BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY
·
2023
·
IF 2.8
Shijin Wu,Qiang Wang,Xiaojing Ma,Lequan Qiu,Hongde Yan
The amino acid residues at the entrance of the catalytic pocket may impose steric hindrance on the substrate to enter the active center of the enzyme. Based on the analysis of the three-dimensional structure of the Saccharomyces cerevisiae old yellow enzyme 3 (OYE3), four bulky residues were chosen and mutated to small amino acids. The results showed that mutation of the W116 residue had interesting impacts on the catalytic performance. All four variants became inactive for the reduction of ( R )-carvone and ( S )-carvone, but inverted the stereoselectivity for the reduction of ( E / Z )-citral. The mutation of the F250 residue had a more positive effect on the activity and stereoselectivity. Two variants, F250A and F250S, showed excellent diastereoselectivity and activity for the reduction of ( R )-carvone (de > 99%, c > 99%) and increased diastereoselectivity and activity for the reduction of ( S )-carvone (de > 96%, c > 80%). One variant of the P295 residue, P295G, displayed excellent diastereoselectivity and activity only for the reduction of ( R )-carvone (de > 99%, c > 99%). Mutation of the Y375 residue had a negative impact on the activity of the enzyme. These findings provide some solutions for rational enzyme engineering of OYE3.
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BIOPROCESS AND BIOSYSTEMS ENGINEERING
·
2021
·Vol. 45
·期 2
·353-364
·
IF 3.21
Li Xue-Yong,Xu Meng-Qiu,Liu Hui,Zhou Qiang,Gao Jian,Zhang Ye-Wang
The combined cross-linked enzyme aggregates (combi-CLEAs) containing galactitol dehydrogenase (Gdh) and NADH oxidase (Nox) were prepared for l -tagatose synthesis. To prevent the excess consumption of cofactor, Nox in the combi-CLEAs was used to in situ regenerate NAD + . In the immobilization process, ammonia sulfate and glutaraldehyde were used as the precipitant and cross-linking reagent, respectively. The preparation conditions were optimized as follows: 60% ammonium sulfate, 1:1 (molar ratio) of Gdh to Nox, 20:1 (molar ratio) of protein to glutaraldehyde, and 6 h of cross-linking time at 35 °C. Under these conditions, the activity of the combi-CLEAs was 210 U g −1 . The combi-CLEAs exhibited higher thermostability and preserved 51.5% of the original activity after eight cycles of reuses at 45 °C. The combi-CLEAs were utilized for the preparation of l -tagatose without by-products. Therefore, the combi-CLEAs have the industrial potential for the bioconversion of galactitol to l -tagatose. Graphical abstract
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Frontiers in Bioengineering and Biotechnology
·
2021
·
IF 5.89
Zhou Li-Jian,Guo Li-Bin,Wei Wei,Lv Zhi-Xiang,Zhang Ye-Wang
Chondroitin AC lyase (ChSaseAC) is one of the essential polysaccharides lyases in low molecular chondroitin sulfate production. In this work, a novel PrChSaseAC from Pedobacter rhizosphaerae was successfully cloned, expressed in Escherichia coli. After optimizing the induction, the recombinant PrChSaseAC could be expressed efficiently at 0.1 mM IPTG, 25°C, and 12 h induction. Then, it was purified with Ni-NTA affinity chromatography. The characterization of the purified PrChSaseAC showed that it had high specific activity and good storage stability, which would favor the production of low molecular weight chondroitin sulfate. It also displayed activity toward chondroitin sulfate C and hyaluronic acid. PrChSaseAC had the highest activity at pH 7.5, 37°C, 10 mM Ca2+, and 5 mg/ml of chondroitin sulfate A. Molecular docking of substrate and enzyme showed the interactions between the enzyme and substrate; it revealed that the enzyme showed high activity to CS-A and hyaluronic acid, but lower activity to CS-C attributed to the structure of the binding pocket. The high stability and specific activity of the enzyme will benefit the industrial production or clinical treatment.
