Process Biochemistry最新文献_第6页

Screening and mechanistic study of chicken embryo egg-yolk peptide-Fe²⁺ chelates based on Caco-2 cells 基于Caco-2细胞的鸡胚蛋黄肽- fe 2 +螯合物的筛选及机制研究

IF 4 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Process Biochemistry

Pub Date : 2025-12-01 DOI: 10.1016/j.procbio.2025.12.001

Jia Lin , Shuaishuai Wei , Shijun Li , Donglei Zheng , Ziwei Zhang , Shijian Dong , Shugang Li , Lulu Ma

The iron-chelating peptides were screened from egg yolk, and the structure, and mechanism were investigated. By examining the incubation stages, day 12 had the highest iron chelation rate of 61.55 % ±1.69 %. On this basis, the peptide sequence PQETPPDTT (PT-9) was screened according to its molecular weight, amino acid composition, and molecular docking (binding energy < −4.2 kcal/mol). Compared with PT-9, the surface charge and average size of PT-9-Fe²⁺ changed from −17.07 mV and 1142 nm to −6.64 mV and 444 nm, respectively. Isothermal titration calorimetry (ITC) and Kinetic simulations revealed that the Gln2, Thr4, Asp7, and Thr9 in PT-9 formed a stable eight-coordination bond network spontaneously (ΔH = −1.689 kJ/mol) with Fe²⁺ via CO, N-H, and three water molecules, with an Fe^2 + release rate lower than 25 % in the gastrointestinal digestive tests. PT-9-Fe²⁺ enhanced Fe²⁺ uptake to 2.0-fold in Caco-2 cells compared with FeSO₄. Through a combination of methods, including RNA-seq and inhibitors, it was demonstrated that PT-9-Fe²⁺ up-regulated the expression of genes encoding Divalent Metal Transporter 1 (DMT1) and Transferrin Receptor (TFRC), and down-regulated the expression of genes encoding tight junction proteins and adhesion proteins. The iron uptake pathways mediated by PT-9-Fe²⁺ were predominantly paracellular transport, endocytosis and DMT1.

从蛋黄中筛选铁螯合肽，并对其结构和作用机理进行了研究。通过观察孵育期，第12天铁螯合率最高，为61.55 %±1.69 %。在此基础上，根据肽序列PQETPPDTT （PT-9）的分子量、氨基酸组成、分子对接（结合能<；−4.2 kcal/mol）筛选肽序列PQETPPDTT （PT-9）。与PT-9相比，PT-9- fe 2 +的表面电荷和平均尺寸分别从−17.07 mV和1142 nm变为−6.64 mV和444 nm。等温滴定量热法（ITC）和动力学模拟表明，PT-9中的Gln2、Thr4、Asp7和Thr9以Fe2 +通过CO、N-H和3个水分子自发形成稳定的八配位键网络（ΔH =−1.689 kJ/mol），胃肠道消化测试中Fe2 +的释放率低于25 %。与FeSO4相比，PT-9-Fe2+在Caco-2细胞中将Fe2+的吸收量提高到2.0倍。通过RNA-seq和抑制剂等综合方法，证实PT-9-Fe2+上调编码二价金属转运蛋白1 （DMT1）和转铁蛋白受体（TFRC）的基因表达，下调编码紧密连接蛋白和粘附蛋白的基因表达。PT-9-Fe2+介导的铁摄取途径主要是细胞旁转运、胞吞作用和DMT1。

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引用次数: 0

Pectin-ZnO bionanocomposite membrane based on Annona muricata leaf extract for in vitro treatment of skin and soft tissue infections 基于番荔枝叶提取物的果胶- zno生物纳米复合膜体外治疗皮肤和软组织感染

IF 4 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Process Biochemistry

Pub Date : 2025-11-28 DOI: 10.1016/j.procbio.2025.11.013

Suganya Ilango , Biswaranjan Paital , Nirmaladevi Ramalingam , Tejasweta Bhuyan , Tapas Ranjan Behera , Dipak Kumar Sahoo

