PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY
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2023
·
IF 2.9
Hua-Ping Zhou,Ding-Ran Wang,Chen-Lu Xu,Ye-Wang Zhang
Heparinase I (EC 4.2.2.7), is an enzyme that cleaves heparin, showing great potential for eco-friendly production of low molecular weight heparin (LMWH). However, owing to its poor catalytic activity and thermal stability, the industrial application of heparinase I has been severely hindered. To improve the catalytic activity, we proposed to engineer both the substrate and Ca2+ binding domains of heparinase I. Several heparinases I from different organisms were selected for multiple sequence alignment and molecular docking to screen the key residues in the binding domain. Nine single-point mutations were selected to enhance the catalytic activity of heparinase I. Among them, T250D was the most highly active one, whereas mutations around Ca2+ binding domain yielded two active mutants. Mutant D152S/R244K/T250D with significantly increased catalytic activity was obtained by combined mutation. The catalytic efficiency of the mutant was 118,875.8 min−1·µM−1, which was improved 5.26 times. Molecular modeling revealed that the improved activity and stability of the mutants were probably attributed to the formation of new hydrogen bonds. The highly active mutant had great potential applications in industry and the strategy could be used to improve the performance of other enzymes.
研究领域:
生物化学
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酶工程
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DOI
PubMed
高影响因子
Research
·
2022
·
IF 11.036
Song Liu,Guanghui Li,Lei Ding,Jin Ding,Qian Zhang,Dan Li,Xingguo Hou,Xiangxing Kong,Jing Zou,Shiming Zhang,Hongbin Han,Yakun Wan,Zhi Yang,Hua Zhu
Due to the rapid spread of coronavirus disease 2019 (COVID-19), there is an urgent requirement for the development of additional diagnostic tools for further analysis of the disease. The isolated nanobody Nb11-59 binds to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor-binding domain (RBD) with high affinity to neutralize the virus and block the angiotensin-converting enzyme 2- (ACE2-) RBD interaction. Here, we introduce a novel nanobody-based radiotracer named 68Ga-Nb1159. The radiotracer retained high affinity for the RBD and showed reliable radiochemical characteristics both in vitro and in vivo. Preclinical positron emission tomography (PET) studies of 68Ga-Nb1159 in mice revealed its rapid clearance from circulation and robust uptake into the renal and urinary systems. Fortunately, 68Ga-Nb1159 could specifically reveal the distribution of the RBD in mice. This study also helped to evaluate the pharmacodynamic effects of the neutralizing nanobody. Moreover, 68Ga-Nb1159 may be a promising tool to explore the distribution of the RBD and improve the understanding of the virus. In particular, this study identified a novel molecular radioagent and established a reliable evaluation method for specifically investigating the RBD through noninvasive and visual PET technology.
研究领域:
医学影像学
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病毒学
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药理学
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DOI
高影响因子
BIORESOURCE TECHNOLOGY
·
2018
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IF 5.807
Jian Du,Xiu Zhang,Xuezhi Li,Jian Zhao,Guodong Liu,Baoyu Gao,Yinbo Qu
Commercial cellulase preparations for lignocellulose bioconversion are mainly produced by the fungus Trichoderma reesei . The maximum cellulose conversion of T. reesei cellulase mixture was 15%–20% higher than that of Penicillium oxalicum in the hydrolysis of corncob residue and Avicel. Nevertheless, both preparations hydrolyzed more than 92% of cellulose in NaOH-mercerized Avicel. When added to Avicel hydrolysis residue that was less reactive to P. oxalicum cellulases, cellobiohydrolase I (CBH I) from T. reesei resulted in a higher cellulose conversion than its homologous proteins from P. oxalicum and Aspergillus niger at the same protein loadings. Further domain exchange experiment attributed the high hydrolytic efficiency of T. reesei CBH I to its inter-domain linker and cellulose-binding domain. The results in part explained the superior performance of T. reesei cellulases on the degradation of native crystalline cellulose, and highlighted the important role of cellulose-binding region in determining the degree of hydrolysis by cellulases.
