Novel Synthesis Strategy for Biocatalyst: Fast Purification and Immobilization of His- and ELP-Tagged Enzyme from Fermentation Broth
Novel Synthesis Strategy for Biocatalyst: Fast Purification and Immobilization of His- and ELP-Tagged Enzyme from Fermentation Broth
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生物催化剂的新型合成策略：从发酵液中快速纯化和固定 His 和 ELP 标记的酶
DOI：
10.1021/acsami.9b09071
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发表时间：
2019
影响因子：
9.5
通讯作者：
Wang Yun
中科院分区：
文献类型：
--
作者：
Zhao Man;Rong Junhui;Han Juan;Zhou Yang;Li Chunmei;Wang Lei;Mao Yanli;Wang Yun
受天然生物矿化过程的启发，无机磷酸盐体系被选为酶包封的候选体系；然而，在长期的制造过程中，酶活性的丧失是不可避免的，仿生矿化机制尚不清楚。同时，纯化工艺在制备高酶载量和高活性的固定化酶中起着关键作用，而从粗发酵液中快速、低成本、环保地提纯生物催化剂仍然是生物化学工程中的一个关键挑战。本文首次提出了由弹性蛋白样多肽（ELP）和His-tag组成的二元标签，将其与β-葡萄糖苷酶（Glu）融合构建重组的Glu-linker-ELP- his (GLEH)，旨在开发一种结合纯化和固定化工艺的快速合成策略，以获得稳定性和可回收性更好的生物催化剂。在25℃条件下，单次反过渡循环10 min， GLEH的纯化率和活性回收率分别达到18.1%和95.2%。然后，利用his标签和超声辅助反应法，在15 min内实现了cu3 (PO4)2纳米花的高效生物矿化。活性恢复和相对活性分别在90.3和111.0%达到最大值。研究表明，杂化纳米花的晶体生长过程包括明显的成核、自组装和奥斯特瓦尔德成熟过程，GLEH酶作为“粘合剂”组装Cu3(PO4)2纳米片。固定化的glh纳米花具有良好的操作稳定性和可回收性，其催化效率接近游离Glu。
Inspired by natural biomineralization process, inorganic phosphates system has been selected as a candidate for the encapsulation of enzyme; however, during the long-term fabrication process, the loss of enzyme activity is unavoidable, and the biomimetic mineralization mechanism is still poorly understood. Meanwhile, the purification process plays a key role in the preparation of immobilized enzyme with high enzyme loading and activity, while the rapid, low-cost, and eco-friendly purification of biocatalyst from crude fermentation broth remains a critical challenge in biochemical engineering. Here, a binary tag composed of elastin-like polypeptide (ELP) and His-tag was presented for the first time to be fused with β-glucosidase (Glu) to construct a recombinant Glu-linker-ELP-His (GLEH) with the aim of developing a fast synthesis strategy combining purification and immobilization processes for a biocatalyst with better stability and recyclability. The purification fold and activity recovery of GLEH reached 18.1 and 95.2%, respectively, once a single inverse transition cycling was conducted at 25 °C for 10 min. Then, efficient biomineralization of hybrid enzyme-Cu3(PO4)2nanoflowers was realized in 15 min by the action of His-tag and ultrasonic-assisted reaction method. The activity recovery and relative activity reached the maximum at 90.3 and 111.0%, respectively. We demonstrate that the crystal growth process of a hybrid nanoflower involves obvious nucleation, self-assembly, and the Ostwald ripening process, and the enzyme GLEH acts as a “binder” to assemble Cu3(PO4)2nanoflakes. The immobilized GLEH nanoflowers show outstanding operation stability and recyclability, and their catalytic efficiency is close to that of free Glu.