Enzyme and Microbial Technology最新文献_第7页

Developing a urea biosensor and safe blood cleaning method utilizing a novel high throughput La-doped CeO2 nanosized artificial urease with high biocompatibility and enzyme-like activity 利用具有高生物相容性和酶样活性的新型高通量la掺杂CeO2纳米人工脲酶开发尿素生物传感器和安全血液清洁方法

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-08-15 DOI: 10.1016/j.enzmictec.2025.110736

Naushad Ahmad , Ashok Kumar Bishoyi , Suhas Ballal , Aman Shankhyan , Shaker Al-Hasnaawei , Karthikeyan Jayabalan , Laxmidhar Maharana , Madan Lal

Herein, a urea biosensor and safe blood cleaning method utilizing a novel high throughput La-doped CeO₂ nanosized artificial urease with high biocompatibility and enzyme-like activity were developed. The urease-like activity, stability composition, morphological characteristics, size, biocompatibility, and crystalline characteristics of the artificial urease were assessed. Considering its high urease-like activity, it is applied for both urea biosensing and safe blood cleaning. The effective factors on the sensing conditions were optimized, providing a wide linear range over 1–10 µM and a low detection limit of 0.5 µM. The repeatability and reproducibility assessments revealed a %RSD of 1.7 % and 2.7 %, in order. The selectivity studies and real blood analysis revealed highly selective, accurate, and reliable determination of urea in biosamples using the developed method, providing a %recovery of 97.8–99.5 % with a low %RSD range of 1.4–3.3 %. Besides, the safe blood cleaning experiments were also performed by urea mineralization from blood. The results exhibited a high blood cleaning yield of 99.5 % at a short time of 30 min, revealing 1.8-fold higher cleaning efficiency for the artificial urease compared to native urease. The reusability studies showed that the blood cleaning yield was saved for about 10 cycles and then it decreased by increasing the operational cycles, reaching 78.6 % after 17 cycles. The shelf-life of the urease was also evaluated within 30 days, revealing a high shelf-stability for the as-prepared nanosized artificial urease. Consequentially, the nanosized artificial urease can be practically applied for both urea detection and blood cleaning in the real world.

本研究利用高生物相容性和酶样活性的新型高通量la掺杂CeO2纳米人工脲酶，开发了一种尿素生物传感器和安全血液清洁方法。对人工脲酶的类脲酶活性、稳定性组成、形态特征、大小、生物相容性和结晶特性进行了评价。由于其具有高的类脲酶活性，因此可用于尿素生物传感和安全血液清洁。对影响传感条件的因素进行了优化，在1-10 µM范围内具有较宽的线性范围，检测限低至0.5 µM。重复性和再现性评估显示，RSD分别为1.7 %和2.7 %。选择性研究和实际血液分析表明，该方法具有较高的选择性、准确性和可靠性，回收率为97.8 ~ 99.5 %，RSD范围为1.4 ~ 3.3 %。此外，还进行了血液尿素矿化的安全血液清洗实验。结果表明，在30 min的短时间内，人工脲酶的血液清洁率高达99.5 %，比天然脲酶的清洁效率高1.8倍。可重复使用性研究表明，血液清洁率可保存约10个循环，然后随着操作周期的增加而降低，17个循环后达到78.6% %。在30天内对脲酶的保质期进行了评估，表明制备的纳米级人工脲酶具有很高的货架稳定性。因此，纳米级人工脲酶可以在现实世界中用于尿素检测和血液清洁。

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引用次数: 0

Breaking the CREB–CBP alliance: Progress, challenges, and therapeutic promise of small-molecule and peptide disruptors of the pKID–KIX interaction 打破CREB-CBP联盟：pKID-KIX相互作用的小分子和肽干扰物的进展、挑战和治疗前景

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-08-14 DOI: 10.1016/j.enzmictec.2025.110734

