MICROCHEMICAL JOURNAL
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2025
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IF 5.1
Mengjie Lin,Wenxin Chen,Cunshan Zhang,Jiewei He,Lina Wu,Ailin Liu,Xinhua Lin,Liqing Lin
Specific-Species enzyme detection serves as a crucial method in foodborne pathogen surveillance. Particularly, endogenous β-galactosidase (β-Gal) enzyme has been widely applied to determine the concentration of Escherichia coli ( E. coli ). The main challenges include reducing analysis time and improving detection sensitivity. A novel strategy for biosensing β-Gal activity and E. coli are developed based on BSA‑manganese dioxide nanosheets (BSA-MnO 2 NS) with the oxidase-mimicking activity to regulate the fluorescence of thiamine. BSA-MnO 2 NS can catalyze the oxidation of non-fluorescent thiamine to generate strong fluorescent dehydrothiamine. When β-Gal enzymes is introduced, p -aminophenyl-β-D-galactopyranoside (PAPG) could be hydrolyzed to form para-aminophenol (PAG), which efficiently reduced BSA-MnO 2 NS into Mn 2+ , resulting in the loss of their oxidase-like activity and the decrease of dehydrothiamine fluorescence. Since endogenous β-Gal in E. coli cells can directly hydrolyze PAPG without destroying bacteria, which greatly reduced the analysis time. The reduction product PAG leads to MnO 2 NS decomposition and thus reduced dehydrothiamine fluorescence. According to this mechanism, fluorescence biosensor for β-Gal activity and E. coli are constructed utilizing thiamine luminescence modulated by BSA-MnO 2 NS, without requiring an additional oxidizer. β-Gal and E. coli showed good linearity in the range of 0.01–2.0 μg/mL and 1.0 × 10 2 –1.0 × 10 4 CFU/mL, and the detection limits reached 8.55 ng/mL and 89.45 CFU/mL, respectively. The proposed enzyme-nanozyme cascade reaction significantly improved the sensitivity. It is highly specific and can be used to distinguish E. coli from other pathogenic bacteria. The strategy has also been successfully applied to the detection of E. coli in real samples.
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ACS Applied Materials & Interfaces
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2025
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IF 8.2
Yu Liu,Shiyue Bi,Zhanxin Song,Ziyi Song,Chao Xu,Mo Xian,Miaomiao Jin
Metal–enzyme cascade catalysis effectively combines the broad reactivity of chemical catalysis with the high selectivity of biocatalysis, improving reaction efficiency and simplifying the process flow through multiple sequential reactions in the same system. The introduction of exogenous palladium nanoparticles (Pd NPs) into Escherichia coli (E. coli) cells can significantly broaden the range of catalytic reactions facilitated by biological enzymes. Additionally, the targeted cytoplasmic synthesis of Pd NPs enhances their utilization efficiency in intracellular catalytic reactions while also eliminating the need for separating and purifying metals and enzymes. However, current methods largely enable the intracellular synthesis of Pd NPs in the periplasmic space and outer membrane. Moreover, the hydrogen sources commonly used in these methods─such as hydrogen (H2) and sodium borohydride (NaBH4)─carry safety risks. In this study, the mechanism of targeted synthesis of Pd NPs on the cytoplasmic side and its process were deeply investigated using a mild hydrogen source, sodium formate, in combination with genetic engineering and preparation conditions. And the constructed functional cell (Pd@E. coli) could catalyze benzaldehyde hydrogenation, with a conversion rate of 41.41% and benzyl alcohol yield of 17.68%, demonstrating considerable catalytic and loading stability. This study provides a reference for constructing catalytic systems for intracellular metal–enzyme cascades. Thus, it could bolster the development opportunities in the areas of non-natural products and drug development and provide ideas for addressing the drawbacks of existing biosynthetic technologies.
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APPLIED BIOCHEMISTRY AND MICROBIOLOGY
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2024
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IF 1
Zhu Y. B.,Zhang W.,Fan Y. L.,Chen Y. Y.,Zhu J. L.,Lu H. X.