Bionanocomposite membranes with increased durability are emerged as promising therapeutic candidates for the treatment of skin and soft tissue infections (SSTIs). In this study, a pectin-ZnO bionanocomposite membrane was synthesized using Annona muricata leaf extract. The formulation was examined with 2.3 % pectin, 5 % glycerol, and green-synthesized ZnO nanoparticles at concentrations of 50, 100, 150, and 200 mg mL⁻¹. The structural and functional characterization of the nanoparticles were confirmed using UV–visible spectroscopy (peak at 378 nm), FTIR, XRD (particle size: 18.41 nm), and SEM (particle size: 16.4 nm). The membrane exhibited strong antibacterial activity, being the highest inhibition observed against Streptococcus spp. (with the observed zone of inhibition of 24–26 mm at 200 mg mL-¹ ZnO and minimum inhibitory concentration of 65.68 µg/mL⁻¹), followed by Staphylococcus aureus and S. epidermidis. Solubility tests revealed excellent hygroscopic properties, enhancing its potential as a bioadhesive plaster. These results indicate that the eco-friendly biodegradable membrane can be used as a promising topical formulation for the treatment for SSTIs.

生物纳米复合膜具有更高的耐久性，是治疗皮肤和软组织感染（SSTIs）的有希望的治疗候选者。本研究以番荔枝叶提取物为原料，合成了果胶- zno生物纳米复合膜。用2.3 %果胶、5 %甘油和绿色合成的氧化锌纳米颗粒（浓度分别为50、100、150和200 mg mL−1）对该配方进行了检测。采用紫外可见光谱（峰值为378 nm）、FTIR、XRD（粒径为18.41 nm）和SEM（粒径为16.4 nm）对纳米颗粒的结构和功能进行了表征。该膜具有较强的抑菌活性，对链球菌的抑菌效果最好（在200 mg mL-1 ZnO下，抑菌范围为24-26 mm，最低抑菌浓度为65.68 µg/mL−1），其次是金黄色葡萄球菌和表皮葡萄球菌。溶解度测试显示了优异的吸湿性能，增强了其作为生物胶粘剂的潜力。这些结果表明，生态友好的生物可降解膜可以作为治疗SSTIs的一种有前景的外用制剂。

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引用次数: 0

Optimization of C-phycocyanin purification by aqueous two-phase system based on a thermo-sensitive copolymer with mild critical solution temperature 基于温和临界温度的热敏共聚物的两水相体系纯化c -藻蓝蛋白的优化

IF 4 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Process Biochemistry

Pub Date : 2025-11-19 DOI: 10.1016/j.procbio.2025.11.010

Shadi Azar, Alireza Habibi , Farshad Rahimpour

This study focuses on the purification of phycocyanin from spirulina platensis (C-PC) by a novel aqueous two-phase system (ATPS) including poly(EO₅₀-ran-PO₅₀) (Mn ∼ 12000 Da) + phosphate salts + water. The advance of this system was the separation of C-PC in the polymer phase after reaching the equilibrium state where 77.5 % of the polymer could be recovered by heating at a temperature above 48 °C. Optimization of the process was carried out by the response surface methodology and setting pH at 6, temperature of 25 °C, phosphate salt solution content of 30 wt%, and polymer content of 28.9 wt% obtained the best condition for achievement of the highest purification fold (PF), purity index (PI) and C-PC recovery yield (R) equaled to 1.68, 1.06 and 95.51 %, respectively. The reusability of the recovered polymer at the three next purifications showed reasonable sustainability with the preservation of more than 90 % of the original performance. The main advantage of this ATPS for C-PC purification is a fast partitioning of C-PC into the polymer phase after 3 h, where the mild critical solution temperature of the copolymer offers a safe thermal recovery of the polymer from purified C-PC without the need to centrifugation or filtration.

采用新型双水相体系（ATPS），包括聚（EO50-ran-PO50）（Mn ~ 12000 Da） + 磷酸盐+ 水，对螺旋藻（C-PC）中的藻蓝蛋白进行了纯化。该体系的先进之处在于，在达到平衡状态后，在48℃以上的温度下加热，可以回收77.5 %的聚合物，从而使聚合物相中的C- pc分离。采用响应面法对工艺进行优化，pH为6，温度为25℃，磷酸盐溶液含量为30 wt%，聚合物含量为28.9 wt%，可获得最高纯化倍数（PF），纯度指数（PI）和C- pc回收率(R)分别为1.68、1.06和95.51 %。在接下来的三次提纯中，回收的聚合物的可重复使用性表现出合理的可持续性，保留了超过90% %的原始性能。该ATPS用于C-PC净化的主要优点是在3 h后C-PC快速分解为聚合物相，共聚物的温和临界溶液温度提供了从纯化的C-PC中安全的热回收聚合物，而无需离心或过滤。