Hassan A. Rudayni

The kinase-inducible domain (pKID) of the transcription factor CREB engages the KIX domain of the co-activator CBP/p300 to drive signal-dependent gene expression that underpins cell proliferation, metabolism and survival. Aberrant CREB–CBP signaling is now implicated in numerous cancers and metabolic disorders, yet until recently the shallow, dynamic pKID–KIX interface was viewed as chemically intractable. This review integrates two decades of progress that overturns that paradigm. We first dissect the structural and allosteric features of KIX that enable coupled folding-and-binding of pKID and reveal hidden ligandable pockets. We then survey the expanding inhibitor repertoire—from early micromolar naphthols (KG-501) through nanomolar naphthamides (666−15) and orally tractable pro-drugs, to high-affinity stapled and D-peptide mimetics—highlighting the assays, structure–activity relationships and pharmacokinetic optimization that have driven each advance. Biophysical and computational insights, including ¹⁹F NMR ligandability maps and millisecond-scale molecular-dynamics trajectories, are shown to guide next-generation design and machine-learning pipelines. Pre-clinical data demonstrate that disrupting CREB–CBP selectively suppresses tumor growth with favorable tolerability, and we outline opportunities for combination therapies, degrader strategies and indication expansion into metabolic and neurocognitive disease. Collectively, these findings position the CREB–CBP interaction as a tractable, multi-modal drug target poised for first-in-human exploration.

转录因子CREB的激酶诱导结构域（pKID）与共激活因子CBP/p300的KIX结构域结合，驱动信号依赖性基因表达，支持细胞增殖、代谢和存活。异常的CREB-CBP信号现在与许多癌症和代谢紊乱有关，但直到最近，浅的动态pKID-KIX界面被认为是化学上难以处理的。这篇综述整合了20年来推翻这一范式的进展。我们首先剖析了KIX的结构和变构特征，这些特征使pKID能够耦合折叠和结合，并揭示了隐藏的可配体口袋。然后，我们调查了不断扩大的抑制剂品种——从早期的微摩尔萘酚（KG-501）到纳摩尔萘胺（666−15）和口服可处理的前药，再到高亲和力的钉状和d肽模拟物——强调了推动每一项进展的分析、构效关系和药代动力学优化。生物物理和计算洞察力，包括19F核磁共振配位性图和毫秒级分子动力学轨迹，将指导下一代设计和机器学习管道。临床前数据表明，破坏CREB-CBP选择性抑制肿瘤生长，具有良好的耐受性，我们概述了联合治疗、降解策略和适应症扩展到代谢和神经认知疾病的机会。总的来说，这些发现将CREB-CBP相互作用定位为一种易于处理的多模式药物靶点，有望首次在人体中进行探索。

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引用次数: 0

Isolation, expression and characterization of a novel thermo-acid/alkali-stable GH10 xylanase BsXynA from Bacillus safensis L7 and its potential for xylooligosaccharide production and animal feed saccharification 一种新型热酸/碱稳定型GH10木聚糖酶BsXynA的分离、表达和鉴定及其在低聚木糖生产和动物饲料糖化中的应用前景

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-08-13 DOI: 10.1016/j.enzmictec.2025.110735

Ting Zhang, Zhong Cheng, YuMei Fan, YuXin Lan, HuiLan Shu, JinHua Chen, FengCheng Jin, LiYuan Qin, DongPing Feng

Xylanases have wide applications in agro-industrial processes. This study reports the discovery and characterization of a novel thermo-acid/alkali-stable GH10 xylanase (BsXynA) from Bacillus safensis L7. The xylanase gene (BsxynA) was cloned and expressed in Escherichia coli BL21 (DE3), yielding a protein of approximately 64 kDa. BsXynA exhibited optimal activity (17.33 U/mg) on beechwood xylan at pH 6.0 and 60°C. Moreover, BsXynA exhibited remarkable thermo-acid/alkali stability, retaining over 60 % activity at pH 5.0–8.0 after 60 min at 60°C and over 80 % activity after 14 days at 4°C within pH 6.0–9.0. Additionally, the enzyme tolerated 50°C and various chemicals, with a half-life of over 16 days. It was activated by K⁺, Na⁺, Ca²⁺, Ba²⁺, and Mg²⁺ ions but inhibited by Zn²⁺, Cu²⁺, and SDS. BsXynA hydrolyzed various xylans but not glucose-based polysaccharides. K_m, V_max, k_cat, and k_cat/K_m for beechwood xylan hydrolysis were found to be 6.61 mg/mL, 24.24 µmol·min⁻¹·mg⁻¹, 15.71 s⁻¹, and 2.38 mL·s⁻¹·mg⁻¹ respectively. Thin-layer chromatography (TLC) analysis showed that BsXynA is an endo-type xylanase, which hydrolyzes beechwood xylan to produce mainly xylobiose (X2) and xylotetraose (X4), with no xylose detected. Furthermore, BsXynA improved animal feed saccharification, making it a promising biocatalyst for biotechnological applications.