研究领域:
生物化学
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微生物学
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DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2017
·
IF 3.671
Shuqin Xu,Meiling Qiu,Xuanyue Zhang,Jinghua Chen
Heparinase I (Hep I) can efficiently depolymerize heparin and heparin sulfate to oligosaccharides or unsaturated disaccharides, which resulted in loss of physiological function such as blood coagulation. In order to realize the immobilization of Hep I on chitin carriers, we cloned Hep I with the chitin binding domain (ChBD) as a chitin-affinity tag, and the Small Ubiquitin-like MOdifier (SUMO) linker as a solvation enhancer in different fusion sequence. DNA and protein gels suggested that 4 kinds of recombinants were successfully constructed and expressed in Escherichia coli ( E. coli ). And the triple functional heparinases isolated from cell lysate could be efficiently purified by chitin beads. After optimizing fermentation conditions, it gave the specific enzyme activities of 1.88 ± 0.11, 3.69 ± 0.45, 3.44 ± 0.38, and 2.73 ± 0.29 IU/mg total proteins for ChBD-Hep I, ChBD-SUMO-Hep I, SUMO-ChBD-Hep I, and ChBD-Hep I-SUMO, respectively, with unfractionated heparin as substrate. The optimal reaction temperature and pH were determined to be 30 °C and 7.0 for all the fusion enzymes. ChBD-SUMO-Hep I exhibited the maximum half-life (48 min) at 30 °C and best thermo-stability under 15–50 °C. All the fusion enzymes showed broad pH-stability in the range of 5.4–9.0.
研究领域:
生物化学
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分子生物学
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产品关联
DOI
PubMed
高影响因子
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
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2019
·Vol. 411
·期 27
·7177-7185
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IF 3.286
Yi Zhengjun,Wang Shuhui,Meng Xiangying,Wu Anqi,Li Qian,Song Yongjie,Zhao Ronglan,Qiao Jinjuan
Accurate and rapid identification of Staphylococcus aureus ( S. aureus ) is of great significance for controlling the food poisoning and infectious diseases caused by S. aureus . In this study, a novel strategy that combines lysin cell-binding domain (CBD)-based magnetic separation with fluorescence detection was developed for the specific and sensitive quantification of S. aureus in authentic samples. The S. aureus cells were separated from the sample matrix by lysin CBD-functionalized magnetic beads. Following lysis by lysostaphin, intracellular catalase was released from S. aureus cells and detected by a fluorometric system composed of horseradish peroxidase (HRP), hydrogen peroxide (H 2 O 2 ), and Amplex Red. S. aureus was quantified via the inhibitory effect of the released intracellular catalase on the fluorometric system since the catalase could decompose the H 2 O 2 . Optimized conditions afforded a calibration curve for S. aureus ranging from 1.0 × 10 2 to 1.0 × 10 7 CFU mL −1 . The detection limit was as low as 78 CFU mL −1 in phosphate-buffered saline (PBS), and the total detection process could be completed in less than 50 min. Other bacteria associated with common food-borne and nosocomial infections negligibly interfered with S. aureus detection, except for Staphylococcus epidermidis , which may have slightly interfered. Moreover, the potential of this proposed method for practical applications has been demonstrated by detection assays of sterilized milk and human serum.
Graphical abstract
研究领域:
微生物学
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分析化学
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食品安全
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DOI
PubMed
高影响因子
Biosensors-Basel
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2022
·Vol. 12
·期 2
·103
·
IF 5.743
Zhanwei Liang,Tao Peng,Xueshima Jiao,Yang Zhao,Jie Xie,You Jiang,Bo Meng,Xiang Fang,Xiaoping Yu,Xinhua Dai
Neutralizing antibody (NAb) is a family of antibodies with special functions, which afford a degree of protection against infection and/or reduce the risk of clinically severe infection. Receptor binding domain (RBD) in the spike protein of SARS-CoV-2, a portion of the S1 subunit, can stimulate the immune system to produce NAb after infection and vaccination. The detection of NAb against SARS-CoV-2 is a simple and direct approach for evaluating a vaccine’s effectiveness. In this study, a direct, rapid, and point-of-care bicolor lateral flow immunoassay (LFIA) was developed for NAb against SARS-CoV-2 detection without sample pretreatment, and which was based on the principle of NAb-mediated blockage of the interaction between RBD and angiotensin-converting enzyme 2. In the bicolor LFIA, red and blue latex microspheres (LMs) were used to locate the test and control lines, leading to avoidance of erroneous interpretations of one-colored line results. Under the optimal conditions, NAb against SARS-CoV-2 detection carried out using the bicolor LFIA could be completed within 9 min, and the visible limit of detection was about 48 ng/mL. Thirteen serum samples were analyzed, and the results showed that the NAb levels in three positive serum samples were equal to, or higher than, 736 ng/mL. The LM-based bicolor LFIA allows one-step, rapid, convenient, inexpensive, and user-friendly determination of NAb against SARS-CoV-2 in serum.Keywords:neutralizing antibody;latex microspheres;lateral flow immunoassay;SARS-CoV-2;receptor binding domain
