Enzyme and Microbial Technology最新文献_第4页

Enhanced degradation of thermoplastic polyurethane plastics based on engineering lignin peroxidase 基于工程木质素过氧化物酶的热塑性聚氨酯塑料的强化降解

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2026-03-01 Epub Date: 2025-11-20 DOI: 10.1016/j.enzmictec.2025.110788

Huan Li, Shengjie Yuan, Ling Chen, Bing Wu

Thermoplastic polyurethane (TPU) with complex physical crosslinking is difficult to degrade under natural environmental conditions. Current degradation methods, particularly for aromatic TPU, suffer from poor degradation efficiency. This study investigated the degradation performance of lignin peroxidase (LiP) on aromatic TPU through protein engineering and multi-enzyme system construction. Results indicated that wild-type LiP (WT-LiP), expressed from the recombinant Pichia pastoris GS115, exhibited a certain degradation effect on aromatic TPU. By using molecular docking techniques to identify key mutation sites, three LiP mutants (F46W, H47W, and H175R) were successfully constructed. Under optimal conditions (30°C, pH 2.5, 1 mM H₂O₂, and 5 U/mg enzyme), the F46W mutant achieved a molecular weight degradation rate of 11.97 % after 3 days of degradation, which is 2.2 times higher than that of the WT-LiP with a weight loss of 2.22 %, and the degradation efficiency in 28 days was 26.59 %. Furthermore, the constructed multi-enzyme systems (LiP-manganese peroxidase-laccase and LiP-carboxylesterase) substantially improved the degradation efficiency of TPU. Specifically, the LiP-carboxylesterase system demonstrated superior performance, achieving molecular weight degradation rates of 29.20 % and weight loss of 5.07 % after 3 days of treatment. This study provides a green enzymatic approach for efficient aromatic TPU plastics degradation and offers more sustainable solutions for plastic waste management.

热塑性聚氨酯（TPU）具有复杂的物理交联，在自然环境条件下难以降解。现有的降解方法，尤其是芳香族TPU的降解效率较差。通过蛋白质工程和多酶体系构建，研究了木质素过氧化物酶（LiP）对芳香TPU的降解性能。结果表明，重组毕赤酵母GS115表达的野生型LiP （WT-LiP）对芳香型TPU具有一定的降解作用。通过分子对接技术确定关键突变位点，成功构建了3个LiP突变体F46W、H47W和H175R。在最佳条件（30°C、pH 2.5、1 mM H₂O₂、5 U/mg酶）下，F46W突变体降解3 d后的分子量降解率为11.97 %，是WT-LiP的2.2倍，失重率为2.22 %，28 d降解效率为26.59 %。此外，构建的多酶体系（lip -锰过氧化物酶-漆酶和lip -羧酸酯酶）大大提高了TPU的降解效率。具体来说，lip -羧酸酯酶系统表现出优异的性能，经过3天的处理，其分子量降解率为29.20 %，失重率为5.07 %。本研究为芳香族TPU塑料的高效降解提供了一种绿色酶法，并为塑料废物管理提供了更可持续的解决方案。

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引用次数: 0

Overexpression of Keratinase Candidate from Bacillus velezensis LPL061 in Escherichia coli BL21(DE3) 韦氏芽孢杆菌候选角化酶LPL061在大肠杆菌BL21（DE3）中的过表达

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2026-03-01 Epub Date: 2025-11-22 DOI: 10.1016/j.enzmictec.2025.110789

Desi Sagita , Tirza Jeli Ping , Aluicia Anita Artarini , Rachmat Mauluddin , Edwin Setiawan , Anto Budiharjo , Catur Riani

Proteases have diverse industrial applications including keratinase, which degrade keratin-rich substrates, such as hair, nails, feathers, and skin. This study aimed to express a protease of Bacillus velezensis LPL061 (WP_003155195.1) in Escherichia coli BL21(DE3) and to evaluate its keratinolytic as a new source of keratinase. Two gene constructs were designed, one containing the propeptide domain (kerfull) and one without it (kerhalf). Expression was induced with 0.05 mM IPTG at 20°C for 20 h. KerFull was partially purified using sequential ultrafiltration. SDS-PAGE analysis showed that KerHalf as a 32.2 kDa protein, while KerFull appeared as a 38 kDa fusion and a 28.5 kDa mature form. QTOF-MS confirmed the amino acid sequence. Only KerFull exhibited proteolytic and keratinolytic, highlighting the importance of the I9 domain for proper folding and enzyme activation. The partially purified mature KerFull protein (P50 fraction) retained activity despite low yield. KerFull showed a broad pH stability (6−11) with optimum at pH 9 and active over a wide temperature range (37–70℃), with an optimum at 60℃. Protein remained stable at 20–40℃ and pH 8. Specific activity reached 16.67 U/mg in the crude and 34.14 U/mg of P50 fraction. When combined with DTT, KerFull effectively degraded chicken feather barbules within 4 h at 37 °C. These findings suggest that one of the proteases from B. velezensis LPL061 (WP_003155195.1) is a robust keratinase candidate, offering broad operational pH and temperature, and efficient keratin degradation even at low concentrations, making it a promising candidate for industrial keratinase applications.