木聚糖酶在农工生产过程中有着广泛的应用。本研究报道了一种新的热酸/碱稳定木聚糖酶（BsXynA）的发现和鉴定。在大肠杆菌BL21 （DE3）中克隆并表达了木聚糖酶基因（bxyna），得到了一个约64 kDa的蛋白。在pH 6.0和60℃条件下，BsXynA对山毛榉木聚糖的活性为17.33 U/mg。此外，BsXynA表现出显著的热酸/碱稳定性，在pH 5.0-8.0下，在60°C下60 min后，活性保持在60% %以上，在pH 6.0-9.0下，在4°C下14 d后活性保持在80% %以上。此外，该酶耐受50°C和各种化学物质，半衰期超过16天。它被K +、Na +、Ca 2 +、Ba 2 +和Mg 2 +激活，但被Zn 2 +、Cu 2 +和SDS抑制。BsXynA能水解多种木聚糖，但不能水解葡萄糖基多糖。山毛榉木聚糖水解的Km、Vmax、kcat和kcat/Km分别为6.61 mg/mL、24.24µmol·min−1·mg−1、15.71 s−1和2.38 mL·s−1·mg−1。薄层色谱（TLC）分析表明，bxyna是一种内型木聚糖酶，主要水解山毛榉木聚糖生成木糖二糖（X2）和木糖四糖（X4），未检测到木糖。此外，BsXynA改善了动物饲料的糖化，使其成为一种有前景的生物技术应用的生物催化剂。

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引用次数: 0

Ethyl lactate synthesis in organic media using a magnetic supported CALB 磁性负载CALB在有机介质中合成乳酸乙酯

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-08-12 DOI: 10.1016/j.enzmictec.2025.110732

Paula Nicolás , Verónica L. Lassalle , María Luján Ferreira

The growing demand for sustainable chemical processes has spurred interest in enzymatic synthesis, particularly for valuable compounds like ethyl lactate. Traditional chemical methods often suffer from drawbacks, highlighting the potential of enzymatic catalysis using immobilized lipases. This study evaluated the performance of magnetic biocatalyst, prepared by immobilizing Candida antarctica lipase B (CALB) on magnetic nanoparticles, for the batch synthesis of ethyl lactate in hexane. Initial experiments using free CALB and commercial Novozym435 proved problematic due to enzyme denaturation and support instability, respectively. While titration-based methods for monitoring the reaction were found to be unreliable due to lactic acid's complex behavior in the reaction medium, titratable acidity reduction suggested an optimal lactic acid to ethanol molar ratio of 1/10. Subsequent HPLC analysis revealed that the magnetic biocatalyst maintained a consistent conversion (%) at higher lactic acid concentrations (up to 17 mg/mL at 45°C, with conversion above 60 % in 5 h), demonstrating its potential for processing larger amounts of substrate. The initial reaction rate was estimated to be 3.8 mM/h. The study also identified experimental challenges in accurate lactic acid quantification and potential catalyst degradation. In conclusion, the magnetic CALB biocatalyst shows promising activity and stability for ethyl lactate synthesis, especially at higher substrate loads, paving the way for further optimization and application in sustainable production.