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引用次数: 0

Enhanced ergosterol biosynthesis in Saccharomyces cerevisiae via rational multigenetic modification and two-stage fermentation 通过合理的多基因修饰和两段发酵增强酿酒酵母麦角甾醇的生物合成

IF 4 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Process Biochemistry

Pub Date : 2025-11-19 DOI: 10.1016/j.procbio.2025.11.011

Yufeng Xie , Chenxu Fan , Minhao Piao , Longgang Jia

Ergosterol is a valuable precursor for synthesis of vitamin D2. However, low-cost production of ergosterol by yeast cell fermentation is still a major challenge due to low ergosterol content in production strains. Here, we screened a high-yield ergosterol producing Saccharomyces cerevisiae strain YX5 that shown to accumulate the highest contents of ergosterol [9.6 mg/g dry cell weight (DCW)]. Overexpression in strain YX5 of the truncated tHMG1 encoding the rate-limiting enzyme HMG-CoA reductase 1 in the ergosterol biosynthesis pathway resulted in a 64 % increase in ergosterol yield (15.7 mg/g DCW). Combined overexpression of tHMG1 and ERG1 encoding the squalene epoxidase or ERG11 encoding the sterol C14-demethylase further enhanced the ergosterol yields by 71 % (26.8 mg/g DCW), 34 % (21.0 mg/g DCW), respectively. Simultaneous overexpression of tHMG1, ERG1 and ERG11 in strain YX5 led to ergosterol yield of 36.2 mg/g DCW, a 276.8 % increase compared to the parental strain. High-cell-density fermentation for strain YX5-tHMG1/ERG1/ERG11 based on a two-stage feeding strategy led to ergosterol titers of 2077.0 mg/L and ergosterol contents of 35.9 mg/g DCW. The engineered strain YX5-tHMG1/ERG1/ERG11 from this study could be a useful chassis for further engineering of more efficient ergosterol production strains.

麦角甾醇是合成维生素D2的重要前体。然而，由于生产菌株中麦角甾醇含量低，通过酵母细胞发酵低成本生产麦角甾醇仍然是一个主要挑战。在这里，我们筛选了一株产麦角甾醇的酿酒酵母YX5菌株，其麦角甾醇含量最高[9.6 mg/g干细胞重（DCW）]。在菌株YX5中过表达编码麦角甾醇生物合成途径中限制性酶HMG-CoA还原酶1的截断tHMG1，导致麦角甾醇产量增加64 %（15.7 mg/g DCW）。编码角鲨烯环氧化酶的tHMG1和编码甾醇c14 -去甲基化酶的ERG11联合过表达可进一步提高麦角甾醇产量，分别提高71 %（26.8 mg/g DCW）和34 %（21.0 mg/g DCW）。菌株YX5同时过表达tHMG1、ERG1和ERG11导致麦角甾醇产量为36.2 mg/g DCW，比亲本菌株提高了276.8 %。菌株YX5-tHMG1/ERG1/ERG11采用两段饲喂策略进行高密度发酵，其麦角甾醇滴度为2077.0 mg/L，麦角甾醇含量为35.9 mg/g DCW。本研究获得的工程菌株YX5-tHMG1/ERG1/ERG11可以为进一步设计更高效的麦角甾醇生产菌株提供有用的基础。

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引用次数: 0

Optimization of pretreatment methods for banana leaf waste and its kinetics studies for effective biogas generation by anaerobic co-digestion with food waste 香蕉叶废弃物预处理方法优化及与厨余厌氧共消化产气动力学研究