研究领域:
免疫学
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病毒学
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诊断技术和程序
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DOI
PubMed
高影响因子
CURRENT ISSUES IN MOLECULAR BIOLOGY
·
2024
·Vol. 46
·期 9
·9916-9927
·
IF 2.8
Miao Tian,Yuan-Yuan Xu,Yang-Nan Li,Shen Yu,Yi-Lin Wang,Xiao-Lai Ma,Ye-Wang Zhang
Dermatan sulfate and chondroitin sulfate are dietary supplements that can be utilized as prophylactics against thrombus formation. Low-molecular-weight dermatan sulfate (LMWDS) is particularly advantageous due to its high absorbability. The enzymatic synthesis of low-molecular-weight dermatan sulfates (LMWDSs) using chondroitin B lyase is a sustainable and environmentally friendly approach to manufacturing. However, the industrial application of chondroitin B lyases is severely hampered by their low catalytic activity. To improve the activity, a semi-rational design strategy of engineering the substrate-binding domain of chondroitin B lyase was performed based on the structure. The binding domain was subjected to screening of critical residues for modification using multiple sequence alignments and molecular docking. A total of thirteen single-point mutants were constructed and analyzed to assess their catalytic characteristics. Out of these, S90T, N103C, H134Y, and R159K exhibited noteworthy enhancements in activity. This study also examined combinatorial mutagenesis and found that the mutant H134Y/R159K exhibited a substantially enhanced catalytic activity of 1266.74 U/mg, which was 3.21-fold that of the wild-type one. Molecular docking revealed that the enhanced activity of the mutant could be attributed to the formation of new hydrogen bonds and hydrophobic interactions with the substrate as well as neighbor residues. The highly active mutant would benefit the utilization of chondroitin B lyase in pharmaceuticals and functional foods.
研究领域:
生物工程
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酶学
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医药生物技术
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DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2025
·
IF 8.5
Yuan Yu,Kailiang Leng,Rong Cao,Min Yang,Junkui Miao,Xixi Wang,Xiaoyan Tang,Xiaofang Liu
Chitinase catalyzes the β-(1,4) glycosidic bonds in chitin, demonstrating antifungal and insecticidal properties. Some chitinases contain multiple chitin-binding domains (ChBDs), which enhance catalysis on α-chitin. However, the specific roles of each ChBD in chitinase function remain poorly understood. Herein, Chitiniphilus eburneus Ce 0303 and ChBD-truncated chitinases were expressed to investigate the contribution of its ChBDs to binding, stability, and catalytic properties. Conserved aromatic residues including Trp-5, Tyr-19, Trp-119 and Trp-132 within ChBDs are crucial for substrate binding. The interaction between substrate and ChBD1 is primarily mediated by hydrogen bonds, while ChBD2 relies predominantly on hydrophobic interactions. Truncated chitinase Chi-2 containing ChBD1 exhibited enhanced catalytic efficiency toward α-chitin, with specific activity of 2.62 U mg −1 and k cat /K m value of 1.16 mL mg −1 s −1 , whereas Ce 0303 and truncated Chi-1 containing ChBD2 demonstrated superior thermal stability, with half-lives of 3.36 and 3.22 h at 55 °C, respectively. ChBDs reduced the crystallinity of α-chitin to as low as 70.55 %, thus enhancing catalytic domain's accessibility, while the endo - and exo -catalytic activities of Ce 0303 remained unchanged. This study advances the understanding of ChBDs function, providing a theoretical foundation for improving α-chitin catalysis and expanding the applications of chitinase in agriculture and aquaculture.
研究领域:
微生物学
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酶学
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分子生物学
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蛋白质工程
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农业生物技术
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产品关联
DOI
PubMed
高影响因子
FOOD CHEMISTRY
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2020
·
IF 6.306
Lu Qiao,He Wang,Junlin He,Shuming Yang,Ailiang Chen
17β-estradiol (E2) residues could enrich in organisms via food chain and lead to harmful biological effects for human body. To ascertain the binding domain of original E2 aptamer (E00) with long-sequence (76-mer), we developed novel truncated aptamers from E00, through rationally designed truncation by intercepting a single ring or a combination of rings (containing hairpin loop, interior loop or multiloop) at different sites and retaining appropriate double helix regions. Through comparison, 15-mer E09 presented improved affinity and higher specificity, indicating the hairpin loop near to 3′ end of E00 served on the binding domain to E2. E09 was used for gold nanoparticles (AuNPs)-based colorimetric determination of E2, achieved the detection limit of 0.02 μg/mL. The truncated aptamer (only 15-mer) first proposed in this study has great application potential in E2 determination, and this work provides proof-of-concept study for truncation of other long-sequence aptamers.