对可持续化学过程日益增长的需求激发了人们对酶合成的兴趣，特别是对有价值的化合物，如乳酸乙酯。传统的化学方法往往遭受的缺点，突出潜力的酶催化使用固定化脂肪酶。研究了磁性纳米颗粒固定化南极念珠菌脂肪酶B （CALB）制备的磁性生物催化剂在正己烷中批量合成乳酸乙酯的性能。使用游离CALB和商用Novozym435进行的初步实验分别证明由于酶变性和支持不稳定性而存在问题。由于乳酸在反应介质中的复杂行为，基于滴定的反应监测方法被认为是不可靠的，可滴定的酸度还原表明乳酸与乙醇的最佳摩尔比为1/10。随后的HPLC分析显示，磁性生物催化剂在较高的乳酸浓度下保持一致的转化率（%）（在45°C下高达17 mg/mL，转化率在5 h内超过60 %），表明其处理大量底物的潜力。初始反应速率估计为3.8 mM/h。该研究还确定了准确乳酸定量和潜在催化剂降解的实验挑战。综上所述，磁性CALB生物催化剂在乳酸乙酯合成中表现出良好的活性和稳定性，特别是在高底物负荷下，为进一步优化和可持续生产应用铺平了道路。

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引用次数: 0

Elucidation of biochemical attributes and enzymatic activity of agarase from Saccharophagus degradans 2–40 食糖菌降解2-40琼脂酶的生化特性及酶活性的研究

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-08-08 DOI: 10.1016/j.enzmictec.2025.110733

Anoth Maharjan , Beom Soo Kim

Saccharophagus degradans 2–40 exhibits agarolytic activity, effectively degrading agar into galactose. Both endo- and exo-agarase, as well as neoagarobiose hydrolase (NABH), play important roles in agar saccharification for the production of monosugars. This study characterizes a novel agarase enzyme from S. degradans 2–40, a marine bacterium renowned for its exceptional polysaccharide-degrading capabilities. We hypothesized that this strain would harbor an efficient and robust agarase with desirable properties for saccharification processes. Following isolation and purification, the agarase underwent biochemical analysis, revealing optimal activity at moderate temperatures and a broad pH range. Furthermore, the fusion of Aga50D with NABH enhanced the catalytic efficiency from 1.873 ± 0.22 (mg/mL)⁻¹s⁻¹ to 4.826 ± 0.19 (mg/mL)⁻¹s⁻¹. In contrast to chemical hydrolysis, enzymatic treatment using agarase offers a more selective, eco-friendly, and high-yield alternative, minimizing by-product formation and preserving functional sugar moieties. The enzyme's ability to produce neoagarobiose (NA2) as its primary product, without any intermediates, makes it an attractive biocatalyst for the production of bioactive oligosaccharides. This study highlights the potential of S. degradans 2–40 as a valuable source of enzymes for industrial biotechnology applications, particularly in the sustainable conversion of marine biomass into high-value products.

食糖降解菌2-40具有降解琼脂的活性，能有效地将琼脂降解为半乳糖。内、外琼脂酶以及新琼脂糖水解酶（NABH）在琼脂糖化生产单糖过程中起着重要作用。本研究表征了一种来自S.降解2-40的新型琼脂酶，这是一种以其特殊的多糖降解能力而闻名的海洋细菌。我们假设，这种菌株将有一个有效的和强大的琼脂酶具有理想的性质，糖化过程。分离纯化后，对该琼脂酶进行了生化分析，结果表明该酶在中等温度和较宽的pH范围内具有最佳活性。此外，Aga50D与NABH的融合使催化效率从1.873 ± 0.22 (mg/mL)−1s−1提高到4.826 ± 0.19 (mg/mL)−1s−1。与化学水解相比，使用琼脂酶的酶处理提供了一种更具选择性，环保和高产的替代方法，最大限度地减少了副产物的形成并保留了功能糖部分。该酶的主要产物是新琼脂糖（NA2），不需要任何中间体，这使其成为生产生物活性低聚糖的有吸引力的生物催化剂。这项研究强调了S.降解2-40作为工业生物技术应用的有价值的酶来源的潜力，特别是在将海洋生物质可持续转化为高价值产品方面。

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引用次数: 0

NADPH regeneration for efficient biosynthesis of indigo by flavin-containing monooxygenase and formate dehydrogenase 含黄素单加氧酶和甲酸脱氢酶对靛蓝高效生物合成的NADPH再生

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-08-05 DOI: 10.1016/j.enzmictec.2025.110731