IF 4 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Process Biochemistry

Pub Date : 2025-11-19 DOI: 10.1016/j.procbio.2025.11.009

Athithyan Ilangovan , Sri Bala Kameswari Kanchinadham

Enhancing enzymatic hydrolysis and biofuel production requires effective biomass pretreatment. This study systematically optimised four pretreatment methods like dilute acid, alkali, molecular solvent, and thermal to maximise reducing sugars yield from lignocellulosic biomass. Key parameters like concentration, temperature, and residence time were varied. The highest reducing sugars yield (8000–10000 mg/L) was achieved with alkali pretreatment (4 % w/v at 100 °C for 30 min), indicating significant cellulose breakdown. molecular solvent and thermal pretreatments produced moderate yields but were promising. The kinetic constant (k) calculated for dilute acid, alkali, molecular solvent, and thermal pretreatments were 0.02726 ± 0.00643 min⁻¹ , 0.02235 ± 0.00584 min⁻¹ , 0.02077 ± 0.0088 min⁻¹ and 0.02218 ± 0.01672 min⁻¹ respectively, using first order kinetics helped to understand the reaction rate and degree of hydrolysis. Biogas generation was modelled using the modified Gompertz equation. Kinetic modelling studies revealed that co-digestion of food waste with alkali-pretreated banana leaf waste led to maximum biogas yield, with a maximum production rate (R_m) of 322.06 ± 51.51 mL/day. This study provides a foundation for scalable biomass valorisation, offering key insights into tailoring pretreatment strategies to enhance hydrolysis, sugar release, and biogas production from lignocellulosic waste.

提高酶解和生物燃料生产需要有效的生物质预处理。本研究系统优化了稀酸、碱、分子溶剂和热四种预处理方法，以最大限度地提高木质纤维素生物质的还原糖产量。关键参数如浓度、温度和停留时间是不同的。碱预处理（4 % w/v, 100°C, 30 min）可获得最高的还原糖产率（8000-10000 mg/L），表明纤维素分解显著。分子溶剂和热预处理产率适中，但前景广阔。动力学常数(k)计算稀酸、碱、溶剂分子,和热预处理0.02726 ±0.00643  分钟⁻¹ , 0.02235±0.00584  分钟⁻¹ , 0.02077±0.0088  分钟⁻¹  和0.02218±0.01672  分钟⁻¹ 分别使用一阶动力学帮助理解反应速率和水解度。利用修正的Gompertz方程对沼气生成进行了建模。动力学模型研究表明，食物垃圾与经碱预处理的香蕉叶垃圾共消化可产生最大的沼气量，最大产气量（Rm）为322.06±51.51 mL/d。该研究为可扩展的生物质增值提供了基础，为定制预处理策略提供了关键见解，以增强木质纤维素废物的水解，糖释放和沼气生产。

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引用次数: 0

Design and optimization of a cyanide dihydratase-cross-linked enzyme aggregate system for enhanced biodegradation of cyanide: Integrating statistical and machine learning approaches 氰化物二水合酶-交联酶聚合系统的设计与优化，以增强氰化物的生物降解：整合统计和机器学习方法

IF 4 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Process Biochemistry

Pub Date : 2025-11-19 DOI: 10.1016/j.procbio.2025.11.012

Ademakinwa A. Nelson , Agunbiade M. Oladele , Adeyanju M. Moronkeji , Alli Kazeem , Ayinla Z. Adenike

This study developed a robust cyanide biodegradation system using cross-linked enzyme aggregates (CLEAs) of cyanide dihydratase from Aureobasidium pullulans NAC8 (ApCynD). The intracellular enzyme was immobilized as cynD-CLEA and evaluated alongside the free enzyme. Immobilization significantly improved operational stability, with over 90 % activity retained after seven reuse cycles and 80 % after six weeks of storage. Structural analysis showed increased α-helix (29.4–38.8 %) and β-turn (12.6 % to 18.4 %) contents, with reductions in β-sheet and random coil, indicating a more ordered conformation. Kinetic profiling revealed elevated V_max and catalytic efficiency despite a higher K_m. Process conditions were optimized using response surface methodology (RSM, R² = 0.902) and artificial neural networks (ANN, R² = 0.958). RSM identified cyanide concentration as the most significant factor, with optimal conditions (1000 ppm, 150 rpm, 1000 mg CLEA, 3 h) yielding 82 % removal. ANN-predicted optimums (240 ppm, 200 rpm, 865 mg CLEA, 6 h) achieved 90.5 % removal, confirming better performance in modeling nonlinear interactions. SHAP analysis enhanced model interpretability. The integrated RSM-ANN approach offers a powerful strategy for optimizing enzyme-based bioremediation, with cynD-CLEA showing strong potential for industrial application. Further studies are recommended to assess broader conditions and extended operational cycles.

