Complete Oxidation of Methanol in Biobattery Devices Using a Hydrogel Created from Three Modified Dehydrogenases
Complete Oxidation of Methanol in Biobattery Devices Using a Hydrogel Created from Three Modified Dehydrogenases
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DOI：
10.1002/anie.201207423
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发表时间：
2013-01-01
影响因子：
16.6
通讯作者：
Banta, Scott
中科院分区：
文献类型：
--
作者：
Kim, Yang Hee;Campbell, Elliot;Banta, Scott
蛋白质工程涉及操纵氨基酸以改善蛋白质的性质。在酶的设计和改进以及为生物材料应用改进结构蛋白的努力中，仍有突破性进展。各种蛋白质和肽结构域已被工程化以产生用于各种应用的新功能材料。[1]在这里，我们报告了这种方法的进展，我们创造了一种新的催化生物材料的工程三脱氢酶自组装。当结合时，所产生的新的催化生物材料能够将甲醇完全氧化为二氧化碳，并且我们展示了这种材料作为两种类型的酶生物电池装置中的阳极改性的应用。[2]水凝胶可以由蛋白质和肽通过用交联结构域装备它们而产生。Tirrell及其同事的开创性工作表明，α-螺旋亮氨酸拉链结构域可用于创建通过卷曲螺旋相互作用自组装成水凝胶的肽，[3]并且我们通过证明这些结构域可以附加到球状蛋白质来扩展这一研究路线。[4]这些水凝胶构建体通过由附加的亮氨酸拉链结构域形成的卷曲螺旋基序和由于蛋白质的四级结构而通过另外的蛋白质/蛋白质相互作用交联。到目前为止，我们已经描述了在荧光蛋白中添加螺旋附件，[4a]一种热稳定的醇脱氢酶，[4 b]一种有机磷酸水解酶，[4c]和一种小漆酶。[4d]当后一种酶与锇修饰的肽结合时，形成了生物电催化水凝胶，可以将氧气还原为水，并可以作为生物电池或酶生物燃料电池的阴极修饰。[4d]几乎在每一种情况下，除了螺旋附件的酶的催化活性的影响很小，和强大的水凝胶已被证明。在这里，我们扩展这种方法来创建一个酶的水凝胶，支持功能合成代谢途径。对三种不同来源的NAD（H）依赖性脱氢酶进行了修饰，使其能够自组装。第一种酶是来自嗜热脂肪芽孢杆菌的四聚醇脱氢酶（ADH），其将甲醇氧化成甲醛。[5]第二种酶是四聚体人醛脱氢酶（ALDH 2），其将甲醛氧化成甲酸盐。[6]最后一种酶是来自酿酒酵母的二聚甲酸脱氢酶（FDH 1），将甲酸氧化为CO2。[7]当这些酶结合时，产生能够完全氧化甲醇的合成代谢途径。[8]该反应的示意图如图1a所示。将α-螺旋亮氨酸拉链结构域（H）和随机结构的可溶性肽结构域（S）遗传地附加到三种脱氢酶基因中的每一种的N-末端。三种新的双功能酶构建体（HSADH、HSALDH 2和HSFDH 1）在E.大肠杆菌中，并按照支持性信息中的描述进行纯化。HSADH和HSFDH 1易于表达和纯化，而HSALDH 2酶需要添加麦芽糖结合蛋白（MBP）才能实现功能性表达。在MBP和HSALDH 2之间添加内含肽结构域，使得其在细胞内表达后自发切割，因此HSALDH 2蛋白可以被纯化，就像不包括融合蛋白一样。[9]第一章
Protein engineering involves the manipulation of amino acids to improve the properties of proteins. Breakthroughs are still being reported in the design and improvement of enzymes as well as efforts to improve structural proteins for biomaterials applications. Various protein and peptide domains have been engineered to create new functional materials for a variety of applications.[1] Here we report an advancement of this approach where we create a new catalytic biomaterial by engineering of three dehydrogenase enzymes for self-assembly. When combined, the resulting new catalytic biomaterial is able to fully oxidize methanol to carbon dioxide and we demonstrate the application of this material as an anode modification in two types of enzymatic biobattery devices.[2] Hydrogels can be created from proteins and peptides by outfitting them with cross-linking domains. Pioneering work by Tirrell and co-workers demonstrated that alpha-helical leucine zipper domains could be used to create peptides that self-assemble into hydrogels through coiled-coil interactions,[3] and we have expanded on this line of research by demonstrating that these domains can be appended to globular proteins.[4] These hydrogel constructs are crosslinked through both the coiled-coil motifs formed by the appended leucine zipper domains and through additional protein/protein interactions because of the quaternary structure of the proteins. So far, we have described the addition of helical appendages to fluorescent proteins,[4a] a thermostable alcohol dehydrogenase,[4b] an organophosphate hydrolase enzyme,[4c] and a small laccase enzyme.[4d] When the latter enzyme was combined with osmium-modified peptides, a bioelectrocatalytic hydrogel was formed that could reduce oxygen to water and could function as a cathode modification for a biobattery or enzymatic biofuel cell.[4d] In almost every case, the addition of the helical appendages has had a minimal impact on the catalytic activity of the enzymes, and robust hydrogels have been demonstrated.Here we extend this approach to create an enzymatic hydrogel that supports a functional synthetic metabolic pathway. Three NAD (H)-dependent dehydrogenase enzymes from different sources were modified for self-assembly. The first enzyme was a tetrameric alcohol dehydrogenase (ADH) from Bacillus stearothermophilus which oxidizes methanol to formaldehyde.[5] The second enzyme was a tetrameric human aldehyde dehydrogenase (ALDH2) which oxidizes formaldehyde to formate.[6] The final enzyme, a dimeric formate dehydrogenase (FDH1) from Saccharomyces cerevisiae, oxidizes formate to CO2.[7] When combined these enzymes produce a synthetic metabolic pathway capable of the complete oxidation of methanol.[8] A schematic diagram of this reaction is as shown in Figure 1a. An alpha-helical leucine zipper domain (H) and randomly structured soluble peptide domain (S) were genetically appended to the N-termini of each of the three dehydrogenase genes. The three new bifunctional enzyme constructs (HSADH, HSALDH2, and HSFDH1) were overexpressed in E. coli and purified as described in the Supporting Information. HSADH and HSFDH1 were readily expressed and purified, while the HSALDH2 enzyme required the addition of the maltose binding protein (MBP) to enable functional expression. An intein domain was added between the MBP and HSALDH2 such that it spontaneously cleaved after expression within the cells and thus the HSALDH2 protein could be purified as though no fusion protein had been included.[9]