Enzyme and Microbial Technology最新文献_第2页

Efficient production of active recombinant cholesterol oxidase from Rhodococcus erythropolis in Escherichia coli via his-tag assisted refolding strategy for cholesterol oxidation 利用his标签辅助胆固醇氧化的重折叠策略在大肠杆菌中高效生产重组红红球菌活性胆固醇氧化酶

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-12-24 DOI: 10.1016/j.enzmictec.2025.110805

Meka Saima Perdani , Dwini Normayulisa Putri , Dita Ariyanti , Cintiya Septa Hasannah , Fitri Yuliasari , Ibnu Maulana Hidayatullah , Heri Hermansyah , Yosuke Fukutani , Masafumi Yohda

This study reports the cloning, expression, and characterization of recombinant cholesterol oxidase (ChOx) from Rhodococcus erythropolis in Escherichia coli BL21(DE3). The ChOx gene was inserted into the pET23b(+) vector using primers designed with site-specific modifications that enabled His-tag activation, expression, and purification by nickel-affinity chromatography to yield a protein of ∼55 kDa. A urea-gradient dialysis refolding protocol was employed to recover active enzyme from inclusion bodies. The purified enzyme exhibited a specific activity of 13.68 U/mg, representing a 2.5-fold improvement over previously reported recombinant ChOx yields with a recovery yield of 76.16 %. Cholesterol oxidation assays optimized enzyme concentration, substrate concentration, and temperature. Optimum activity occurred at 0.4 mg/mL enzyme and 20 mM cholesterol, achieving > 80 % substrate conversion, while the enzyme retained stability up to 50 °C. This work demonstrates a His-tag-assisted refolding strategy that efficiently produces active recombinant Rhodococcus erythropolis ChOx (RhoChOx). The optimization of catalytic parameters highlights the enzyme’s robustness under diverse conditions. These findings establish RhoChOx as a promising biocatalyst for industrial cholesterol oxidation and provide a methodological framework applicable to other recombinant oxidases.

本研究报道了红红红球菌重组胆固醇氧化酶（ChOx）在大肠杆菌BL21（DE3）中的克隆、表达和特性分析。将ChOx基因插入pET23b（+）载体，使用经过位点特异性修饰设计的引物，通过镍亲和层析使his标签激活、表达和纯化，产生约55 kDa的蛋白。采用尿素梯度透析复折叠方法从包涵体中回收活性酶。纯化酶的比活性为13.68 U/mg，比先前报道的重组ChOx产量提高2.5倍，回收率为76.16 %。胆固醇氧化试验优化酶浓度、底物浓度和温度。酶的最佳活性为0.4 mg/mL和20 mM胆固醇，达到>； 80 %底物转化率，酶在50°C下保持稳定性。这项工作证明了his标签辅助的重折叠策略，有效地产生活性重组红polis红球菌ChOx （RhoChOx）。催化参数的优化突出了酶在不同条件下的鲁棒性。这些发现确立了RhoChOx作为工业胆固醇氧化生物催化剂的前景，并为其他重组氧化酶提供了适用的方法框架。

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引用次数: 0

Cellulose biosynthesis in nature and In Vitro: mechanisms and challenges 纤维素在自然界和体外的生物合成：机制和挑战

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-12-24 DOI: 10.1016/j.enzmictec.2025.110804

Shijing Sun , Huasha Liang , Zehai Lei , Jialei Xu , Siqi Wang , Zhongyuan Zhao , Tongming Yin

This review systematically compares the biosynthetic mechanisms of cellulose in plants and bacteria, focusing on the structure and function of their distinct enzymatic complexes. In bacteria, cellulose synthesis is driven by the BcsAB transmembrane complex alongside auxiliary proteins like BcsC and BcsD. In plants, rosette-shaped cellulose synthase complexes (CSCs), which are composed of multiple CesA catalytic subunits, synthesize cellulose microfibrils. This process is critically coordinated by auxiliary proteins, such as KOR1 and COBRA-LIKE proteins, which associate with or regulate the CSCs to ensure proper microfibril formation and crystallization. A central challenge in the field is the in vitro production of cellulose I, the native crystalline form with superior mechanical properties. While strategies such as enzyme extraction and heterologous expression in Escherichia coli enable cellulose production, they typically yield the less desirable cellulose II allomorph and face issues of low yield and instability. This review synthesizes current knowledge on key enzymes, in vitro synthesis methods, and resulting cellulose characteristics to identify key obstacles and future pathways toward the efficient bio-manufacturing of cellulose I for sustainable applications.