蛋白酶有多种工业应用，包括角化酶，它能降解富含角蛋白的底物，如头发、指甲、羽毛和皮肤。本研究目的是在大肠杆菌BL21（DE3）中表达一种名为velezensis Bacillus LPL061 （WP_003155195.1）的蛋白酶，并评价其角化酶作为角化酶的新来源。设计了两个基因结构，一个包含前肽结构域（kerfull），一个不包含前肽结构域（kerhalf）。0.05 mM IPTG在20℃下诱导表达20 h。采用序贯超滤法对KerFull进行部分纯化。SDS-PAGE分析显示KerHalf为32.2 kDa蛋白，而KerFull为38 kDa融合蛋白和28.5 kDa成熟蛋白。QTOF-MS证实了氨基酸序列。只有KerFull表现出蛋白水解和角蛋白水解，这突出了I9结构域对正确折叠和酶激活的重要性。部分纯化的成熟KerFull蛋白（P50部分）尽管产量低，但仍保持活性。KerFull具有较宽的pH稳定性（6−11），其中pH值为9时的稳定性最佳；在较宽的温度范围（37 ~ 70℃）内具有活性，60℃时的稳定性最佳。蛋白在20 ~ 40℃、pH值8条件下保持稳定。粗馏分比活性为16.67 U/mg， P50馏分比活性为34.14 U/mg。当KerFull与DTT联合使用时，在37℃下，在4 h内可有效降解鸡毛小管。这些发现表明，velezensis中的一种蛋白酶LPL061 （WP_003155195.1）是一种强大的角化酶候选物，具有广泛的工作pH和温度，即使在低浓度下也能有效降解角化酶，使其成为工业角化酶应用的有希望的候选物。

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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 : 2026-03-01 Epub 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

PhcK mediates transport of a β-5-type lignin-derived dimer in Sphingobium lignivorans SYK-6 PhcK介导木质素鞘SYK-6中β-5型木质素衍生二聚体的转运

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2026-03-01 Epub Date: 2025-11-22 DOI: 10.1016/j.enzmictec.2025.110784

Mitsuru Kawazoe , Masaya Fujita , Shojiro Hishiyama , Naofumi Kamimura , Eiji Masai

Lignin is a major component of plant cell walls; however, its heterogeneous and complex structure has hindered its efficient utilization. Recently, strategies that combine chemical pretreatment with microbial conversion to produce valuable chemicals have attracted attention. To develop an ideal microbial platform for this purpose, it is essential to elucidate the complete bacterial catabolic system for lignin-derived aromatic compounds. Here, we identified an inner membrane transporter gene involved in the uptake of a metabolite of dehydrodiconiferyl alcohol (DCA), a lignin-derived β-5 dimer, in Sphingobium lignivorans SYK-6. SLG_12820 (phcK) was found to encode a major facilitator superfamily transporter belonging to the endosomal spinster family and is regulated by PhcR, a transcriptional regulator of DCA catabolism genes. Through mutant analysis and a specific uptake assay based on PhcR effector recognition, we demonstrated that PhcK functions as an inner membrane transporter that specifically imports the DCA metabolite, 3-(2-(4-hydroxy-3-methoxyphenyl)-3-(hydroxymethyl)-7-methoxy-2,3-dihydrobenzofuran-5-yl)acrylic acid.