Yingying Zhu , Dawei Ni , Zeyu Li , Zhebin Hao , Liang Wang , Wanmeng Mu

Indigo is an important blue pigment widely used in textile, food, and medicine industries. Biological production of indigo attracts increasing attention recently. Cell factory production of indigo encounters the problem of the toxicity of the precursor indole. Enzymatic production is the alternative biological approach, however, NADPH regeneration should be solved. In this study, flavin-containing monooxygenase from Methylophaga aminisulfidivorans was used for enzymatic production of indigo from indole and formate dehydrogenase from Pseudomonas sp. 101 was co-expressed for NADPH regeneration. Indigo production was enhanced by combination of molecular modification, promoter engineering, and translation initiation region engineering. Finally, 0.183 g/L of indigo was produced from 0.5 g/L of indole and 0.5 mM of sodium formate, with the conversion ratio of 32.5 %. This study demonstrates a feasible and effective strategy for enzymatic production of indigo.

靛蓝是一种重要的蓝色颜料，广泛用于纺织、食品、医药等行业。近年来，靛蓝的生物生产越来越受到人们的关注。细胞工厂生产靛蓝遇到的问题是前体吲哚的毒性。酶促生产是替代的生物途径，但NADPH再生应解决。本研究利用甲基噬菌（Methylophaga aminisulfidivorans）的含黄素单加氧酶从吲哚中产生靛蓝，并利用假单胞菌（Pseudomonas sp. 101）的甲酸脱氢酶进行NADPH再生。通过分子修饰、启动子工程和翻译起始区工程的结合，提高了靛蓝的产量。最后，以0.5 g/L吲哚和0.5 mM甲酸钠为原料，可制得0.183 g/L靛蓝，转化率为32.5 %。本研究证明了一种可行和有效的酶促生产靛蓝的策略。

引用次数: 0

Preparation of ginsenoside Rd using a novel α-L-arabinofuranosidase BpAbf51A from Bacillus pumilus 利用矮芽孢杆菌α- l -阿拉伯糖糠糖苷酶BpAbf51A制备人参皂苷Rd

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-08-04 DOI: 10.1016/j.enzmictec.2025.110729

Yuzhu Shen , Yue Yang , Yudi Song , Yakun Shan , Jiaxin Liu , Yanbo Hu

α-L-Arabinofuranosidase has been widely used in the fields of enhancing pasta production quality, fruit juice clarification, enhancing wine flavor, increasing feed utilization rate, and developing special drug ingredients, playing a pivotal role in the food processing, feed, and medical care industries. In this study, a novel α-L-arabinofuranosidase (BpAbf51A), belonging to glycoside hydrolase family 51 (GH51), was cloned from the Bacillus pumilus strain 145 and expressed in Escherichia coli BL21 (DE3), with a molecular weight of approximately 56.0 kDa. BpAbf51A comprises two characteristic domains of the GH51 family: an N-terminal (β/α)₈-barrel catalytic domain and a C-terminal jelly-roll domain. The enzyme exhibits high substrate specificity for p-nitrophenyl-α-L-arabinofuranoside and demonstrates optimal catalytic activity at 50°C and pH 8.0, suggesting its potential for industrial applications under moderate conditions. Notably, BpAbf51A specifically hydrolyzes the arabinofuranosyl moiety at the C-20 position of ginsenoside Rc to produce ginsenoside Rd. Molecular docking and two-dimensional interaction diagrams further revealed that the key amino acid residues, Ser213 and Asn214 of BpAbf51A, form strong hydrogen bonds with ginsenoside Rc. In this study, a novel α-L-arabinofuranosidase, BpAbf51A, has demonstrated significant potential for industrial applications in the production of rare saponins and other glycoside-based natural products, providing new research directions for the development of efficient biocatalysts.