本文系统地比较了植物和细菌中纤维素的生物合成机制，重点介绍了它们不同的酶复合物的结构和功能。在细菌中，纤维素的合成是由BcsAB跨膜复合体以及BcsC和BcsD等辅助蛋白驱动的。在植物中，由多个纤维素合成酶催化亚基组成的玫瑰状纤维素合成酶复合物（CSCs）可合成纤维素微原纤维。这一过程由辅助蛋白（如KOR1和COBRA-LIKE蛋白）进行关键协调，它们与CSCs相关或调节CSCs以确保适当的微纤维形成和结晶。该领域的一个核心挑战是纤维素I的体外生产，这是一种具有优越机械性能的天然晶体形式。虽然酶提取和在大肠杆菌中异种表达等策略能够生产纤维素，但它们通常会产生不太理想的纤维素II异型，并面临低产量和不稳定的问题。本文综述了目前在关键酶、体外合成方法和纤维素特性方面的知识，以确定纤维素I的高效生物制造和可持续应用的关键障碍和未来途径。

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引用次数: 0

The potential and innovative applications of CRISPR gene editing technology in enzyme gene development CRISPR基因编辑技术在酶基因开发中的潜力和创新应用。

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-12-20 DOI: 10.1016/j.enzmictec.2025.110799

Youmin Zhu

The CRISPR gene editing technology is simple in design and highly efficient, making it the most widely used gene editing tool today. At present, CRISPR gene editing technology has shown a certain application value in enzyme development, but its application potential has not been fully developed. CRISPR gene editing technology can not only be used to knockin enzyme genes and knockout genes that are not conducive to enzyme expression, but can also be applied to single-base editing of enzyme genes, tandem sgRNA for multi-enzyme gene editing, sgRNA library for enzyme screening, endogenous enzyme gene modification, transcriptional activation or inhibition of enzyme gene expression, and fluorescence imaging of enzyme genes. Especially, this review innovatively proposes for the first time that CRISPR gene editing technology can be used for site specific fusion of enzyme genes, cell surface display of endogenous enzymes, and knockin of super long DNA for simultaneous expression of multiple enzymes, providing new ideas for maximizing the value of CRISPR gene editing technology in enzyme development in the future.

CRISPR基因编辑技术设计简单，效率高，是当今应用最广泛的基因编辑工具。目前，CRISPR基因编辑技术在酶的开发中已显示出一定的应用价值，但其应用潜力尚未得到充分开发。CRISPR基因编辑技术不仅可以用于敲入酶基因和敲除不利于酶表达的基因，还可以用于酶基因的单碱基编辑、串联sgRNA用于多酶基因编辑、sgRNA文库用于酶筛选、内源性酶基因修饰、酶基因表达的转录激活或抑制、酶基因的荧光成像等。特别是，本文创新性地首次提出了CRISPR基因编辑技术可用于酶基因的位点特异性融合、内源性酶的细胞表面展示、超长DNA的敲入以同时表达多种酶，为未来最大化CRISPR基因编辑技术在酶开发中的价值提供了新的思路。

引用次数: 0

Levansucrase from Gluconacetobacter diazotrophicus. Key residues involved in levan synthesis 重氮养糖醋杆菌的左旋蔗糖酶。利凡合成中涉及的关键残基

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-12-12 DOI: 10.1016/j.enzmictec.2025.110802

Ana G. Martínez , Yamira Quintero , Duniesky Martínez , Alexis Musacchio , Odet Céspedes , Carmen Menéndez

