Heterologous expression and characterization of a fumarate reductase from Lactobacillus panisapium with activity toward trans-10-hydroxy-2-decenoic acid.

IF 3.3 2区农林科学 Q1 AGRICULTURE, MULTIDISCIPLINARY

Journal of the Science of Food and Agriculture Pub Date : 2025-06-04 DOI:10.1002/jsfa.14416

Hayate Itatani, Nao Takeda, Yukihiro Kimura, Ayanori Yamaki, Shinji Takenaka

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引用次数: 0

Abstract

Background: Lactobacillus panisapium, isolated from the gastrointestinal tract of a queen honeybee, converts trans-10-hydroxy-2-decenoic acid (10H2DA) into 10-hydroxydecanoic acid (10HDAA) in royal jelly. 10HDAA has shown significant anti-inflammatory potential. This study aimed to identify the reductase responsible for this bioconversion and characterize its properties.

Results: An enzyme was partially purified from L. panisapium cell extracts based on its 10H2DA-reducing activity. Liquid chromatography-mass spectrometry and phylogenetic analyses identified the enzyme as a water-soluble fumarate reductase (~40% identity to reductases from Shewanella frigidimarina and Klebsiella pneumoniae). The reductase was heterologously expressed in Escherichia coli. The recombinant enzyme displayed activity toward fumarate and 10H2DA in the presence of flavin adenine dinucleotide and nicotinamide adenine dinucleotide hydride. The enzyme exhibited optimal stability at 50 °C and pH 6.0. An active-site glycine residue in the L. panisapium reductase likely facilitates the binding of large substrates such as 10H2DA. Substituting this residue with threonine decreased activity toward 10H2DA while maintaining activity toward fumarate. Structural modeling revealed that, compared with the homologous reductase from S. frigidimarina, the enzyme from L. panisapium lacks a heme-binding domain, again consistent with enhanced access for larger substrates. Whole cells of E. coli expressing the recombinant reductase effectively converted 10H2DA to 10HDAA, offering a scalable alternative to using purified recombinant enzyme.

Conclusion: This study provides insights into the structure-function relationship of a novel fumarate reductase-family enzyme from L. panisapium and demonstrates its potential for biocatalytic applications in producing 10HDAA-enriched royal jelly, contributing to the development of functional foods. © 2025 Society of Chemical Industry.

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具有反式-10-羟基-2-十烯酸活性的富马酸还原酶的异源表达和鉴定。

背景：从蜂王胃肠道中分离出的panisapium乳杆菌可将蜂王浆中的反式10-羟基-2-癸酸（10H2DA）转化为10-羟基癸酸（10HDAA）。hdaa具有显著的抗炎潜力。本研究旨在鉴定负责这种生物转化的还原酶并表征其性质。结果：根据其还原10h2da的活性，从全草细胞提取物中部分纯化出一种酶。液相色谱-质谱分析和系统发育分析鉴定该酶为水溶性富马酸还原酶（与冷希瓦氏菌和肺炎克雷伯菌还原酶的同源性约为40%）。还原酶在大肠杆菌中异种表达。重组酶在黄素腺嘌呤二核苷酸和烟酰胺腺嘌呤二核苷酸存在下对富马酸盐和10H2DA具有活性。酶在50℃、pH 6.0条件下稳定性最佳。L. panisapium还原酶中的活性位点甘氨酸残基可能促进大型底物（如10H2DA）的结合。用苏氨酸取代该残基降低了对10H2DA的活性，但保持了对富马酸的活性。结构模型显示，与S. frigidimarina的同源还原酶相比，L. panisapium的酶缺乏血红素结合结构域，再次与更大底物的可及性相一致。表达重组还原酶的大肠杆菌全细胞有效地将10H2DA转化为10HDAA，为使用纯化的重组酶提供了一种可扩展的替代方法。结论：本研究揭示了L. panisapium中新型富马酸还原酶家族酶的结构-功能关系，并证明了其在生产富含10hdaa的蜂王浆中的生物催化应用潜力，为功能性食品的开发提供了依据。©2025化学工业协会。

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来源期刊

Journal of the Science of Food and Agriculture 工程技术-农业综合

CiteScore

8.10

自引率

4.90%

发文量

634

审稿时长

3.1 months

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