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WATER RESEARCH
·
2015
·
IF 5.528
Yujiao Long,Jinren Ni,Zuhui Wang
Although the identification of effective oxidant species has been extensively studied, yet the subcellular mechanism of bacterial inactivation has never been clearly elucidated in electrochemical disinfection processes. In this study, subcellular mechanism of Escherichia coli inactivation during electrochemical disinfection was revealed in terms of comprehensive factors such as cell morphology, total organic components, K + leakage , membrane permeability, lipid peroxidation, membrane potential, membrane proteins, intracellular enzyme, cellular ATP level and DNA. The electrolysis was conducted with boron-doped diamond anode in three electrolytes including chloride, sulfate and phosphate. Results demonstrated that cell inactivation was mainly attributed to damage to the intracellular enzymatic systems in chloride solution. In sulfate solution, certain essential membrane proteins like the K + ion transport systems were eliminated. Thus, the pronounced K + leakage from cytosol resulted in gradual collapse of the membrane potential, which would hinder the subcellular localization of cell division-related proteins as well as ATP synthesis and thereby lead to the bacterial inactivation. Remarkable lipid peroxidation was observed, while the intracellular damage was negligible. In phosphate solution, the cells sequentially underwent overall destruction as a whole cell with no captured intermediate state, during which the organic components of the cells were mostly subjected to mineralization . This study provided a thorough insight into the bacterial inactivation mechanism on the subcellular level.
研究领域:
微生物学
·
环境工程
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PROTEIN EXPRESSION AND PURIFICATION
·
2024
·
IF 1.4
Dezheng Peng,Yang Li,Linlin Si,Bo Zhu,Peng Wu,Yibang Li,Dongfang Tang,Yu Liu,Yunxiao Zhang
Semaglutide is currently the most promising antidiabetic drug, especially for the treatment of type 2 diabetes mellitus, due to its excellent efficacy in glycemic control and weight loss. However, the production of semaglutide remains high cost, and high yield, low cost, and high purity still remains a challenge. Herein, we reported a convenient and high-yield strategy for the preparation of semaglutide through fragmented condensation coupling, involving solid-phase peptide synthesis of tetrapeptide and on-column refolding and on-column enzyme cleavage based inclusion body expression of Lys 26 Arg 34 GLP-1 (11–37) with fused protein tags in an X-Y-D4K-G pattern. The optimized N-terminal protein tag significantly boosts inclusion body expression level, while on-column refolding and on-column enzyme cleavage avoid precipitation, enhancing efficiency and yield together with one-step purification. The successful preparation of semaglutide is expected to achieve large-scale industrial production with low cost, high yield and high purity.
研究领域:
药学
·
生物技术
·
化学工程
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MICROCHIMICA ACTA
·
2024
·Vol. 192
·期 1
·1-13
·
IF 5.3
Liu Junjie,Wu Shengxi,Zhang Lu,Zhang Mingjun,Wu Haotian,Shi Miwan,Weng Yuan,Ye Wenqiao,Gu Kui
A novel proposal is introduced with an unlabeled electrochemical immunosensor for the detection of tumor broad-spectrum biomarker vascular endothelial growth factor (VEGF165) Copper-based metal organic frameworks (Cu MOFs)-carbon nanotubes (MWCNTs) were employed as its substrates, functionalized with methylene blue (MB) for signal enhancement. Cu-MOFs-MWCNTs nanocomposites were synthesized successfully via a solvothermal method and were then deposited on the surface of a glassy carbon electrode (GCE), with the addition of methylene blue to amplify the signal. Due to the expansive specific surface area provided by the carbon nanotubes and the amino groups facilitated by the metal–organic framework nanomaterials, the anti-VEGF165 monoclonal antibody was immobilized on the electrochemical immunosensor through covalent bonding, which could bind specifically to VEGF165, thereby causing a detectable change in the current. The developed immunosensor exhibited a linear detection range for VEGF165 spanning from 1 to 100,000 pg/ml, with the R 2 value of 0.997 by differential pulse voltammetry (DPV) method, and the limit of detection was as low as 0.9686. Additionally, the electrochemical immunosensor presented commendable selectivity, availability, and reproducibility, along with an effective capacity for detecting VEGF165 in clinical samples. Consequently, the immunosensor is expected to revolutionize early screening for VEGF165-related diseases, which offer a promising, cost-effective, and precise diagnostic tool for clinical utility. Graphical
研究领域:
生物医学工程
·
肿瘤学
·
分析化学
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NUCLEIC ACIDS RESEARCH
·
2024
·
IF 16.6
Wang Qing,Zhang Jie,Zhao Zhe,Li Yichen,You Jiajia,Wang Yi,Li Xiangfei,Xu Meijuan,Rao Zhiming
High spontaneous mutation rate is crucial for obtaining ideal phenotype and exploring the relationship between genes and phenotype. How to break the genetic stability of organisms and increase the mutation frequency has become a research hotspot. Here, we present a practical and controllable evolutionary tool (oMut-Cgts) based on dual genetic level modification engineering for Corynebacterium glutamicum. Firstly, the modification engineering of transcription and replication levels based on RNA polymerase α subunit and DNA helicase Cgl0854 as the ‘dock’ of cytidine deaminase (pmCDA1) significantly increased the mutation rate, proving that the localization of pmCDA1 around transient ssDNA is necessary for genome mutation. Then, the combined modification and optimization of engineering at dual genetic level achieved 1.02 × 104-fold increased mutation rate. The genome sequencing revealed that the oMut-Cgts perform uniform and efficient C:G→T:A transitions on a genome-wide scale. Furthermore, oMut-Cgts-mediated rapid evolution of C. glutamicum with stress (acid, oxidative and ethanol) tolerance proved that the tool has powerful functions in multi-dimensional biological engineering (rapid phenotype evolution, gene function mining and protein evolution). The strategies for rapid genome evolution provided in this study are expected to be applicable to a variety of applications in all prokaryotic cells.