Abstract Two immunochromatographic assays based on hematite (α-Fe 2 O 3 ) nanoparticles were developed for rapid detection of aflatoxin M1 (AFM1) and Escherichia coli O157:H7 in the milk. Hematite nanoparticles were prepared and utilized as colored signal labels. The competitive format was applied for AFM1 detection, while the sandwich format for E. coli O157:H7 determination. Under optimal conditions, the assay exhibited a visual detection limit of 0.10 ng/mL and quantitative determination limit of 0.017 ng/mL for AFM1. For E. coli O157:H7, a visual detection limit of 2.9 × 10 5 CFU/mL was achieved. The entire determination process could be accomplished within 15 min. Compared with traditional nanolabels, hematite nanoparticles showed advantages of facile preparation, high stability and easy bioconjugation. The developed assays provided effective and rapid screening strategies for AFM1 and E. coli O157:H7 in milk samples with high sensitivity and specificity.
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Journal of Biological Engineering
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2026
·Vol. 20
·期 1
·29
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IF 6.5
Ye Changchuan,Zhang Yan,Zhang Jie,Shi Menglei,Nie Feixue,Liu Qinghua
Background Vitamin K 2 (VK 2 ), as a derivative of the menaquinone family, plays an important role in the prevention of osteoporosis and cardiovascular calcification. The realization of the industrialization of VK 2 and the reduction of its production cost have become the focus of attention. Results In this work, an E. coli strain with high VK 2 accumulation was constructed through rational metabolic engineering and stepwise improvement based on regulatory metabolic information and CRISPR/Cas9-mediated gene knockout. We first constructed a recombinant E. coli strain BW-T7/MU to produce menaquinol-8 (MKH 2 -8, a reduced form of VK 2 ) by overexpressing menA and ubiE genes, which encoding the rate-limiting enzymes of the menaquinol pathway. After 24 h and 48 h of fermentation, this strain BW-T7/MU reach a titer of 303 mg/L and 232 mg/L. Secondly, we overexpressed different related genes wrbA (oxidative stress mitigation), qorB (reduction of quinones) and menF (conversion of chorismate to isochorismate), respectively. Among these recombinant strains, the strain BW-T7/MUW (overexpressing menA , ubiE and wrbA genes) reached the highest titer of VK 2 after 48 h of fermentation. The optimization of the medium led to an increase in the accumulation of VK 2 . Subsequently, the rational metabolic engineering of gene knockout further increased the titer of VK 2 . The recombinant strain ΔB/MUW was selected as the dominant strain for further optimization, with a high VK 2 titer of 724 mg/L. A final attempt is to overexpress ispB gene to increased flux of isoprenoid side chain synthesis, resulting in strain ΔB/MUWI with a titer of 859 mg/L in a shake flask and 1360 mg/L in a 5 L fermenter after 48 h cultivation. Conclusions The stepwise engineering strategy raised the VK 2 titer from the initial 303 mg/L to 859 mg/L through rational pathway modification and systematic gene expression. Further optimization in batch fermentation increased the VK 2 titer to 1360 mg/L, which highlights the strong engineering impact of our strategy.