Levansucrase from Gluconacetobacter diazotrophicus (LsdA, EC 2.4.1.10) produces short-chain fructooligosaccharides, mainly 1-kestotriose, and levan polysaccharide from sucrose. By random mutagenesis, twenty-five LsdA residues were identified as being crucial for levan synthesis, with the substitution of these residues resulting in a decrease or complete elimination of levan synthesis. Fourteen residues (R171, H172, S228, R229, T243, F304, N306, E314, E327, R334, A369, D398, H419, and G432) were identified within the active site cavity, while eleven were dispersed across the protein. Saturation mutagenesis of H172 and R171 (-1 subsite) showed that the H172E/P mutant primarily exhibited sucrose hydrolysis, while the H172S and R171K-H172S variants were less affected in transfructosylation. Furthermore, HPAEC-PAD analysis revealed that the H172S and R171K-H172S variants synthesized 1-kestotriose, 6-kestotriose, 6G-kestotriose, and 1,1-kestotetraose. These variants had specific activity values similar to those of the native LsdA. However, the synthesis of 1,6-kestotetraose was found to be compromised, indicating a loss of the processive mechanism, suggesting that these variants have lost the ability to elongate via β-(2→6) links.

重氮营养葡萄糖醋杆菌（glucconacetobacter diazotrophicus, LsdA, EC 2.4.1.10）的左旋蔗糖酶（Levansucrase）产生短链低聚果糖，主要是1-酮三糖，并从蔗糖中产生左旋多糖。通过随机诱变，鉴定出25个对levan合成至关重要的LsdA残基，这些残基的替代导致levan合成的减少或完全消除。在活性位点空腔内鉴定出14个残基（R171、H172、S228、R229、T243、F304、N306、E314、E327、R334、A369、D398、H419和G432）， 11个残基分散在整个蛋白中。H172和R171（-1亚位）的饱和诱变表明，H172E/P突变体主要表现为蔗糖水解，而H172S和R171K-H172S突变体在转果糖基化方面受影响较小。此外，HPAEC-PAD分析显示，H172S和R171K-H172S变体合成1-酮三糖、6-酮三糖、6g -酮三糖和1,1-酮四糖。这些变体具有与本地LsdA相似的特定活性值。然而，1,6-酮四糖的合成被发现受到损害，表明过程机制的丧失，这表明这些变体已经失去了通过β-（2→6）连接延长的能力。

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引用次数: 0

Microbial-enzyme co-fermentation of low-grade tobacco: Metagenomics and metabolomic insights into flavor formation 低品位烟草的微生物-酶共发酵：元基因组学和代谢组学对风味形成的见解

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-12-11 DOI: 10.1016/j.enzmictec.2025.110803

Ming Shu , Huijie Xue , Yang Yang , Xiao Zhang , Shitou Li , Tengfei Bian , Kailong Yuan , Chunping Xu

Microbial-enzyme co-fermentation effectively enhances the quality of low-grade tobacco leaves quality, but the underlying mechanisms of flavor formation remain unclear. This study investigated the dynamics and relationships of microbial communities and volatile aroma metabolites during low-grade tobacco leaves fermentation through metagenomics and headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME-GC-MS). Results showed that during microbial-enzyme co-fermentation, the tobacco leaves fermented for four days (D4) exhibited the highest levels of total sugars and reducing sugars, the peak total content of aroma metabolites, and the best sensory quality. Pseudomonadota, Bacillota, and Ascomycota were dominant microorganisms during fermentation. During the initial stage (D1–D4), Saccharomyces was the dominant genus, which was subsequently displaced by Pantoea at D5. This microbial succession coincided with a decline in sensory quality, indicating its crucial role in shaping flavor evolution during co-fermentation. During microbial-enzyme co-fermentation process, a total of 46 volatile metabolites were detected in low-grade tobacco leaves. Among them, seven esters with high variable important in projection values and strong microbial correlations were identified as characteristic aroma metabolites, including ethyl phenylacetate, benzyl acetate, phenylethyl acetate, ethyl myristate, ethyl palmitate, ethyl oleate, and methyl linolenate. Gene function annotation revealed carbohydrate metabolism was the most abundant, followed by amino acid metabolism. Spearman correlation analysis elucidated the formation mechanism of characteristic ester metabolites. Specifically, short-chain esters correlated with glycerolipid and amino acid metabolism, while long-chain esters linked to glycolysis and fatty-acid biosynthetic pathways.