木质素是植物细胞壁的主要成分；然而，其异质复杂的结构阻碍了其高效利用。近年来，将化学预处理与微生物转化相结合以生产有价值化学品的策略引起了人们的关注。为了开发一个理想的微生物平台，有必要阐明木质素衍生芳香族化合物的完整细菌分解代谢系统。在这里，我们发现了一个参与木质素衍生的β-5二聚体脱氢二硝基醇（DCA）代谢物摄取的内膜转运基因。SLG_12820 （phcK）编码一个属于内体spinster家族的主要促进体超家族转运蛋白，并受DCA分解代谢基因的转录调节因子PhcR调控。通过突变体分析和基于PhcR效应识别的特异性摄取实验，我们证明PhcK作为一种内膜转运体，特异性地进口DCA代谢物3-（2-（4-羟基-3-甲氧基苯基）-3-（羟甲基）-7-甲氧基-2,3-二氢苯并呋喃-5-基）丙烯酸。

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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 : 2026-03-01 Epub 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

Structure and functional role of the SLHs-CBM54 tandem in bacterial multimodular glycoside hydrolases SLHs-CBM54串联在细菌多模糖苷水解酶中的结构和功能作用

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2026-03-01 Epub Date: 2025-11-21 DOI: 10.1016/j.enzmictec.2025.110787

Oksana V. Berezina , Alina I. Selimzyanova , Kirill V. Gordeev , Wolfgang H. Schwarz , Natalia A. Lunina

Carbohydrate-binding modules (CBMs) enhance enzymatic degradation of polysaccharides by improving substrate binding. CBM54, a family of carbohydrate-binding modules, is found in surface-displayed multimodular glycoside hydrolases (MGHs) of gram-positive bacteria. These modules bind insoluble polysaccharides, including chitosan, chitin, xylan, cellulose, and fungal cell wall β-glucans. In MGHs, the CBM54 module is invariably positioned downstream of three S-layer homology modules (SLHs), which anchor the enzyme to the bacterial cell surface. The SLHs-CBM54 tandem promotes bacterial adherence to substrates, concentrating hydrolytic enzymes at the interface and facilitating efficient uptake of soluble degradation products. CBM54 contains a cleavage site that divides it into two structurally distinct parts, which remain associated via hydrogen bonding. The processing of CBM54 promotes bacterial detachment from the substrate and their migration toward new nutrient sources. Beyond MGHs, the SLHs-CBM54 tandem occurs in many other bacterial proteins with uncharacterized functions. SLH modules may self-assemble into 2D arrays on synthetic supports, enabling their use as matrices for protein nucleation and crystal growth. The SLHs-CBM54 tandem fusion with functional modules offers an innovative approach to surface modification, with broad biomedical and biotechnological applications. Due to its versatile architecture, this system holds promise for antigen display, vaccine development, and enhanced polysaccharide degradation processes.

碳水化合物结合模块（CBMs）通过改善底物结合来促进多糖的酶降解。CBM54是一个碳水化合物结合模块家族，存在于革兰氏阳性细菌的表面显示的多模块糖苷水解酶（MGHs）中。这些模块结合不溶性多糖，包括壳聚糖、几丁质、木聚糖、纤维素和真菌细胞壁β-葡聚糖。在MGHs中，CBM54模块总是位于三个s层同源模块（SLHs）的下游，SLHs将酶固定在细菌细胞表面。SLHs-CBM54串联促进细菌粘附在底物上，在界面上集中水解酶，促进可溶性降解产物的有效吸收。CBM54含有一个裂解位点，将其分成两个结构不同的部分，它们通过氢键保持联系。CBM54的处理促进了细菌脱离基质并向新的营养源迁移。除了MGHs外，SLHs-CBM54串联还存在于许多其他功能未知的细菌蛋白中。SLH模块可以在合成支架上自组装成二维阵列，使其能够用作蛋白质成核和晶体生长的基质。SLHs-CBM54串联融合功能模块提供了一种创新的表面改性方法，具有广泛的生物医学和生物技术应用。由于其多功能结构，该系统有望用于抗原展示，疫苗开发和增强多糖降解过程。

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引用次数: 0

Sensitive and specific fluorometric assay for assessment of glyoxalase II enzymatic activity in microbial samples and biological tissue 用于评估微生物样品和生物组织中乙二醛酶活性的灵敏和特异性荧光测定法

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2026-03-01 Epub Date: 2025-11-20 DOI: 10.1016/j.enzmictec.2025.110783

