equivalents has been an area of intense research in the last decades. Methods explored include chemical, electrochemical, and photochemical approaches. None of the methods to regenerate NADH has reached an efficiency comparable with enzymatic regeneration (e.g. formate dehydrogenase) which remains the method of choice for most applications. In this review, we summarize primarily organometallic-based approaches for NADH regeneration methods which include non-enzymatic steps, before moving on to the most recent developments in synthetic NADH related transformations. We highlight the frequent problem of mutual inactivation between the organometallic catalyst for NADH regeneration and the corresponding NADH dependent downstream enzyme. Potential remedies are discussed, such as the compartmentalization of the organometallic complex.     

Improving Biocatalytic Properties of an Azoreductase via the N-Terminal Fusion of Formate Dehydrogenase

Azoreductases require NAD(P)H to reduce azo dyes but the high cost of NAD(P)H limits its application. Formate dehydrogenase (FDH) allows NAD(P)⁺ recycling and therefore, the fusion of these two biocatalysts seems promising. This study investigated the changes to the fusion protein involving azoreductase (AzoRo) of Rhodococcus opacus 1CP and FDH (FDH_C23S and FDH_{C23SD195QY196H}) of Candida boidinii in different positions with His-tag as the linker. The position affected enzyme activities as AzoRo activity decreased by 20-fold when it is in the N-terminus of the fusion protein. FDH_C23S+AzoRo was the most active construct and was further characterized. Enzymatic activities of FDH_C23S+AzoRo decreased compared to parental enzymes but showed improved substrate scope – accepting bulkier dyes. Moreover, pH has an influence on the stability and activity of the fusion protein because at pH 6 (pH that is suboptimal for FDH), the dye reduction decreased to more than 50 % and this could be attributed to the impaired NADH supply for the AzoRo part.     

Engineering a Formate Dehydrogenase for NADPH Regeneration**

Nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) constitute major hydrogen donors for oxidative/reductive bio-transformations. NAD(P)H regeneration systems coupled with formate dehydrogenases (FDHs) represent a dreamful method. However, most of the native FDHs are NAD⁺-dependent and suffer from insufficient reactivity compared to other enzymatic tools, such as glucose dehydrogenase. An efficient and competitive NADP⁺-utilizing FDH necessitates the availability and robustness of NADPH regeneration systems. Herein, we report the engineering of a new FDH from Candida dubliniensis (CdFDH), which showed no strict NAD⁺ preference by a structure-guided rational/semi-rational design. A combinatorial mutant CdFDH-M4 (D197Q/Y198R/Q199N/A372S/K371T/▵Q375/K167R/H16L/K159R) exhibited 75-fold intensification of catalytic efficiency (k_cat/K_m). Moreover, CdFDH-M4 has been successfully employed in diverse asymmetric oxidative/reductive processes with cofactor total turnover numbers (TTNs) ranging from 135 to 986, making it potentially useful for NADPH-required biocatalytic transformations.     

Removal and recovery of ammonium ion from wastewater by adsorption on natural zeolite

The Removal test of ammonium ion from cokes wastewater by natural zeolite was performed in two point of view. One is to examine the adsorption capacity stability for ammonium ion in recyclic adsorption-desorption. The other is to find out the effective chemical regeneration process used by experiments and mathematical method in connection with regeneration cost and the recovery of ammonium ion.     

碳酸酐酶和甲酸脱氢酶的稳定性研究进展

化石燃料的大量燃烧使温室气体CO₂的排放量不断增加,对环境造成恶劣影响,将CO₂捕集并转化为高附加值化学品是实现节能减排和变废为宝的一种双赢策略。酶催化CO₂捕集和转化具有高效、高选择性、反应条件温和、环境友好等优点。碳酸酐酶（CA）可大大加速CO₂水合反应,而甲酸脱氢酶（FDH）可催化CO₂还原为甲酸,二者协同可增强CO₂还原动力学。但酶促反应的工业化应用过程中,酶所处环境的温度、酸碱度以及其他离子的种类和浓度等因素均可能导致酶失活,因此,酶的稳定性研究至关重要。本文从热稳定性、酸碱稳定性和离子稳定性的角度,综述了CA和FDH的稳定性研究进展。改善酶稳定性的手段包括使用极端微生物、酶分子设计与改造、固定化等,重点讨论了固定化对酶稳定性的提升效果,为未来的工业化应用提供参考。     