微生物-酶共发酵可有效提高低质烟叶品质，但其风味形成机制尚不清楚。本研究采用宏基因组学、顶空固相微萃取-气相色谱-质谱联用技术研究了低度烟叶发酵过程中微生物群落与挥发性香气代谢物的动态变化及其相互关系。结果表明，发酵4 d （D4）烟叶总糖和还原糖含量最高，香气代谢产物总含量最高，感官品质最佳。假单胞菌、杆状杆菌和子囊菌是发酵过程中的优势菌群。在初始阶段（D1-D4）， Saccharomyces是优势属，随后在D5被Pantoea取代。这种微生物演替与感官品质的下降相吻合，表明其在共发酵过程中形成风味演变的关键作用。在微生物-酶共发酵过程中，低品位烟叶共检测到46种挥发性代谢物。其中，7种具有高投影值重要变量和强微生物相关性的酯类被鉴定为特征香气代谢物，包括苯基乙酸乙酯、乙酸苄酯、乙酸苯乙酯、肉豆酸乙酯、棕榈酸乙酯、油酸乙酯和亚麻酸甲酯。基因功能注释显示碳水化合物代谢最丰富，其次是氨基酸代谢。Spearman相关分析阐明了特征性酯代谢产物的形成机制。具体来说，短链酯类与甘油脂和氨基酸代谢相关，而长链酯类与糖酵解和脂肪酸生物合成途径相关。

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引用次数: 0

A unique highly efficient, thermostable and multi-substrate specific galactanase (AtGH53) from Acetivibrio thermocellus cleaving both β (1,4)- and β (1,6)- linked galactans 一种独特的高效、耐热和多底物特异性半乳糖酶（AtGH53），来自热细胞活动弧菌，可切割β(1,4)-和β(1,6)-连接的半乳糖

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-12-08 DOI: 10.1016/j.enzmictec.2025.110801

Shreya Biswas , Ardhendu Mandal , Carlos M.G.A. Fontes , Arun Goyal

The gene encoding endo-β-1,4-galactanase, AtGH53 from Acetivibrio thermocellus was cloned, expressed and the encoded soluble protein was biochemically characterized. Purified AtGH53 showed molecular mass of approximately, 36 kDa, an optimum temperature 70°C and half-life of 15 h at 70°C. AtGH53 displayed stability in acidic and alkaline pH ranges, with an optimum pH 7.5. AtGH53 activity increased over 30 % by Ni or Co²⁺ ions. AtGH53 exhibited broad substrate specificity, displaying the highest activity with potato pectic-galactan with V_max of 1432 U.mg⁻¹ and K_M of 1.2 mg.mL⁻¹ . TLC and HPLC analyses of potato galactan hydrolysis by AtGH53 showed initially the endo-lytic cleaving property and later shifting to exo-lytic mode. This was confirmed by the release of β-1,4-linked galacto-oligosaccharides of higher degrees of polymerization (DP>3) from potato galactan in first 2 h, followed by accumulation of galactobiose and galactose up to 24 h. In contrast, hydrolysis of larch-arabinogalactan by AtGH53 (specific activity, 148.6 U.mg⁻¹) resulted in the release of β-1,6-galactobiose from branches of the polymer. This indicated that AtGH53 also exhibits lower efficiency in hydrolyzing β-1,6-galactan of arabinogalactan via an exo-mode of action. The broad substrate specificity, pH stability and thermostability of AtGH53 make it a versatile enzyme for biotechnological applications.

克隆、表达了热细胞活动弧菌内切-β-1,4-半乳糖酶（endo-β-1,4-半乳糖酶）编码基因AtGH53，并对编码的可溶性蛋白进行了生化表征。纯化后的AtGH53分子量约为36 kDa，最适温度为70℃，70℃时半衰期为15 h。AtGH53在酸性和碱性范围内均表现出稳定性，最适pH值为7.5。Ni 或Co2+离子使AtGH53活性增加30% %以上。AtGH53具有广泛的底物特异性，对马铃薯果胶半乳的活性最高，Vmax为1432 U。mg¹ 和KM 1.2 mg. ml⁻¹ 。AtGH53对马铃薯半乳氨酸水解的TLC和HPLC分析显示，半乳氨酸最初具有内解裂解性质，随后转变为外解模式。马铃薯半乳糖在前2 h内释放出较高聚合度的β-1,4-连接半乳糖低聚糖（DP>3），随后在24 h内积累半乳糖和半乳糖，证实了这一点。相比之下，AtGH53水解落叶松阿拉伯半乳聚糖(比活性，148.6 U。Mg−1)导致β-1,6-半乳糖二糖从聚合物的分支中释放。这表明AtGH53通过外显作用模式水解阿拉伯半乳聚糖中的β-1,6-半乳聚糖的效率也较低。AtGH53广泛的底物特异性、pH稳定性和热稳定性使其成为生物技术应用的多功能酶。

