BIOTECHNOLOGY AND BIOPROCESS ENGINEERING
·
2013
·Vol. 17
·期 6
·1128-1134
·
IF 1.277
Li Bin,Wang Lei,Su Lingqia,Chen Sheng,Li Zhaofeng,Chen Jian,Wu Jing
OmpA signal peptide mediated cgt gene from Paenibacillus macerans JFB05-01 was cloned and expressed in E. coli BL21 (DE3). The effects of glycine and Triton X-100 on extracellular production of α-cyclodextrin glycosyltransferase (α-CGTase) were investigated. When supplemented with Gly or Triton X-100 to the culture media individually, the secreted extracellular enzyme reached 32 or 33 U/mL at 48 h of cultivation, respectively. When supplemented with Gly and Triton X-100 together, the extracellular α-CGTase activity reached 48 U/mL after 48 h cultivation, which was 20-fold of the control group without any additives. Analysis of membrane permeability demonstrated that addition of glycine and Triton X-100 enhanced the permeability of both outer and inner membrane. The potential mechanism of the enhanced protein secretion was discussed.
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BIOSENSORS & BIOELECTRONICS
·
2026
·
IF 10.5
Qi Feng,Jiaying Wang,Zileng Yu,Zhipeng Zhang,Huizi Zheng,Jiao Qu,Xiaolin Zhu
To address the growing environmental risks associated with emerging contaminants and limited rapid toxicity assessment tools, we developed a mediator-free microbial electrochemical biosensor using Escherichia coli nucleotide metabolism. This biosensor enables the direct detection of electroactive metabolites without exogenous mediators by utilizing the oxidation peak current as a sensitive toxicity indicator. An electrode was modified with a nanocomposite of carboxylated multi-walled carbon nanotubes and zinc oxide (MWCNT-COOH/ZnO), which significantly enhanced the electrochemical signals. Mechanistic studies revealed that the detection signal originated from the oxidation of guanine and xanthine. The biosensor was used to detect four bisphenol analogues and two per-/polyfluoroalkyl substances. The toxicity followed the order of bisphenol AF > bisphenol A > bisphenol F > bisphenol S, suggesting that bisphenol AF posed a higher toxicity risk than bisphenol A. The half-maximal inhibitory concentration (IC 50 ) for perfluorooctanesulfonic acid was 1011.13 μM, and its substitute, 6:2 fluorotelomer sulfonic acid, exhibited significantly lower toxicity at equivalent concentrations. The biosensor was further applied to actual water samples from the Yitong River, revealing generally low toxicity (inhibition rate <30 %) with higher inhibition rates observed at densely populated urban sites. This biosensor is characterized by simple operation, fast response, low cost, and independence from water turbidity or color, thereby offering an efficient and sensitive analytical platform for both toxicity assessment of emerging contaminants and safety evaluation of real water samples.
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ENVIRONMENTAL POLLUTION
·
2023
·
IF 8.9
Shuxin Li,Wenjing Zhang,Dayi Zhang,Wei Xiu,Shengyu Wu,Juanfen Chai,Jincai Ma,Muhammad Yousuf Jat Baloch,Simiao Sun,Yuesuo Yang
The vadose zone is a critical zone for microbial entry into the subsurface environment, and various types of inorganic and organic colloids can affect the migration of pathogenic bacteria. In the study, we explored the migration behavior of Escherichia coli O157:H7 with humic acids (HA), iron oxides (Fe 2 O 3 ) or their mixture, uncovering their migration mechanisms in the vadose zone. The effect of complex colloids on the physiological properties of E. coli O157:H7 was analyzed based on the measured particle size, zeta potential and contact angle. HA colloids significantly promoted the migration of E. coli O157:H7, where Fe 2 O 3 was opposite. The migration mechanism of E. coli O157:H7 with HA and Fe 2 O 3 is obviously different. Multiple colloids dominated by organic colloid will further highlight its promoting effect on E. coli O157:H7 under the guidance of electrostatic repulsion due to the influence of colloidal stability. Multiple colloids dominated by metallic colloid will inhibit the migration of E. coli O157:H7 under the control of capillary force due to the restriction of contact angle. The risk of secondary release of E. coli O157:H7 can be effectively reduced when the ratio of HA/Fe 2 O 3 is ≥ 1. Combining this conclusion with the distribution characteristics of soil in China, an attempt was made to analyse the migration risk of E. coli O157:H7 on a national scale. In China, from north to south, the migration capacity of E. coli O157:H7 gradually decreased, and the risk of secondary release gradually increased. These results provide ideas for the subsequent study of the effect of other factors on the migration of pathogenic bacteria on a national scale and provide risk information about soil colloids for the construction of pathogen risk assessment model under comprehensive conditions in the future.
