远端突变可提高游离和固定 NOV1 二氧合酶合成香兰素的效率。

IF 4.1 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Mario De Simone , Lur Alonso-Cotchico , Maria Fátima Lucas , Vânia Brissos , Lígia O. Martins
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引用次数: 0

摘要

蛋白质工程对于提高工业生物催化酶的效率和稳健性至关重要。NOV1 是一种细菌二氧酶,它催化木质素衍生的异丁香酚一步氧化成香兰素,从而具有生物技术潜力,香兰素是一种常用的调味剂,可用于食品、清洁产品、化妆品和药品。Zymspot 是一种预测远端优势突变的工具,可简化蛋白质工程设计。利用定点突变共构建了 41 个变体,然后对其中 6 个活性最高的酶变体进行重组。与野生型相比,两个变体(分别有两个和三个突变)的活性提高了近10倍,运行稳定性也提高了40倍。此外,这些变体在金属亲和树脂中的固定效率为 90% 至 100%,而野生型的固定效率约为 60%。在生物转化过程中,在 24 小时的周期内逐步加入 50 毫摩尔的异丁香酚,1D2 变体在六个反应周期后产生了约 144 毫摩尔的香兰素,相当于约 22 毫克,表明摩尔转化率为 35%。这一产量比野生型高出约 2.5 倍。我们的研究结果凸显了远端蛋白工程在增强酶的活性、稳定性和金属结合选择性等功能方面的功效,从而满足了工业生物催化剂的标准。这项研究为酶的优化提供了一种新方法,可对各种生物技术应用产生重大影响。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Distal mutations enhance efficiency of free and immobilized NOV1 dioxygenase for vanillin synthesis

Distal mutations enhance efficiency of free and immobilized NOV1 dioxygenase for vanillin synthesis

Protein engineering is crucial to improve enzymes’ efficiency and robustness for industrial biocatalysis. NOV1 is a bacterial dioxygenase that holds biotechnological potential by catalyzing the one-step oxidation of the lignin-derived isoeugenol into vanillin, a popular flavoring agent used in food, cleaning products, cosmetics and pharmaceuticals. This study aims to enhance NOV1 activity and operational stability through the identification of distal hotspots. located at more than 9 Å from the active site using Zymspot, a tool that predicts advantageous distant mutations, streamlining protein engineering. A total of 41 variants were constructed using site-directed mutagenesis and the six most active enzyme variants were then recombined. Two variants, with two and three mutations, showed nearly a 10-fold increase in activity and up to 40-fold higher operational stability than the wild-type. Furthermore, these variants show 90–100 % immobilization efficiency in metal affinity resins, compared to approximately 60 % for the wild-type. In bioconversions where 50 mM of isoeugenol was added stepwise over 24-h cycles, the 1D2 variant produced approximately 144 mM of vanillin after six reaction cycles, corresponding to around 22 mg, indicating a 35 % molar conversion yield. This output was around 2.5 times higher than that obtained using the wild-type. Our findings highlight the efficacy of distal protein engineering in enhancing enzyme functions like activity, stability, and metal binding selectivity, thereby fulfilling the criteria for industrial biocatalysts. This study provides a novel approach to enzyme optimization that could have significant implications for various biotechnological applications.

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来源期刊
Journal of biotechnology
Journal of biotechnology 工程技术-生物工程与应用微生物
CiteScore
8.90
自引率
2.40%
发文量
190
审稿时长
45 days
期刊介绍: The Journal of Biotechnology has an open access mirror journal, the Journal of Biotechnology: X, sharing the same aims and scope, editorial team, submission system and rigorous peer review. The Journal provides a medium for the rapid publication of both full-length articles and short communications on novel and innovative aspects of biotechnology. The Journal will accept papers ranging from genetic or molecular biological positions to those covering biochemical, chemical or bioprocess engineering aspects as well as computer application of new software concepts, provided that in each case the material is directly relevant to biotechnological systems. Papers presenting information of a multidisciplinary nature that would not be suitable for publication in a journal devoted to a single discipline, are particularly welcome.
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