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引用次数: 0

Peach gum-derived immobilization of Klebsiella sp. cells combined with nanoparticle addition for robust enhancement of biohydrogen production 桃胶源克雷伯氏菌细胞的固定化与纳米颗粒的结合增强了生物制氢的能力。

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-12-08 DOI: 10.1016/j.enzmictec.2025.110800

Qin Zhang , Qianzi Cheng , Juanjuan Cao , Cancan Zhang , Bo Wu , Yanbin Li , Sejabodile Precious Moanokeng

This study developed a peach-gum-derived immobilization of Klebsiella sp. cells combined with nanoparticles (NPs) to enhance biohydrogen production. The optimal ratio of peach gum (PG) to sodium alginate was determined to be 1.5:1 for effective immobilization of Klebsiella sp. cells, which was further improved by the addition of magnetite NPs and NiFe₂O₄ NPs (NFNPs). The results showed that the Im + NFNP 20 mg/L treatment was optimal, achieving the highest cumulative hydrogen production of 5996.35 ± 0.29 mL/L at 120 h, which was 1.89 times higher than the control treatment. Notably, this treatment resulted in the maximum Y_H2/S (128.42 ± 2.05 mL/g) and the highest peak number of viable cells ((2.91 ± 0.03) * 10⁹ cfu/mL), as well as enhanced peak activities of hydrogenase and formate-hydrogen lyase. Furthermore, the combination of immobilization and optimum NFNP addition noticeably promoted the expressions of several hydrogenase and formate-hydrogen lyase related genes and altered the distribution of soluble metabolites, which enhanced the hydrogenase- and formate-hydrogen lyase-catalyzed pathways. Thus, we propose a mechanism in which PG-derived immobilization facilitates the entry of NPs into Klebsiella sp. cells, and elucidate the molecular basis for the efficient cellular uptake and distribution of NFNPs in Klebsiella sp. cells.

本研究开发了一种由桃胶衍生的克雷伯氏菌细胞与纳米颗粒（NPs）结合的固定化方法，以提高生物氢的产生。确定桃胶（PG）与海藻酸钠的最佳固定比为1.5:1，通过添加磁铁矿NPs和NiFe2O4 NPs （NFNPs）进一步提高固定化克雷伯氏菌细胞的效果。结果表明，Im + NFNP 20 mg/L处理效果最佳，在120 h时累计产氢量最高，为5996.35 ± 0.29 mL/L，是对照处理的1.89倍。其中，YH2/S最高（128.42 ± 2.05 mL/g），活细胞峰值数最高（2.91 ± 0.03）* 109 cfu/mL)，氢化酶和甲酸解氢酶活性峰值增强。此外，固定化和最佳NFNP添加的结合显著促进了几个氢化酶和甲酸氢裂解酶相关基因的表达，改变了可溶性代谢产物的分布，从而增强了氢化酶和甲酸氢裂解酶催化的途径。因此，我们提出了一种pg衍生的固定化促进NPs进入克雷伯氏菌细胞的机制，并阐明了NFNPs在克雷伯氏菌细胞中有效摄取和分布的分子基础。

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引用次数: 0

Mechanistic investigation of the enhanced catalytic activity of B. sub lipase A mutant I157V and its application in biodiesel production b亚脂肪酶A突变体I157V催化活性增强机理研究及其在生物柴油生产中的应用

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-12-03 DOI: 10.1016/j.enzmictec.2025.110798

Zhong Ni, Jia Zhao, Huimin Zhou, Huayun Jia, E-Bin Gao, ZhongJian Guo, Zhaoyang Hu, Shangshang Ma, Yong Chen