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Frontiers in Immunology
·
2026
·
IF 7
Bingliang Zhou,Xiangshun Meng,Zhengying Yu,Bao Zhang,Wei Zhao,Lei Wang,Liang Peng,Jinhu Zou,Jingyu Chen,Xu Lin,Xuefeng Gao,Sheng-He Huang,Hong Cao
IntroductionIbeA is a critical virulence factor that is present in the GimA island in Escherichia coli (E. coli) K1, the most common cause of Gram-negative bacterial meningitis in newborns. IbeA has multiple virulence functions, including adhesion, invasion, and biofilm formation in the pathogenesis of E. coli K1 meningitis. However, it is unclear how IbeA coordinates its role in bacterial adhesion, invasion, and biofilm formation during crossing the blood-brain barrier (BBB).MethodsAn isogenic ibeA deletion mutant (ZD1) was generated from the neonatal meningitis–associated E. coli K1 strain E44. To define IbeA-mediated virulence, bacterial adhesion, invasion, and biofilm formation were quantified in human brain microvascular endothelial cells (HBMECs) using TEER, confocal imaging, and crystal violet assays. A neonatal rat meningitis model evaluated bacterial dissemination, BBB disruption, neuroinflammation, and neurological deficits. Mechanistic studies focused on Vimentin (VIM) dynamics using lipid-raft fractionation, cytoskeleton extraction, immunofluorescence, vimentin-knockout (Vim-KO) HBMEC and Co-immunoprecipitation to assess its redistribution, post-translational modifications, and interaction with NF-κB. The functional involvement of the IbeA–VIM–NF-κB signaling pathway was further examined by modulating VIM activity with glatiramer acetate (GA) in both in vitro and in vivo experiments.ResultsDeletion of ibeA and VIM significantly impaired bacterial adhesion, invasion, and biofilm formation both on HBMECs and on abiotic surfaces. In neonatal rats, infection with ZD1 resulted in higher survival rates, milder neurological symptoms, and a marked reduction of biofilm structures within the cerebral microvasculature compared with infection by the wild-type strain. IbeA-triggered post-translational modifications and cytoplasmic mobilization of VIM drive its nuclear translocation, which in turn activates NF-κB signaling. Pharmacological inhibition of VIM with GA disrupted IbeA-mediated virulence, lowering bacterial loads in the blood and alleviating neurological injury.ConclusionsIbeA is a key virulence factor that coordinates E. coli K1 adhesion, invasion, and biofilm formation by activating the VIM–NF-κB signaling pathway. Our study establishes for the first time a direct mechanistic link between IbeA’s multiple virulence functions through the above signaling pathway leading to acute BBB disruption and meningitis and identify GA as a potential drug development candidate for this disease.
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PROCESS BIOCHEMISTRY
·
2023
·
IF 4.4
Neng Xiong,Zi-Qi Li,Rui Xu,Kai-Qi Guo,Yu-Xiao Liu,Ya-Ping Xue,Yu-Guo Zheng
Surface-crosslinked hydrogel particles encapsulating E. coli cells harboring nitrilase were constructed and used as a durable biocatalyst in the synthesis of gabapentin intermediate. E. coli cells (50 g/L) expressing nitrilase were encapsulated in 2% (w/v) alginate hydrogel, 0.5%(w/v) glutaraldehyde and 1%(w/v) polyethyleneimine were sequentially used as crosslinker. The constructed alginate particles with GA&PEI composite crosslinking had a similar catalytic capacity compared to equivalent free cells, which could completely hydrolyze 1.0 M substrate 1-cyanocyclohexaneacetonitrile within 4 h at pH 7.5 and 35 °C, and have a specific activity of 134.6 U/g, an optimum temperature at 55 °C and a corresponding half-life up to 19.1 ± 1.9 h. The particles showed excellent storage stability, reusability and mechanical strength, which retained 90.3% activity after 2-month storage at 4 °C, and 83.2% activity after 14-day storage at 35 °C. In batch reactions, 50 g/L particles could be reused for maximum 32 cycles, maintained 63.5% residual activity and 50.7% particle integrity, providing an average product yield of 94.6% in the first 30 batches and a productivity of 1467.03 g/L/day. Besides, the stability and catalytic activity of the particles were almost unaffected in several organic co-solvents and in purified water, showing their good industrial potential.