α- l -阿拉伯糖葡糖苷酶广泛应用于提高面食品质、澄清果汁、增强葡萄酒风味、提高饲料利用率、开发特殊药物成分等领域，在食品加工、饲料、医疗保健等行业中发挥着举足轻重的作用。本研究从短小芽孢杆菌145菌株中克隆出一种新的α- l -阿拉伯糖葡糖苷酶（BpAbf51A），属于糖苷水解酶51家族（GH51），在大肠杆菌BL21 （DE3）中表达，分子量约为56.0 kDa。BpAbf51A包含GH51家族的两个特征结构域：n端（β/α）8桶催化结构域和c端果冻状结构域。该酶对对硝基苯-α- l-阿拉伯糖醛酸苷具有较高的底物特异性，在50°C和pH 8.0条件下具有最佳的催化活性，表明其在中等条件下具有工业应用潜力。值得注意的是，BpAbf51A特异性水解了人参皂苷Rc C-20位的阿拉伯糖糠基片段，生成人参皂苷Rd。分子对接和二维相互作用图进一步揭示了BpAbf51A的关键氨基酸残基Ser213和Asn214与人参皂苷Rc形成了强氢键。在本研究中，一种新型α- l -阿拉伯糖醛酸苷酶BpAbf51A在稀有皂苷和其他糖苷类天然产物的生产中显示出巨大的工业应用潜力，为高效生物催化剂的开发提供了新的研究方向。

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引用次数: 0

DNA box-assisted T7 transcription techniques combined with Cas13a for detection of the influenza A (H1N1) virus DNA盒辅助T7转录技术联合Cas13a检测甲型H1N1流感病毒

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-08-02 DOI: 10.1016/j.enzmictec.2025.110728

Mengyuan Zhou , He Sun , Shengjun Bu , Yao Xu , Hongyu Zhou , Ziqin Song , Zebin Zhang , Zhuo Hao , Songling Yu , Jiayu Wan , Feng Tang

The influenza A (H1N1) virus continues to undergo mutations, posing a serious threat to public health. In this study, an innovative system was developed using a transcriptional isothermal amplification scheme combined with a DNA box for detection of H1N1 RNA. The split T7 promoter was assembled on four edges of the hexahedral DNA box to form two target-capturing robotic arms. Transcribed ssRNA was accurately recognized by the Cas13a system and used the trans-cleavage activity to release a fluorescent signal. As compared to the traditional split T7 technique, the novel DNA box greatly improved the reaction rate and biological stability in complex organisms. The sensor platform strategy enabled stable and accurate detection of H1N1 RNA with detection limits as low as the fM level. In general, the proposed system provided a good sensing tool for virus diagnosis and monitoring with great potential in environmental and public health applications.

甲型H1N1流感病毒继续发生变异，对公众健康构成严重威胁。在这项研究中，我们开发了一种利用转录等温扩增方案结合DNA盒检测H1N1 RNA的创新系统。将分裂的T7启动子组装在六面体DNA盒的四个边缘上，形成两个捕获靶标的机械臂。转录后的ssRNA被Cas13a系统准确识别，并利用其反式裂解活性释放荧光信号。与传统的T7分裂技术相比，新型DNA盒在复杂生物中的反应速度和生物稳定性大大提高。该传感器平台策略能够稳定、准确地检测H1N1 RNA，检测限低至fM水平。总体而言，该系统为病毒诊断和监测提供了良好的传感工具，在环境和公共卫生领域具有很大的应用潜力。

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引用次数: 0

Cell surface display of CAL-B in Escherichia coli using the split GFP system 利用分裂式GFP系统显示CAL-B在大肠杆菌中的细胞表面

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-07-29 DOI: 10.1016/j.enzmictec.2025.110726

Jisoo Lee, Jong-In Won

Bacterial surface display systems enable the immobilization of proteins on the outer membrane for applications such as peptide library screening, biosensing, and bioadsorption. However, the display efficiency of large proteins remains limited, primarily due to challenges in translocating bulky polypeptides across the membrane. To address this, a split Green Fluorescent Protein (split GFP)-based strategy was evaluated. This method employs two non-fluorescent GFP fragments—GFP11 and GFP1–10—that reassemble into a fluorescent complex when co-localized. In this study, the small GFP fragment (GFP11) was genetically fused to Lipoprotein-Outer Membrane Protein A (Lpp-OmpA) to promote membrane anchoring, while the large GFP fragment (GFP1–10) was fused to Candida antarctica lipase B (CAL-B). The CAL-B-GFP1–10 fusion protein was expressed separately and then incubated with cells displaying Lpp-OmpA-GFP11, facilitating potential reassembly on the cell surface. Restoration of fluorescence served as an indirect indicator of successful surface localization. Enzymatic assays were also performed to compare the activity of CAL-B displayed via the split GFP system versus conventional direct fusion to Lpp-OmpA. The results demonstrated that the split GFP approach can enhance surface display and preserve enzymatic function, offering a promising alternative for displaying large or structurally complex proteins. While further optimization is needed, these findings support the potential of split GFP-assisted strategies in expanding the scope of bacterial surface display applications.