Optimizing lipases for the transesterification of long-chain fatty acid esters plays a vital role in enhancing biodiesel yields, especially from recycled cooking oils. In this study, we applied error-prone PCR to create a diverse mutagenesis library based on Bacillus subtilis lipase A(BSLA). The I157V mutant, selected through 96-well high-throughput screening, exhibited over sixfold greater catalytic efficiency (k_cat/K_m) than the wild-type enzyme. Insights from molecular docking and dynamic simulations pointed to the mutation’s effect on aligning substrates more effectively within the enzyme’s catalytic center. This alignment minimized non-productive binding modes and reinforced the enzyme-substrate interactions, which in turn boosted catalytic output. In biodiesel production using waste cooking oil as feedstock, the I157V mutant achieved a methyl ester yield of 87 % (w/w) after 10 h, representing an ∼45 % increase compared to 60 % (w/w) for the wild type. These findings not only advance enzyme engineering for industrial biocatalysis but also underline the potential of targeted mutations in supporting sustainable energy solutions.

优化脂肪酶对长链脂肪酸酯酯的酯交换反应对提高生物柴油的产量，特别是从再生食用油中提取生物柴油具有重要作用。本研究以枯草芽孢杆菌脂肪酶a （Bacillus subtilis lipase a， BSLA）为基础，应用易出错PCR技术建立了一个多样化的诱变文库。通过96孔高通量筛选筛选出的I157V突变体的催化效率（kcat/Km）是野生型酶的6倍以上。来自分子对接和动态模拟的见解指出，突变对酶的催化中心内的底物更有效地排列产生了影响。这种排列最小化了非生产性结合模式，加强了酶与底物的相互作用，从而提高了催化产量。在以废食用油为原料的生物柴油生产中，I157V突变体在10 h后的甲酯产率为87 % (w/w)，比野生型的60 % （w/w）提高了~ 45 %。这些发现不仅推动了工业生物催化的酶工程，而且强调了靶向突变在支持可持续能源解决方案方面的潜力。

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引用次数: 0

Dithionite-supported biohydrogenation of muconic acid to adipic acid by lyophilized Escherichia coli cells expressing recombinant enoate reductase 表达重组乙酸还原酶的冻干大肠杆菌细胞在二硫代酸支持下将粘液酸生物加氢为己二酸。

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-12-02 DOI: 10.1016/j.enzmictec.2025.110797

Khorcheska A. Batyrova , Anna N. Khusnutdinova , Alexander F. Yakunin

Adipic acid is an important six-carbon dicarboxylic acid with numerous industrial applications in polymers (nylon) and the food industry. Traditional manufacturing of adipic acid relies on petroleum feedstocks and involves energy-intensive chemical processes with negative environmental impacts. Consequently, alternative synthesis methods are being developed, including the hydrogenation of biobased muconic acid to adipic acid via chemical catalysis or enzymatic reduction with 2-enoate reductases. This study revealed that purified full-length 2-enoate reductase ERBC from Heyndrickxia (Bacillus) coagulans can reduce the three muconic acid isomers (cis,cis, cis,trans, trans,trans) using NADH as a reductant. Titration of the purified ERBC with different chemical reductants showed that its redox cofactors (FMN, FAD, and [4Fe-4S]) can also be reduced by dithionite and Ti(III)-citrate. However, only dithionite and NADH supported the biohydrogenation of trans-cinnamic acid and cis,cis-muconic acid. The individually expressed and purified large domain of ERBC also catalyzed muconic acid reduction with these reductants, but exhibited lower activity and produced only 2-hexenedioic acid as the product. Efficient conversion of muconic acid to adipic acid was demonstrated using lyophilized E. coli cells expressing full-length ERBC as the catalyst, with dithionite acting as both a reductant and an oxygen scavenger. The use of lyophilized recombinant Escherichia coli cells with dithionite for ERBC-mediated biohydrogenation of muconic acid eliminates the need for protein purification and costly natural cofactors (NAD(P)H), as well as enhances ERBC tolerance to high substrate concentrations and creates anaerobic conditions for ERBC activity. This approach shows promise for biobased adipic acid production and other applications of 2-enoate reductases.