Mohammed Alaa Kadhum, Mahmoud Hussein Hadwan

This study presents a novel fluorescence-based assay for quantifying Glyoxalase II (Glo II) enzymatic activity, using N-(9-Acridinyl)maleimide (NAM) as a fluorescent probe. The assay is designed to measure glutathione (GSH) production resulting from the hydrolysis of S-D-lactoylglutathione by Glo II, providing a sensitive and reliable method for assessing enzyme activity across various biological samples. The protocol involves incubating Glo II samples at 37 °C, then adding NAM, which reacts with thiol groups to form a fluorescent adduct. The fluorescence intensity is measured at excitation and emission wavelengths of 360 nm and 432 nm, respectively, allowing for precise quantification of Glo II activity. The NAM-Glo II method demonstrates exceptional sensitivity and specificity, with limits of detection (LOD) and quantification (LOQ) of 0.01 U/L and 0.033 U/L, respectively. This high sensitivity is crucial for accurately measuring Glo II activity in diverse bacterial strains, where enzyme levels may vary. Comparative studies with established methods reveal that the NAM-Glo II assay consistently yields results comparable to, and in some cases superior to, those obtained using UV-based techniques. Notably, the method effectively minimizes interference from common biomolecules, such as amino acids and carbohydrates, which can confound traditional assays. The NAM-Glo II method is a reliable, sensitive tool for quantifying Glo II activity, crucial for neurological and microbial studies. It enables accurate enzyme measurement, reveals higher activity in E. coli, aids bacterial metabolism research, and supports insights into detoxification, resistance, and targeted antimicrobial therapies.

本研究采用N-（9-吖啶基）马来酰亚胺（NAM）作为荧光探针，提出了一种新的荧光定量测定乙二醛酶（Glo II）酶活性的方法。该试验旨在测量Glo II水解s - d -乳酸谷胱甘肽产生的谷胱甘肽（GSH），为评估各种生物样品中的酶活性提供了一种敏感可靠的方法。该方案包括在37 °C下孵育Glo II样品，然后加入NAM， NAM与巯基反应形成荧光加合物。荧光强度分别在激发波长360 nm和发射波长432 nm处测量，从而可以精确定量Glo II活性。该方法灵敏度高，特异度高，检出限为0.01 U/L，定量限为0.033 U/L。这种高灵敏度对于准确测量不同细菌菌株的Glo II活性至关重要，其中酶水平可能会有所不同。与现有方法的比较研究表明，NAM-Glo II分析的结果与使用基于uv的技术获得的结果一致，在某些情况下优于使用基于uv的技术获得的结果。值得注意的是，该方法有效地减少了常见生物分子（如氨基酸和碳水化合物）的干扰，这些干扰会混淆传统的检测方法。NAM-Glo II方法是定量Glo II活性的可靠、灵敏的工具，对神经学和微生物研究至关重要。它能够精确测量酶，揭示大肠杆菌中更高的活性，有助于细菌代谢研究，并支持解毒，耐药性和靶向抗菌治疗的见解。

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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 : 2026-03-01 Epub 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

Development and optimization of an engineered E. coli platform for nitrotryptophan biosynthesis 工程大肠杆菌合成硝基色氨酸平台的建立与优化

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2026-03-01 Epub Date: 2025-11-20 DOI: 10.1016/j.enzmictec.2025.110785

Haiying Lin , Man Zhang , Wenbin Kang , Jianru Pan

Nitrotryptophan and its derivatives are valuable building blocks for synthesizing bioactive compounds and functional materials. This study reports the development of an efficient and novel bio-catalytic bioreactor in Escherichia coli capable of the direct aromatic nitration of tryptophan, enabling the synthesis of nitrotryptophan isomers. The biosynthetic pathway incorporates a self-sufficient P450 enzyme (TB14, consisting of TxtE-linker14-BM3R) from Streptomyces for the direct insertion of a nitro group into the indole ring of L-tryptophan. This process is supported by a nitric oxide synthetase (BsNOS) from Bacillus subtilis or its chemical alternative, sodium nitroprusside (SNP), to produce nitric oxide (NO) from L-arginine, which facilitates the direct nitration. As both TB14 and BsNOS require the reductant NADPH for their respective biochemical reactions, a glucose dehydrogenase (GDH) from Bacillus subtilis was included in the experimental design to ensure NADPH regeneration within the system.The initial engineered strain produced 133.2 mg/L of nitrotryptophan in TB medium. Through systematic optimization, including pathway balancing, fermentation condition enhancement, and elimination of competing metabolic pathways, the final titer was successfully increased to 209.9 mg/L within 48 h. This work establishes a robust platform for the microbial production of valuable nitroaromatic compounds and provides key insights for future biocatalytic nitration strategies.