本研究利用普鲁兰短毛霉NAC8 （ApCynD）氰化物二水合酶的交联酶聚集体（CLEAs）建立了一个强大的氰化物生物降解系统。将胞内酶固定为cynD-CLEA，并与游离酶一起进行评价。固定化显著提高了操作稳定性，在7个重复使用周期后保持了超过90% %的活性，在6周后保持了80% %的活性。结构分析表明，α-螺旋（29.4 ~ 38.8 %）和β-转（12.6 ~ 18.4 %）含量增加，β-片和随机线圈含量减少，构象更加有序。动力学分析显示，尽管Km增加，但Vmax和催化效率也有所提高。采用响应面法（RSM, R²= 0.902）和人工神经网络（ANN, R²= 0.958）对工艺条件进行优化。RSM发现氰化物浓度是最重要的影响因素，最佳条件为1000 ppm， 150 rpm, 1000 mg CLEA, 3 h，去除率为82% %。ann预测的最优值（240 ppm, 200 rpm, 865 mg CLEA, 6 h）的去除率达到了90.5 %，证实了建模非线性相互作用的更好性能。SHAP分析增强了模型的可解释性。综合RSM-ANN方法为优化酶基生物修复提供了强有力的策略，cynD-CLEA显示出强大的工业应用潜力。建议进行进一步研究，以评估更广泛的条件和延长的业务周期。

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引用次数: 0

Engineering a self-sufficient nitric oxide-generating module (BsNOS-YkuN) for versatile P450-catalyzed nitration platforms 设计一个自给自足的一氧化氮生成模块（BsNOS-YkuN），用于多功能p450催化硝化平台

IF 4 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Process Biochemistry

Pub Date : 2025-11-17 DOI: 10.1016/j.procbio.2025.11.008

Haiying Lin , Yun Chen , Yijie Yu , Yanmei Wang , Jianru Pan

Nitrotryptophan is a high-value compound, yet its production via traditional chemical synthesis suffers from low selectivity and environmental concerns. Biocatalysis using P450 "nitrases" offers a promising green alternative, but its efficiency is often limited by the supply of the crucial co-substrate, nitric oxide (NO), often due to electron transfer limitations in NO synthases. To address this, we engineered a self-sufficient Escherichia coli biocatalyst based on a novel fusion enzyme, BsNOS-YkuN, which links a nitric oxide synthase with its essential reductase to ensure efficient electron transfer. This fusion biocatalyst exhibited superior specific activity (13.5 U/mg) and enabled a robust cell-free nitration system, yielding 86.5 mg/L of product after optimization. When implemented in a whole-cell system with the P450 nitrase TxtE-BM3R, this module proved highly effective. Systematic optimization of fermentation (25°C, pH 7.7) and a fed-batch strategy boosted the titer to 220.6 mg/L. Furthermore, deleting the competing gene pheA accelerated production from endogenous precursors. This work demonstrates a multi-layered engineering strategy, creating a versatile platform for nitroaromatic production proven in both in vitro and in vivo contexts.

硝基色氨酸是一种高价值的化合物，但传统的化学合成方法存在选择性低和环境问题。使用P450“氮化酶”进行生物催化提供了一种很有前途的绿色替代方案，但其效率往往受到关键的共底物一氧化氮（NO）供应的限制，这通常是由于NO合成酶的电子转移限制。为了解决这个问题，我们设计了一种自给自足的大肠杆菌生物催化剂，该催化剂基于一种新型融合酶BsNOS-YkuN，它将一氧化氮合酶与其必需的还原酶连接起来，以确保有效的电子转移。该融合生物催化剂表现出优异的比活性（13.5 U/mg），并实现了稳健的无细胞硝化系统，优化后的产物产量为86.5 mg/L。当使用P450 nitrase TxtE-BM3R在全细胞系统中实施时，该模块被证明是非常有效的。系统优化发酵（25°C, pH 7.7）和补料分批策略，使滴度达到220.6 mg/L。此外，删除竞争基因pheA加速了内源性前体的产生。这项工作展示了一个多层次的工程策略，创造了一个多功能的平台，在体外和体内环境中都证明了硝基芳烃的生产。