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Pathogens
·
2022
·Vol. 11
·期 9
·1020
·
IF 4.531
Julio Abel Alfredo dos Santos Simone Come,Yibin Zhuang,Tianzhen Li,Simone Brogi,Sandra Gemma,Tao Liu,Edson Roberto da Silva
Arginase is a metalloenzyme that plays a central role inLeishmaniainfections. Previously, rosmarinic and caffeic acids were described as antileishmanial agents and asLeishmania amazonensisarginase inhibitors. Here, we describe the inhibition of arginase inL. amazonensisby rosmarinic acid analogs (1–7) and new caffeic acid-derived amides (8–10). Caffeic acid esters and amides were produced by means of an engineered synthesis inE. coliand tested againstL. amazonensisarginase. New amides (8–10) were biosynthesized inE. colicultured with 2 mM of different combinations of feeding substrates. The most potent arginase inhibitors showed Ki(s) ranging from 2 to 5.7 μM. Compounds2–4and7inhibitedL. amazonensisarginase (L-ARG) through a noncompetitive mechanism whilst compound9showed a competitive inhibition. By applying an in silico protocol, we determined the binding mode of compound9. The competitive inhibitor of L-ARG targeted the key residues within the binding site of the enzyme, establishing a metal coordination bond with the metal ions and a series of hydrophobic and polar contacts supporting its micromolar inhibition of L-ARG. These results highlight that dihydroxycinnamic-derived compounds can be used as the basis for developing new drugs using a powerful tool based on the biosynthesis of arginase inhibitors.Keywords:arginase;Leishmania amazonensis;biosynthesis;natural products;molecular modeling
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ENVIRONMENTAL POLLUTION
·
2022
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IF 9.988
Long Li,Haoran Dong,Yue Lu,Haoxuan Zhang,Yangju Li,Junyang Xiao,Shuangjie Xiao,Zilan Jin
The bacteria toxicity of nanoscale zero-valent iron (nZVI) can be changed during its application in water treatment but the toxicity mechanism is still not well understood, particularly under anaerobic conditions . Here, the toxicity of nZVI and its aging products towards Escherichia coli ( E. coli ) and the mechanisms of extracellular and intracellular reactive oxygen species (ROS) damage were deeply probed in the presence and absence of oxygen in ultrapure water. Under aerobic conditions , the ROS damage primarily caused by the generation of extracellular free •OH can be a major contributor to the toxicity of nZVI to E. coli . By contrast, in anaerobic nZVI treatment system, the intracellular •OH can be quenched by benzoic acid which is a cell permeable quencher and the electron spin resonance (ESR) signals of 5,5-dimethy-1-pyrroline (DMPO)- •OH were evidently observed in system with the addition of F − which could desorb the surface •OH into solution. It indicated that the intracellular •OH adsorbed on the particle surface can also play an indispensable role in inactivating cells under anaerobic conditions. Moreover, nZVI can steeply decline the membrane potential, causing severe membrane disruption and therefore resulting in the stronger toxicity in anaerobic conditions. Furthermore, the chemical composition transformation of nZVI and generation of benign iron corrosion products (e.g., Fe 3 O 4 , γ-Fe 2 O 3 , γ-FeOOH) are mainly responsible for the reduced toxicity with the increasing aging time. These results provide insights into the extracellular and intracellular ROS damage occurred in aerobic and anaerobic nZVI treatment systems, offering more perspective to the risk assessment of nZVI application.
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INTERNATIONAL DAIRY JOURNAL
·
2022
·
IF 3.572
Yang Song,Lihan Wang,Jiayuan Zhao,Hongxuan Li,Xinyan Yang,Shiqian Fu,Xue Qin,Qing Chen,Yujun Jiang,Chaoxin Man
Escherichia coli O157:H7 is a pathogenic foodborne bacterium of public health concern. A simple, rapid, and environmentally friendly method was developed to synthesise papain-modified gold nanoclusters ( [email protected] ). Following this, aptamers-modified [email protected] ( [email protected] @AuNCs) were constructed by reacting Au–S covalent bonds between thiolated aptamers and the [email protected] , enhancing the peroxidase activity. E. coli O157:H7 could be captured through the [email protected] @AuNCs, significantly improving the catalytic activity toward the peroxidase substrate 3,3′,5,5′-tetramethylbenzidine, and the blue products could be generated. Papain was used for the first time as a template for the synthesis of AuNCs of peroxidase-like activity for detection. The quantitative detection for E. coli O157:H7 was 39 cfu mL −1 in pure culture. The detection limit of E. coli O157:H7 was 5.6 × 10 2 , 5 × 10 2 and 4.9 × 10 2 cfu mL −1 in ultra-high temperature sterilised, pasteurised, and raw milk samples, respectively, after 2 h pre-incubation, indicating that the developed sensor has potential for pathogen detection in foods.