硝基色氨酸及其衍生物是合成生物活性化合物和功能材料的重要组成部分。本研究报道了在大肠杆菌中开发一种高效的新型生物催化生物反应器，该反应器能够对色氨酸进行直接芳香硝化，从而合成硝基色氨酸异构体。该生物合成途径包含链霉菌中的一种自给自足的P450酶（TB14，由txte - link14 - bm3r组成），用于将硝基直接插入l -色氨酸的吲哚环中。该过程由枯草芽孢杆菌或其化学替代品硝普钠（SNP）的一氧化氮合成酶（BsNOS）支持，由l -精氨酸产生一氧化氮（NO），这有利于直接硝化。由于TB14和BsNOS各自的生化反应都需要还原剂NADPH，因此实验设计中加入枯草芽孢杆菌的葡萄糖脱氢酶（GDH）来保证系统内NADPH的再生。初始工程菌株在TB培养基中产生133.2 mg/L的硝基色氨酸。通过平衡途径、改善发酵条件、消除竞争代谢途径等系统优化，在48 h内成功将最终滴度提高到209.9 mg/L。这项工作为微生物生产有价值的硝基芳香族化合物建立了一个强大的平台，并为未来的生物催化硝化策略提供了关键的见解。

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引用次数: 0

Recombinant expression and nucleotide hydrolysis activity of NTPDase 4 from Trichomonas vaginalis 阴道毛滴虫ntpase 4的重组表达及核苷酸水解活性研究

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2026-03-01 Epub Date: 2025-11-20 DOI: 10.1016/j.enzmictec.2025.110786

Magno Sinval Pereira Ribeiro , Laura Cipolatto da Rosa , Giulia Bongiorni Galego , Mateus Dias de Oliveira , Carolina Wicteky Totti , Rogerio Margis , Geancarlo Zanatta , Tiana Tasca

Trichomonas vaginalis is the etiologic agent of trichomoniasis, the non-viral sexually transmitted infection most prevalent in world. It is important to investigate biochemical aspects of the parasite that contribute to our understanding of the biology and applications in the treatment and diagnosis of the infection. The nucleoside triphosphate diphosphohydrolase (NTPDase) is an enzyme that hydrolyses extracellular adenine and guanine nucleotides, forming nucleosides adenosine and guanosine. This is important for parasite survival through the purine salvage pathway, since adenosine is the precursor for the entire purine nucleotides pool in T. vaginalis. Herein we expressed TvNTPDase4 in the bacterial system Escherichia coli. Our data demonstrate that the enzyme is active, being able to hydrolyze ATP, ADP and AMP at a concentration of 10 μg of purified protein/reaction. The inhibitors gadolinium and adenosine 5′-[α,β-methylene]diphosphate (AMPCP) inhibited the hydrolysis of rTvNTPDase4. The inhibition of ATPase/ADPase activity was more effective with gadolinium, while the inhibition of AMPase activity was more effective with AMPCP. The enzyme rTvNTPDase4 was not cytotoxic to HMVII cells. In molecular dynamics, we observed that the ability of TvNTPDase4 to hydrolyze ATP, ADP, and AMP substrates occurs through direct interactions with the apyrase-conserved regions (ACR), especially ACR1 and ACR4. In this work, we did not find any candidate sequence for ecto-5′-nucleotidase (E-5′-N) in T. vaginalis, which leads us to believe that the parasite does not have this enzyme in its proteomic repertoire. Finally, we report that rTvNTPDase4 expressed and purified from a bacterial is active and has potential for biotechnological applications.

阴道毛滴虫是世界上最常见的非病毒性性传播感染——毛滴虫病的病原。重要的是研究寄生虫的生化方面，有助于我们对生物学的理解以及在感染的治疗和诊断中的应用。核苷三磷酸二磷酸水解酶（ntpase）是一种酶，水解胞外腺嘌呤和鸟嘌呤核苷酸，形成核苷腺苷和鸟嘌呤。由于腺苷是阴道绦虫整个嘌呤核苷酸库的前体，这对于通过嘌呤回收途径存活的寄生虫是重要的。我们在大肠杆菌系统中表达了TvNTPDase4。我们的数据表明该酶具有活性，能够在纯化蛋白/反应浓度为10 μg时水解ATP、ADP和AMP。抑制剂钆和腺苷5′-[α，β-亚甲基]二磷酸（AMPCP）抑制rTvNTPDase4的水解。钆对ATPase/ADPase活性的抑制更有效，而AMPCP对AMPase活性的抑制更有效。酶rTvNTPDase4对HMVII细胞无细胞毒性。在分子动力学中，我们观察到TvNTPDase4水解ATP、ADP和AMP底物的能力是通过与apyase -保守区域（ACR），特别是ACR1和ACR4的直接相互作用发生的。在这项工作中，我们没有在阴道绦虫中发现任何外5′-核苷酸酶（E-5′-N）的候选序列，这使我们相信寄生虫的蛋白质组库中没有这种酶。最后，我们报道了从细菌中表达和纯化的rTvNTPDase4具有活性，并且具有生物技术应用的潜力。

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引用次数: 0