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Analytical Methods
·
2023
·Vol. 16
·期 4
·496-502
·
IF 3.1
Hangli Yan,Linghao Wu,Jingyu Wang,Yi Zheng,Fengxia Zhao,Qinqin Bai,Hongmei Hu,Hao Liang,Xiangheng Niu
The detection of foodborne pathogens is crucial for food hygiene regulation and disease diagnosis. Colorimetry has become one of the main analytical methods in studying foodborne pathogens due to its advantages of visualization, low cost, simple operation, and no complex instrument. However, the low sensitivity limits its applications in early identification and on-site detection for trace analytes. In order to overcome such a limitation, herein we propose a joint strategy featuring dual signal amplification based on the hybridization chain reaction (HCR) and DNA-enhanced peroxidase-like activity of gold nanoparticles (AuNPs) for the sensitive visual detection of Escherichia coli. Target bacteria bound specifically to the aptamer domain in the capture hairpin probe, exposing the trigger domain for HCR and forming the extended double-stranded DNA (dsDNA) structures. The peroxidase-like catalytic capacity of AuNPs can be enhanced significantly by dsDNAs with the sticky ends of dsDNAs being adsorbed on AuNPs and the rigidity of dsDNAs causing the spatial regulation of AuNP concentration. The intensity of the enhancement was linearly related to the number of target bacteria. With the above strategy, the detection limit of our colorimetric method for Escherichia coli was down to 28 CFU mL−1 within a short analytical time (50 min). This study provides a new perspective for the sensitive and visual detection of early bacterial contamination in foods.
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LWT-FOOD SCIENCE AND TECHNOLOGY
·
2023
·
IF 6
Junyan Liu,Tengyi Huang,Riyi Xu,Zhufang Xiang,Thanapop Soteyome,Xiangyi Chen,Qianfei Zhang,Qiurong Huang,Zixu Wu,Yunyi Huang,Gongliang Liu,Chunyun Qu,Gamini Seneviratne,Birthe V. Kjellerup,Zhenbo Xu
Escherichia coli O157:H7 is a major foodborne pathogen causing food poisoning. Standard detection method for foodborne E. coli is plate counting, which is time consuming and omits VBNC cells. PSR is an isothermal amplification technique identifying trace amount DNA. However, its effectiveness is influenced by residual DNA from dead cells. This study aimed at designing a PMA-PSR system for accurate determination of viable cells of E. coli O157:H7. Firstly, PSR was optimized in fluorescence indicator, betaine concentration and reaction time. The optimized system could successfully identify rfbE, stx1 and stx2 genes within 45 min at 65 °C. Secondly, sensitivity and specificity were tested in pure culture and different food samples. With 100% specificity, PSR could determine DNA amount as low as 1.12 pg/μL. Thirdly, a PMA-PSR system was designed for accurate determination of viable cells. Cells were treated with 5 μg/mL PMA to remove residual DNA from dead cells. Pretreated cells were adapted to PSR, thus viable cells were specifically detected. Fourthly, the system was applied in food samples with VBNC cells. Viable cells could be accurately determined in accordance with regular assay. In conclusion, the PMA-PSR system was an efficient tool for accurate determination of E. coli O157:H7 viable cells.