己二酸是一种重要的六碳二羧酸，在聚合物（尼龙）和食品工业中有着广泛的工业应用。传统的己二酸制造依赖于石油原料，涉及能源密集型的化学过程，对环境有负面影响。因此，人们正在开发其他合成方法，包括通过化学催化或用2-烯酸还原酶将生物基粘膜酸加氢成己二酸。本研究发现，从海因德里克氏杆菌（芽孢杆菌）凝固物中纯化的全长2-烯酸还原酶ERBC可以用NADH作为还原剂还原三种粘膜酸异构体（顺式、顺式、顺式、反式、反式、反式）。用不同的化学还原剂对纯化的ERBC进行滴定，结果表明其氧化还原辅助因子（FMN、FAD和[4Fe-4S]）也可以被二亚硝酸盐和柠檬酸钛还原。然而，只有二亚硝酸盐和NADH支持反式肉桂酸和顺式、顺式粘膜酸的生物加氢。单独表达纯化的ERBC大结构域也能催化这些还原剂还原黏液酸，但活性较低，产物仅为2-己烯二酸。用表达全长ERBC的冻干大肠杆菌细胞作为催化剂，二亚硝酸盐同时作为还原剂和氧气清除剂，证明了粘液酸向己二酸的有效转化。利用冻干重组大肠杆菌细胞和二亚硝酸盐进行ERBC介导的粘膜酸生物加氢，消除了蛋白质纯化和昂贵的天然辅助因子（NAD(P)H）的需要，同时增强了ERBC对高底物浓度的耐受性，并为ERBC活性创造了厌氧条件。这种方法显示了生物基己二酸生产和2-烯酸还原酶的其他应用前景。

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引用次数: 0

Effect of lipopolysaccharide on ganglioside expression in human induced pluripotent stem cell-derived kidney organoids. 脂多糖对人诱导多能干细胞衍生肾类器官中神经节苷脂表达的影响。

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-12-01 Epub Date: 2025-08-06 DOI: 10.1016/j.enzmictec.2025.110730

Jae-Sung Ryu, Jin Ok Yu, Ki Kwan Kim, Eun-Jeong Jeong, Min Young Kim, Hyo Gyeong Yun, Eun Bin Song, Ji-Su Kim, Young-Woock Noh, Young-Kug Choo

An organoid is a self-organizing, three-dimensional (3D), stem cell-derived structure that closely mimics the structural, cellular, and functional properties of specific organs or tissues. Organoids are widely utilized for assessing drug efficacy, safety, and industrial chemical toxicity. The purpose of this study was to generate a kidney organoid from human induced pluripotent stem cells (iPSCs) and establish a sepsis-associated acute kidney injury (SA-AKI) model by treatment with lipopolysaccharide (LPS). We further analyzed changes in ganglioside expression following LPS treatment in kidney organoids. As a result, we observed that the expression of kidney-specific markers was significantly increased during differentiation. Next, we confirmed that the levels of inflammation-related markers and reactive oxygen species (ROS) were significantly increased, whereas mitochondrial membrane potential (MMPΨ) was significantly reduced in LPS-treated kidney organoids. Interestingly, ganglioside GM3, GM2, GD3, and GD1a expression, as well as their biosynthesis, was notably decreased in LPS-treated kidney organoids. These findings suggest that gangliosides play critical roles in inflammation and may contribute to the pathophysiology of SA-AKI, highlighting the potential of kidney organoids as a valuable model system for studying kidney injury and associated inflammatory responses.

类器官是一种自组织的三维（3D）干细胞衍生的结构，它密切模仿特定器官或组织的结构、细胞和功能特性。类器官被广泛用于评估药物的有效性、安全性和工业化学毒性。本研究的目的是利用人诱导多能干细胞（iPSCs）生成肾类器官，并通过脂多糖（LPS）处理建立脓毒症相关急性肾损伤（SA-AKI）模型。我们进一步分析了脂多糖处理后肾类器官中神经节苷脂表达的变化。结果，我们观察到在分化过程中肾脏特异性标志物的表达显著增加。接下来，我们证实了炎症相关标志物和活性氧（ROS）水平显著增加，而在lps处理的肾类器官中，线粒体膜电位（MMPΨ）显著降低。有趣的是，神经节苷脂GM3、GM2、GD3和GD1a的表达及其生物合成在lps处理的肾类器官中显著降低。这些发现表明神经节苷在炎症中起关键作用，并可能参与SA-AKI的病理生理，突出了肾类器官作为研究肾损伤和相关炎症反应的有价值的模型系统的潜力。

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