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引用次数: 0

Chitosan-modified carboxylated magnetic nanoparticles immobilized glycosidases for the production of icaritin 壳聚糖修饰羧化磁性纳米颗粒固定化糖苷酶制备淫羊藿苷

IF 4 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Process Biochemistry

Pub Date : 2025-11-15 DOI: 10.1016/j.procbio.2025.11.007

Yuxuan Zhao , Ye Li , Wenting Fei , Hao Liang

In recent years, nanomaterials have emerged as prominent carriers in biomedical applications. This trend aims to address limitations inherent in traditional enzyme immobilization methods and reduce the costs associated with enzyme biocatalysts in industrial processes. In this study, we selected non-toxic magnetic nanoparticles (MNPs), distinguished by their exceptional biocompatibility, as immobilization carriers to overcome the inherent instability and non-reusability of free enzymes. The MNPs were coated with chitosan to form a protective layer, thereby significantly enhancing enzyme stability. This approach also circumvented the need for precipitants, thereby avoiding the associated damage observed in conventional cross-linking. Building upon this foundation, glucosidase and rhamnosidase were immobilized onto these modified MNPs for converting Epimedin C into Icaritin. Compared to conventional cross-linked enzyme aggregation techniques, this system exhibited superior catalytic efficiency. Furthermore, the immobilized enzyme system enables rapid magnetic separation of the reaction product. Notably, the immobilized enzymes retained approximately 70 % of their initial activity after eight reuse cycles, demonstrating significant potential for industrial-scale applications. These findings demonstrate the potential of chitosan-modified magnetic nanoparticles as efficient, reusable enzyme carriers for sustainable biocatalytic processes in pharmaceutical and industrial applications.

近年来，纳米材料已成为生物医学领域的重要载体。这一趋势旨在解决传统酶固定化方法固有的局限性，并降低工业过程中与酶生物催化剂相关的成本。在这项研究中，我们选择无毒磁性纳米颗粒（MNPs）作为固定载体，以其独特的生物相容性来克服游离酶固有的不稳定性和不可重复使用性。壳聚糖包被MNPs形成保护层，从而显著提高酶的稳定性。这种方法还避免了对沉淀剂的需要，从而避免了在传统交联中观察到的相关损害。在此基础上，将葡萄糖苷酶和鼠李糖苷酶固定在这些修饰的MNPs上，将淫羊藿苷C转化为淫羊藿苷。与传统的交联酶聚合技术相比，该体系具有更高的催化效率。此外，固定化酶系统可以实现反应产物的快速磁分离。值得注意的是，经过8次重复使用后，固定化酶保持了大约70% %的初始活性，显示出工业规模应用的巨大潜力。这些发现证明了壳聚糖修饰的磁性纳米颗粒作为高效、可重复使用的酶载体在制药和工业应用中的可持续生物催化过程的潜力。

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引用次数: 0

Removal selectivity of His6-SUMO-Tag in recombinant protein production based on skin cell behaviors 基于皮肤细胞行为的His6-SUMO-Tag在重组蛋白生产中的去除选择性

IF 4 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Process Biochemistry

Pub Date : 2025-11-13 DOI: 10.1016/j.procbio.2025.11.006

Zilong Zhao , Weigang Yuwen , Linlin Qu , Daidi Fan

His₆-SUMO-tag, widely used in recombinant protein production for enhancing solubility and folding, has seen increased use due to its removable nature via SUMO proteases. However, the necessity of removing the His₆-SUMO-tag during the production of recombinant proteins remains controversial in various studies, primarily due to differing views on whether the His₆-SUMO-tag may impede the functional activity of the recombinant protein. This study aims to validate His6-SUMO-tag's effects on cell proliferation, adhesion, and migration, and explore its role in skin cell metabolism using transcriptomics. His₆-SUMO-tag produced in Escherichia coli was purified with Ni-IDA Magbeads, characterized, and yielded 0.25 ± 0.08 g/L. Optimal proliferative concentrations for HaCaT and HSF cells were 1 mg/mL and 0.5 mg/mL, respectively, upregulating MKI67 and PCNA gene expression. His₆-SUMO-tag may have complex adhesion mechanisms with varying effects on cell types and no significant migratory effect on skin cells. Its impact on gene transcription in HaCaT and HSF cells was similar, focusing on immune and inflammatory responses. This indicates that the influence of the His₆-SUMO-tag on the activity of recombinant proteins that do not relate to immune-activation is minimal, suggesting that it may optionally be retained without removal. This study underscores His₆-SUMO-tag's potential in assessing cellular activity and supports its use in protein and fermentation engineering.

