Coupling a recombinant oxidase to catalase through specific noncovalent interaction to improve the oxidation of 5-hydroxymethylfurfural to 2,5-furandicarboxylic acid
Coupling a recombinant oxidase to catalase through specific noncovalent interaction to improve the oxidation of 5-hydroxymethylfurfural to 2,5-furandicarboxylic acid
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通过特异性非共价相互作用将重组氧化酶与过氧化氢酶偶联，以改善 5-羟甲基糠醛氧化为 2,5-呋喃二甲酸
DOI：
10.1016/j.enzmictec.2021.109895
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发表时间：
2021-08-20
影响因子：
3.4
通讯作者：
Feng, Wei
中科院分区：
文献类型：
--
作者：
Han, Yining;Qu, Wenxin;Feng, Wei
5-羟甲基糠醛氧化酶（HMFO）可以催化羟基和醛的氧化。它催化5羟甲基糠醛生成2，5-呋喃二甲酸。然而，HMFO的应用遇到了两个问题：HMFO在大肠杆菌中的表达。主要是以包涵体的形式，并且H2 O2的副产物对HMFO稳定性具有负面影响。为了解决这些问题，通过将C-末端融合到弹性蛋白样多肽（ELP）来产生重组HMFO。ELP-HMFO可以表达显著减少的包涵体。ELP-HMFO表现出改善的稳定性和对H2 O2的耐受性。通过将HMFO的N-末端融合到富含谷氨酸的亮氨酸拉链基序（ZE）来进行进一步的重组。类似地，重组过氧化氢酶（CAT）通过将N-末端融合至ELP并将C-末端融合至富含亮氨酸的亮氨酸拉链基序（ZR）来产生。ELP-HMFO-ZE能与ZR-CAT-ELP特异性相互作用，这归因于ZE和ZR的卷曲螺旋缔合。ELP-HMFO-ZE#ZR-CAT-ELP协调两种酶各自的催化活性。ELP-HMFO-ZE催化HMF的氧化，生成的过氧化氢被Zr-CAT-ELP分解为H2O和氧气。在HMF的氧化过程中，HMFO的辅因子FAD被还原，并且需要分子氧来再氧化还原的FAD。H_2O_2分解放出的氧气正好满足需要，由于两种酶之间距离较短，可以有效地从Zr-CAT-ELP扩散到ELP-HMFOZE。
5-Hydroxymethylfurfural oxidase (HMFO) can catalyze both hydroxyl and aldehyde oxidations. It catalyzes 5hydroxymethylfurfural into 2,5-furandicarboxylic acid. However, the application of HMFO encountered two problems: the expressed HMFO in Escherichia coli. is largely in the form of inclusion bodies, and the by-product of H2O2 has a negative effect on HMFO stability. To solve these problems, recombinant HMFO was generated by fusing the C-terminus to an elastin-like polypeptide (ELP). ELP-HMFO can be expressed with significantly reduced inclusion bodies. ELP-HMFO exhibited improved stability and tolerance toward H2O2. Further recombination is carried out by fusing the N-terminus of HMFO to a glutamic acid-rich leucine zipper motif (ZE). Similarly, recombinant catalase (CAT) is generated by fusing the N-terminus to ELP and fusing the C-terminus to an arginine-rich leucine zipper motif (ZR). ELP-HMFO-ZE can interact specifically with ZR-CAT-ELP, ascribing to the coiled-coil association of ZE and ZR. ELP-HMFO-ZE#ZR-CAT-ELP coordinates the respective catalytic activities of the two enzymes. ELP-HMFO-ZE catalyzes the oxidation of HMF, and the generated hydrogen peroxide is decomposed by ZR-CAT-ELP into H2O and oxygen. During the oxidation of HMF, the cofactor FAD of HMFO is reduced, and molecular oxygen is needed to reoxidize the reduced FAD. The evolved oxygen from the decomposing of H2O2 can just meet the requirement, which can be diffused efficiently from ZR-CAT-ELP to ELP-HMFOZE due to the short distance between the two enzymes.