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Journal of Global Antimicrobial Resistance
·
2022
·
IF 4.349
Wenting Zhang,Tengfei Zhang,Chen Wang,Guixing Liang,Qin Lu,Guoyuan Wen,Yunqing Guo,Yiluo Cheng,Zui Wang,Huabin Shao,Qingping Luo
ABSTRACT Objectives This study investigated the prevalence and characteristics of mcr-1- harbouring Escherichia coli isolated from chickens in central China from 2014 to 2019. Methods A total of 1132 E. coli isolated from 1647 chicken swabs were analysed for colistin susceptibility by broth microdilution method and prevalence of mcr-1 gene by PCR. The colistin-resistant E. coli isolates were typed by multi-locus sequence typing (MLST) and tested with 12 antimicrobial agents. The transconjugation assay was conducted for the mcr-1 -positive isolates using the transconjugant E. coli C600. Results Of the 1132 E. coli isolated from chickens, 131 isolates (11.6%) exhibited colistin resistance, and 51 isolates (4.5%) were mcr-1 positive. The mcr-1 -positive rate was quite low in 2014 (2.3%) and 2015 (1.7%), increased to peak in 2016 (12.6%) and 2017 (11.4%), and then decreased significantly in 2018 (1.7%) and 2019 (0.9%). The 131 colistin resistant isolates were assigned to 66 unique sequence types (STs), 27 of which contained mcr-1 -positive isolates. Compared with mcr-1 -negative E. coli, mcr-1 -positive E. coli showed higher resistance rates to nalidixic acid, ciprofloxacin, ceftriaxone, cefotaxime, and tetracycline. Furthermore, 30 of the 51 mcr-1 positive isolates transduced their mcr-1 gene into E. coli C600, and 13 of the 30 transconjugants carried more than one replicon types. Conclusion The mcr-1 positive rate varied enormously during 2014–2019 in central China. The ban on colistin likely decreased the dissemination of mcr-1 in E. coli isolates from chickens. Multidrug-resistant trait is observed in mcr-1 positive E. coli isolates and can be transferred into other transconjugants.
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Jove-Journal of Visualized Experiments
·
2023
·
IF 1.2
Wenwen Dong,Chunxian Tao,Bo Yang,Erika Miyake,Zhenqing Li,Dawei Zhang,Yoshinori Yamaguchi
Polymerase chain reaction (PCR) is a traditional method employed for the amplification of a target gene that has played an important role in biomolecular diagnostics. However, traditional PCR is very time-consuming because of the low-temperature variation efficiency. This work proposes a continuous-flow-PCR (CF-PCR) system based on a microfluidic chip. The amplification time can be greatly reduced by running the PCR solution into a microchannel placed on heaters set at different temperatures. Moreover, as capillary electrophoresis (CE) is an ideal way to differentiate positive and false-positive PCR products, a CE system was built to achieve efficient separation of the DNA fragments. This paper describes the process of amplification of Escherichia coli (E. coli) by the CF-PCR system built in-house and the detection of the PCR products by CE. The results demonstrate that the target gene of E. coli was successfully amplified within 10 min, indicating that these two systems can be used for the rapid amplification and detection of nucleic acids.
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FOOD CHEMISTRY
·
2023
·
IF 8.8
Xiaoqing Wang,Huiyi Chen,Jihui Zhang,Hong Zhou,Xiangying Meng,Na Wang,Yishan Fang,Bo Cui
The detection of Escherichia coli ( E. coli ) is of great significance for the environment and human health. Herein, a photoelectrochemical (PEC) detection strategy based on molecularly imprinted polymers (MIPs) was proposed for the sensitive detection of E. coli . 4,4′,4″-Trinitrotriphenylamine (TPA-NO 2 ) was prepared using a simple nitration reaction. Subsequently, MIP films were polymerized on the surface of TPA-NO 2 using 1,3-dihydrothieno[3,2- d ]pyrimidine-2,4-dione as the functional monomer with the dual functions of specific recognition and sensitization. The linear range was 10–10 8 CFU/mL and the limit of detection was 10 CFU/mL. It showed favorable recoveries in real sample tests of milk, orange juice and tomato. Additionally, the ability of functional monomers to bind excellently with E. coli was verified using molecular docking techniques. This research provided broader possibilities for constructing MIPs-PEC sensors and analyzing the interaction mechanism between E. coli and functional monomers.
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FOOD CONTROL
·
2019
·
IF 4.248
Yulin Zhu,Changzhu Li,Haiying Cui,Lin Lin
As a non-thermal sterilization method, pulsed light (PL) has been widely applied in food industries to control the microbial contamination, however little is known about the antimicrobial mechanism of PL. This study deeply investigated the antimicrobial mechanism of PL against Escherichia coli O157:H7 ( E. coli O157:H7). The changes of morphology, endoenzyme, entocyte, respiratory metabolism and DNA of E. coli O157:H7 after PL treatment were emphatically studied. TEM and SEM results exhibited that cell integrity was destroyed heavily after PL treatment. Entocyte including proteins, ATP and DNA were reduced remarkably for PL-treated samples. In addition, the activity of ATPase, β-galactosidase, alkaline phosphatase, topoisomerase, as well as the metabolism were inhibited for E. coli O157:H7 with PL treatment. The electron spin resonance spectrum validated the production of hydroxyl radical by PL, thus resulting in the elevation of intracellular reactive oxygen and the acceleration of cell death. Finally, PL displayed better antibacterial activity in carrot juice without adverse effect on its quality.