研究领域:
分析化学
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纳米技术
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生物技术
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产品关联
DOI
PubMed
高影响因子
BIOTECHNOLOGY LETTERS
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2018
·Vol. 40
·期 5
·837-845
·
IF 1.846
Hui Chang-Ye,Guo Yan,Yang Xue-Qin,Zhang Wen,Huang Xian-Qing
Objectives To improve the Pb 2+ biosorption capacity of the potential E. coli biosorbent, a putative Pb 2+ binding domain (PbBD) derived from PbrR was efficiently displayed on to the E. coli cell surface. Results The PbBD was obtained by truncating the N-terminal DNA-binding domain and C-terminal redundant amino acid residues of the Pb 2+ -sensing transcriptional factor PbrR. Whole-cell sorbents were constructed with the full-length PbrR and PbBD of PbrR genetically engineered onto the surface of E. coli cells using Lpp-OmpA as the anchor. Followed by a 1.71-fold higher display of PbBD than PbrR, the presence of PbBD on the surface of E. coli cells enabled a 1.92-fold higher Pb 2+ biosorption than that found in PbrR-displayed cells. Specific Pb 2+ binding via PbBD was the same as Pb 2+ binding via the full-length PbrR, with no observable decline even in the presence of Zn 2+ and Cd 2+ . Conclusions Since surface-engineered E. coli cells with PbBD increased the Pb 2+ binding capacity and did not affect the adsorption selectivity, this suggests that surface display of the metal binding domain derived from MerR-like proteins may be used for the bioremediation of specific toxic heavy metals.
研究领域:
微生物学
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环境科学
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生物技术
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DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
·
2019
·Vol. 20
·期 3
·744
·
IF 4.183
Tianzhu Guan,Yonghai Sun,Yongjun Wang,Zhuolin Li,Tiezhu Li,Ligang Hou
The rapid analysis of stilbene estrogens is crucially important in the environment, food and health sectors, but quantitation of lower detection limit for stilbene estrogens persists as a severe challenge. We herein described a homologous and sensitive fluorescence polarization (FP) assay based on estrogen receptor α ligand binding domain (ER-LBD) to monitor stilbene estrogens in milk. Under optimal conditions, the half maximal inhibitory concentrations (IC50) of the FP assay were 9.27 nM, 12.94 nM, and 22.38 nM for hexestrol, dienestrol and diethylstilbestrol, respectively. And the corresponding limits of detection (LOD) values were 2.94 nM, 2.89 nM, and 3.12 nM. Finally, the assay was applied to determine the stilbenes in milk samples where the mean recoveries ranged from 95.76% to 112.78% and the coefficients of variation (CV) below 12.00%. Furtherly, we have focused our study on high cross-reactivity phenomena by using two in silico approaches, including molecular docking analysis and topology analysis. Overall, docking results show that several residues in the hydrophobic pocket produce hydrophobic interactions with the tested drug molecules, which contribute to the stability of their binding. In this paper, we conclude that the FP method is suitable for the rapid detection of stilbenes in milk samples, requiring no expensive analytical equipment or time-consuming sample preparation. This work offers a practical approach that applies bioscience technology in food safety testing and improves analytical speed and laboratory efficiency.Keywords:Estrogen receptor α ligand binding domain (ER-LBD);stilbene estrogens;fluorescence polarization;molecular docking
研究领域:
食品安全检测
·
生物科学技术
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产品关联
DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2025
·
IF 6.7
Jiacheng Yang,Jing Wang,Heng Zhang,Shijiao Li,Xiaofeng Wang,Didar Baimanov,Zehao Zhang,Yang Li,Peng Yu,Xinfeng Zhang,Liming Wang
Strategies for the rapid detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are critically needed due to conventional methods’ limitations: narrow range, virus mutation-induced failure, time-consuming, and complex operations. Herein, we propose a method utilizing CuInP2S6 nanosheet probes and Bio-Layer Interferometry (BLI) technology for the rapid (5–10 min), noninvasive, and broad-spectrum detection of the SARS-CoV-2 spike receptor binding domain (RBD) in human saliva. The nanoprobe exhibits a higher binding affinity to the RBD compared to most saliva proteins, allowing it to be immobilized on BLI sensors for easier detection of protein binding and elution. An eluent buffer containing Tween-20 and salts was employed to separate salivary proteins while retaining the RBD on the probes. This system can detect the RBD across a broad spectrum and a low limit of 25 ng/mL (S/N = 3) in less than 10 min. To validate this system, experiments with pseudoviruses showed accurate identification and binding of the RBD. Molecular dynamics simulations elucidated the mechanism for selective binding of probes to RBD. In conclusion, we propose a conceptual study based on an in situ strategy with inorganic nanoprobes alongside BLI techniques for convenient, noninvasive, and rapid detection of SARS-CoV-2 and its variants. This strategy is anticipated to inspire the design and implementation of nanoprobes for the rapid and selective detection of pathogens in the future.
研究领域:
病毒学
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医学诊断
·
纳米技术
✓ 已验证
产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）