二氧化碳还原用甲酸脱氢酶的研究进展

CO2的还原和资源化利用是缓解温室效应的重要手段。生物催化剂对反应和底物具有高选择性,因此被用于构建高效的CO2还原系统。其中甲酸脱氢酶(Formate dehydrogenase, FDH),特别是某些NAD+^依赖性/包含金属(W-或Mo-)辅因子的甲酸脱氢酶,能够可逆地将CO2还原成甲酸盐。本综述总结了最近发现的CO2还原用甲酸脱氢酶的还原活性及催化机制、酶工程改造、全细胞生物催化和基于CO2还原酶的应用进展,为CO2还原用生物催化剂将来的研究提供了较大的启示,为以丰富、廉价和可持续的CO2为原料,构建可行的CO2还原系统,以生产增值的燃料和化学品提供了理论基础。     

A Minimalist Approach to the Design of Complexity‐Enriched Bioactive Small Molecules: Discovery of Phenanthrenoid Mimics as Antiproliferative Agents

Over the last decades, much effort has been devoted to the design of the “ideal” library for screening, the most promising strategies being those which draw inspiration from biogenic compounds, as the aim is to add biological relevance to such libraries. On the other hand, there is a growing understanding of the role that molecular complexity plays in the discovery of new bioactive small molecules. Nevertheless, the introduction of molecular complexity must be balanced with synthetic accessibility. In this work, we show that both concepts can be efficiently merged—in a minimalist way—by using very simple guidelines during the design process along with the application of multicomponent reactions as key steps in the synthetic process. Natural phenanthrenoids, a class of plant aromatic metabolites, served as inspiration for the synthesis of a library in which complexity‐enhancing features were introduced in few steps using multicomponent reactions. These resulting chemical entities were not only more complex than the parent natural products, but also interrogated an alternative region of the chemical space, which led to an outstanding hit rate in an antiproliferative assay: four out of twenty‐six compounds showed in vitro activity, one of them being more potent than the clinically useful drug 5‐fluorouracil.     

Design and optimization of electrochemical microreactors for continuous electrosynthesis

The study focuses on the design and construction, as well as the theoretical and experimental optimization of electrochemical filter press microreactors for the electrosynthesis of molecules with a high added value. The main characteristics of these devices are firstly a high-specific electrochemical area to increase conversion and selectivity, and secondly the shape and size of the microchannels designed for a uniform residence time distribution of the fluid. A heat exchanger is integrated into the microstructured electrode to rapidly remove (or supply) the heat required in exo- or endothermic reactions. The microreactors designed are used to perform-specific electrosynthesis reactions such as thermodynamically unfavorable reactions (continuous NADH regeneration), or reactions with high enthalpy changes.     

Gallium(III) triflate: an efficient and a sustainable Lewis acid catalyst for organic synthetic transformations

Green chemical processes play a crucial role in sustainable development, and efficient recyclable catalysts that can be conveniently applied in various chemical reactions are the key elements for the development of sustainable synthetic processes. Many organic transformations rely on Lewis and Br?nsted acid catalysts, and such molecules have been widely studied in organic synthesis. Over the years, researchers have looked for Lewis acid catalysts that provide high selectivity and high turnover frequency but are also stable in aqueous media and recoverable. Since the first preparation of trifluoromethanesulfonic acid by Hazeldine (triflic acid, HOTf), researchers have synthesized and used numerous metal triflates in a variety of organic reactions. Even though the rare earth metal triflates have played a major role in these studies, the majority of rare earth triflates lack one or more of the primary properties of sustainable catalysts: low cost and easy availability of the metals, easy preparation of triflates, aqueous/thermal stability, recyclability, and catalytic efficiency. In this Account, we describe the synthetic applications of Ga(OTf)(3) and its advantages over similar catalysts. Ga(OTf)(3) can be conveniently prepared from gallium metal or gallium chloride in excess of trifluoromethanesulfonic acid (triflic acid) under reflux. Among many Lewis acid catalysts recently studied, Ga(OTf)(3) is water tolerant and soluble and requires very low catalyst loading to drive various acid-catalyzed reactions including Friedel-Crafts alkylation, hydroxyalkylation, and acylation selectively and efficiently. In many reactions Ga(OTf)(3) demonstrated high chemo- and regioselectivity, high yields, excellent stability, and recyclability. We successfully synthesized many biologically active heterocycles and their fluoroanalogs under mild conditions. Many challenging reactions such as the ketonic Strecker reactions proceed efficiently via Ga(OTf)(3) catalysis. Because it is stable in water, this catalyst provides the opportunity to study substrates and develop new synthetic protocols in aqueous media, significantly reducing the production of hazardous waste from organic solvents and toxic catalyst systems.