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BIOPROCESS AND BIOSYSTEMS ENGINEERING
·
2018
·Vol. 41
·期 9
·1347-1354
·
IF 2.139
Tang Xiao-Ling,Liu Xiao,Suo Hui,Wang Zhi-Chao,Zheng Ren-Chao,Zheng Yu-Guo
The tyrosine phenol lyase (TPL) catalyzed synthesis of L-DOPA was regarded as one of the most economic route for L-DOPA synthesis. In our previous study, a novel TPL from Fusobacterium nucleatum ( Fn -TPL) was exploited for efficient biosynthesis of L-DOPA. However, the catalytic efficiency decreased when the reaction system expanded from 100 mL to 1 L. As such, the bioprocess for scale-up production of L-DOPA was developed in this study. To increase the stability of substrate and product, as well as decrease the by-product formation, the optimum temperature and pH were determined to be 15 °C and pH 8.0, respectively. The initial concentration of pyrocatechol, pyruvate and ammonium acetate was fixed at 8, 5 and 77 g/L and a fed-batch approach was applied with sodium pyruvate, pyrocatechol and ammonium acetate fed in a concentration of 5, 5 and 3.5 g/L, respectively. In addition, L-DOPA crystals were exogenously added to inhibit cell encapsulation by the precipitated product. The final L-DOPA concentration reached higher than 120 g/L with pyrocatechol conversion more than 96% in a 15-L stirred tank, demonstrating the great potential of Fn -TPL for industrial production of L-DOPA.
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APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
·
2018
·Vol. 103
·期 2
·793-806
·
IF 3.34
Yang Haiquan,Hu Jinyuan,Lu Xiao,Wang Fuxiang,Shen Wei,Hu Wei,Wang Lingling,Chen Xianzhong,Liu Long
Most recombinant proteins in Escherichia coli are not efficiently secreted to the extracellular space. Structural stabilisation of the cell wall is essential for extracellular protein production in E. coli , for which D,D-carboxypeptidases are essential. Herein, we perturbed the peptidoglycan structure of the E. coli cell wall by overexpressing D,D-carboxypeptidase genes dacA or dacB , and investigated the effect on extracellular protein production. Overexpression of dacA or dacB promoted the accumulation of intracellular soluble peptidoglycan, altered cell morphology (shape and size) and led to the formation of transparent globular structures in E. coli cells. Compared with controls (CK), extracellular production of recombinant green fluorescent protein (GFP) was increased by 1.7- and 2.3-fold upon overexpression of dacA and dacB , respectively. Similarly, extracellular production of recombinant amylase and α-galactosidase was increased by 4.5- and 2.8-fold, respectively, upon overexpression of dacA , and by 11.9- and 2.5-fold, respectively, upon overexpression of dacB . Overexpression of dacA or dacB enhanced both the outer and inner membrane permeability of E. coli . This cell wall engineering strategy opens up a new direction for enhancing extracellular protein and chemical production in E. coli .
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PROCESS BIOCHEMISTRY
·
2018
·
IF 2.616
Feng Xue,Xiangju Ya,Qi Tong,Yuansong Xiu,He Huang
A new halohydrin dehalogenase-like gene (HHDH) in the genome of Pseudomonas umsongensis YCIT1612 was identified, cloned and over-expressed in Escherichia coli . HHDH-Pu exhibited the highest sequence identity of 45% in comparison with the previously reported HHDHs. The recombinant HHDH-Pu was purified to homogeneity and its catalytic characteristics in epoxide ring opening was explored. Compared to other reported HHDHs, HHDH-Pu showed higher catalytic activity, exhibiting varied activities (2.4–50.4 U/mg) toward all the tested epoxides. It catalyzed the ring opening of epoxides with low to moderate enantioselectivity ( E =1.13-10.89). Rac- PGE was found to be the most selective among the tested epoxides. In the ring opening of rac -PGE, nucleophiles such as Cl − , N 3 − , NO 2 − , Br − or SCN − were accepted. N 3 − was selected as the optimal nucleophile. Some parameters that may alter this bio-resolution, such as the concentration of N 3 − , temperature and pH were evaluated. By optimizing the reaction conditions, the residual ( S )-PGE could be obtained in 23.5% yield and more than 99% ee from 40 mM rac -PGE.
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