细菌表面展示系统使蛋白质固定在外膜上的应用，如肽库筛选，生物传感和生物吸附。然而，大蛋白质的显示效率仍然有限，主要是由于在跨膜转运大体积多肽的挑战。为了解决这个问题，我们评估了一种基于绿色荧光蛋白（GFP）的分裂策略。该方法使用两个非荧光GFP片段gfp11和gfp1 - 10，它们在共定位时重新组装成荧光复合物。在本研究中，小片段GFP （GFP11）与脂蛋白外膜蛋白A （lp - ompa）融合以促进膜锚定，而大片段GFP （GFP1-10）与南极念珠菌脂肪酶B （CAL-B）融合。CAL-B-GFP1-10融合蛋白单独表达，然后与显示lp - ompa - gfp11的细胞孵育，促进细胞表面的潜在重组。荧光的恢复是表面成功定位的间接指标。还进行了酶分析，比较通过分裂GFP系统显示的CAL-B的活性与传统的直接融合到lp - ompa。结果表明，分裂GFP方法可以增强表面显示并保持酶功能，为显示大型或结构复杂的蛋白质提供了有希望的替代方法。虽然需要进一步优化，但这些发现支持了分裂gfp辅助策略在扩大细菌表面显示应用范围方面的潜力。

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引用次数: 0

Application of natively expressed chitinase as a sustainable fungal bioshield 几丁质酶在真菌生物防护中的应用

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-07-29 DOI: 10.1016/j.enzmictec.2025.110727

Farah Deeba , Tarek E. Mazeed , Davita L. Watkins , Chad A. Rappleye , David W. Wood

Postharvest losses of fruits and vegetables due to fungal spoilage pose a significant challenge, compounded by growing consumer concerns about the harmful effects of chemical fungicides and the limited effectiveness of traditional food coatings (such as wax and biopolymer formulations) in controlling microorganisms, particularly fungi. In this study, we developed a formulation-free method using a non-modified chitinase from a thermostable strain of Bacillus cereus isolated from soil. The purified chitinase inhibited in vitro mycelium growth of four food-pathogenic fungi: Penicillium digitatum, Neurospora crassa, Aspergillus fumigatus, and Alternaria alternata. Furthermore, when applied directly on strawberry and onion tissue, the purified chitinase inhibited Aspergillus fumigatus and A. niger colonization of the produce. This demonstrates the addition of chitinase provides a cost-effective and non-toxic alternative as a bio-active food coating material to prevent fungal fruit and vegetable spoilage and extend food shelf life.

由于真菌腐败导致的水果和蔬菜采后损失构成了重大挑战，而消费者对化学杀菌剂有害影响的担忧日益增加，传统食品涂层（如蜡和生物聚合物配方）在控制微生物，特别是真菌方面的有效性有限，这使情况更加复杂。在这项研究中，我们开发了一种无配方的方法，使用从土壤中分离的耐热蜡样芽孢杆菌菌株中提取的未经修饰的几丁质酶。纯化的几丁质酶可抑制四种食源性真菌：指状青霉、粗神经孢子菌、烟曲霉和互交霉的体外菌丝体生长。此外，当直接施用于草莓和洋葱组织时，纯化的几丁质酶可以抑制烟曲霉和黑曲霉的定植。这表明添加几丁质酶作为一种具有生物活性的食品涂层材料提供了一种具有成本效益和无毒的替代品，可以防止真菌水果和蔬菜变质，延长食品保质期。

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