his6 -SUMO-标签广泛用于重组蛋白生产，以提高溶解度和折叠性，由于其可通过SUMO蛋白酶去除的性质，其使用越来越多。然而，在重组蛋白的生产过程中去除his6 - sumo标签的必要性在各种研究中仍然存在争议，主要是因为对his6 - sumo标签是否会阻碍重组蛋白的功能活性存在不同的看法。本研究旨在验证His6-SUMO-tag对细胞增殖、粘附和迁移的影响，并利用转录组学方法探讨其在皮肤细胞代谢中的作用。用Ni-IDA Magbeads纯化大肠杆菌中产生的His6-SUMO-tag，并对其进行了表征，其产率为0.25 ± 0.08 g/L。HaCaT和HSF细胞的最佳增殖浓度分别为1 mg/mL和0.5 mg/mL，上调MKI67和PCNA基因的表达。His6-SUMO-tag可能具有复杂的粘附机制，对细胞类型有不同的影响，对皮肤细胞无明显的迁移作用。它对HaCaT和HSF细胞基因转录的影响相似，主要集中在免疫和炎症反应上。这表明his6 - sumo -标签对与免疫激活无关的重组蛋白活性的影响很小，表明它可以选择性地保留而不去除。这项研究强调了his6 - sumo标签在评估细胞活性方面的潜力，并支持其在蛋白质和发酵工程中的应用。

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引用次数: 0

Immobilization of glutamate decarboxylase on tussah silk modified magnetic nanoparticles via laccase catalyzed oxidative coupling 漆酶催化氧化偶联固定化柞蚕丝磁性纳米颗粒上谷氨酸脱羧酶的研究

IF 4 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Process Biochemistry

Pub Date : 2025-11-11 DOI: 10.1016/j.procbio.2025.11.004

Hanhan Xie , Kaixuan Liu , Yuanrui Gao, Ruofu Shi, Chengli Yang, Dali Li

Gamma-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the central nervous system, exhibits multiple physiological functions. The GABA is synthesized through the decarboxylation of glutamic acid, a reaction catalyzed by the enzyme glutamate decarboxylase (GAD). In this study, magnetic nanoparticles were coated with tussah silk (TS) protein. The GAD was then immobilized via laccase-catalyzed oxidative coupling. Characterization revealed that the TS protein coated magnetic nanoparticles exhibited spherical morphology with rough surfaces, an average particle size of 27 nm, and excellent magnetic separation capability. Compared to the free GAD, the immobilized GAD exhibited significantly enhanced pH and thermal stability. After 15 days at 4 °C, the immobilized GAD retained 76.3 % relative activity, whereas the free enzyme showed a complete loss of activity. Furthermore, the immobilized GAD maintained approximately 50 % residual activity after seven operational recycles. This work successfully established an enzymatic crosslinking strategy for GAD immobilization on magnetic nanoparticles, providing both an efficient approach for GABA production and a novel methodology for enzyme immobilization technology.

γ -氨基丁酸（GABA）是中枢神经系统的主要抑制性神经递质，具有多种生理功能。GABA是由谷氨酸脱羧酶（GAD）催化的谷氨酸脱羧反应合成的。在本研究中，磁性纳米颗粒被柞蚕丝（TS）蛋白包裹。然后通过漆酶催化氧化偶联固定化GAD。表征结果表明，TS蛋白包被的磁性纳米颗粒呈球形，表面粗糙，平均粒径为27 nm，具有优异的磁分离性能。与游离GAD相比，固定化GAD的pH值和热稳定性显著提高。在4°C下放置15天后，固定化GAD保持了76.3% %的相对活性，而游离酶则完全丧失了活性。此外，固定化GAD在七次操作循环后保持了大约50% %的剩余活性。本研究成功地建立了一种磁性纳米颗粒固定GAD的酶交联策略，为GABA的生产提供了一种有效的途径，也为酶固定技术提供了一种新的方法。

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