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Journal of Environmental Chemical Engineering
·
2022
·
IF 7.968
Jinxiu Li,Jinxin Zhao,Bing Li,Huaitian Bu,Weizhao Yin,Sihao Lv,Jinhua Wu
Activated carbon-supported goethite (α-FeOOH@AC) was prepared by a simple impregnation method to catalyze hydrogen peroxide (H 2 O 2 ) for effective inactivation of Escherichia coli ( E. coli ) with α-FeOOH and AC as controls. As expected, α-FeOOH particles were uniformly distributed on AC surfaces and surface-bound Fe(III) was partially converted to Fe(II) by structural hydroxyl groups of AC. The resultant Fe(II) catalyzed H 2 O 2 to generate hydroxyl radicals (·OH) for effective E. coli inactivation by destroying its membrane and intracellular substances. The α-FeOOH@AC-H 2 O 2 system therefore achieved an effective inactivation efficiency of 4.5 log, which was much higher than 1.6 log of the α-FeOOH-H 2 O 2 system and 1.9 log of the AC-H 2 O 2 system. Besides, due to the cycle of Fe(III)/Fe(II) redox on AC surface during reaction, high inactivation efficiency and low leaching iron concentration of 0.29 mg/L could be achieved at neutral condition. In addition, the α-FeOOH@AC showed excellent stability and reusability, and the used α-FeOOH@AC could be refreshed by eluting surface-bound iron oxides and reloading α-FeOOH particles with the above impregnation method. Our study demonstrated that α-FeOOH@AC is an effective and stable heterogeneous Fenton catalyst for the inactivation of E. coli in surface water treatment .
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FOOD CHEMISTRY
·
2023
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IF 8.8
Xiu Wang,Wei Li,Shijie Dai,Menghua Dou,Saisai Jiao,Jing Yang,Weiwei Li,Ya Su,Qianjin Li,Jianlin Li
The aim of this research was to develop a simple, rapid, sensitive, high-throughput detection method for foodborne Escherichia coli (E. coli) O157:H7 based on the aptamer-modified gold nanoparticles@macroporous magnetic silica photonic microsphere (Au@MMSPM). Such Au@MMSPM array system for E. coli O157:H7 not only integrated sample pretreatment with rapid detection, but also showed highly enhanced effect to develop a highly sensitive SERS assay. The established SERS assay platform gave a wide linear detection range (10–10 6 CFU/mL) and low limit of detection (2.20 CFU/mL) for E. coli O157:H7. The whole analysis time including sample pretreatment and detection was 110 min. This SERS-based assay platform provided a new high-throughput, highly sensitive and fast detection technology for monitoring E. coli O157:H7 in real samples from the fields of food industry, medicine and environment.
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INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2023
·
IF 8.2
Meng Shi,Demin Kong,Hui Zhang,Deming Rao,Tianlong Zhao,Jing Yang,Zhanzhi Liu,Sheng Chen,Fengshan Zhang,Jing Wu,Lei Wang
Latex clearing protein from Streptomyces sp. strain K30 (Lcp K30 ) is a natural oxidoreductase that can catalyse the cleavage of rubber through dioxygenation. It has significant potential applications in polymer degradation. However, its limited expression in engineered strains restricts its utility. This study aimed to enhance the soluble expression and enzyme activity of Lcp K30 in E. coli BL21 (DE3) by optimizing fermentation conditions and making molecular modifications. The enzyme activity reached 5.05 U·mL −1 by optimizing the induction conditions, adding cofactors, and using chemical chaperones , which was 237.1 % of the initial case. Further enhancements in soluble expression were achieved through site mutations guided by the PROSS server, resulting in 8 out of 13 mutants with increased protein expression , a high positive mutation rate of 61.5 %. Subsequently, combined mutants were created by merging single mutants with enhanced protein expression and enzyme activity. The top three double mutants, G91D/S149A, G91D/A210H, and G91D/H296P, displayed expression levels at 173.3 %, 173.3 %, and 153.3 % of the wild-type Lcp K30 , respectively. These mutants also exhibited enhanced fermentation enzyme activity, reaching 149.5 %, 250.0 %, and 420.2 % compared to the wild-type, along with improved specific activities. This study provides insights for the efficient production of Lcp K30 and a practical foundation for its application.
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