研究领域:
微生物学
·
遗传学
·
合成生物学
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WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY
·
2024
·Vol. 41
·期 1
·1-12
·
IF 4
Li Yang-Nan,Zhu Chen-Yuan,Xu Chen-Lu,Yu Shen,Huan Tong,Zhang Ye-Wang
A heparinase III ( Ns Hep-III) from Niabella sp . was identified, cloned, and expressed as soluble form in E. coli BL21 (DE3). With heparin as substrate, the maximum activity of Ns Hep-III of 90.0 U·mg − 1 was achieved at pH 7.1 Tris-HCl (containing 15 mM Mg 2+ ) and 30 o C. The half-life of Ns Hep-III was determined to be 5 h at 30 o C. The interactions between Ns Hep-III and substrates were studied by molecular docking. The combination of Ns Hep-III and a heparinase I from Bacteroides eggthii ( Be Hep-I) was employed to cleave heparin. Analysis of the enzymatic products of Ns Hep-III by SAX-HPLC showed seven different modified disaccharides, indicating that Ns Hep-III has a wide range of substrate specificity. The results of GPC analysis demonstrated that the average molecular weight of the product of heparin cleavage by the combination of Ns Hep-III/ Be Hep-I was reduced to 3969 Da, which accounted for 90% of all the components, and complied with the requirements of the European Pharmacopoeia. Ns Hep-III has notable activity and efficiency in cleaving heparin, which is potentially useful for the industrial production of low molecular weight heparin.
研究领域:
生物化学
·
药学
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ACS Sustainable Chemistry & Engineering
·
2024
·
IF 7.1
Yicong Lu,Xiao Zheng,Wan-Qiu Liu,Shengjie Ling,Jian Li
Cell-free protein synthesis (CFPS) has proven invaluable for expressing a wide array of proteins and enzymes, boasting significant advantages, such as facile manipulation, rapid mass transfer, and high productivity. However, traditional cell-free expression systems typically operate in batch format reactions, posing challenges for enzyme reuse in biocatalysis and rendering supplemented gene templates nonrecyclable. To overcome this limitation, we introduce a chitin-functionalized CFPS system designed to immobilize in vitro-expressed enzymes and gene templates for sustainable biocatalysis and protein synthesis. At its core, this system capitalizes on the strong binding affinity between a chitin-binding domain (ChBD) and crystalline chitin nanofibers (ChNFs). Specifically, we engineer ChBD fusion with target proteins and enzymes, allowing for their cell-free expression and in situ immobilization on ChNFs. This facilitates the effortless recycling of enzymes for multiple biocatalytic reactions, while ChNFs-immobilized enzymes can also be deployed in continuous flow biocatalysis setups. Leveraging the ChBD-ChNF pairing, gene templates can likewise be immobilized and recycled for sustained gene expression. Our results demonstrate that the chitin-functionalized cell-free system significantly enhances CFPS performance through immobilization with ChNF materials. This work underscores the flexibility and robustness of cell-free systems, which can seamlessly integrate with advanced techniques from fields such as chemistry and materials science for impactful applications.
研究领域:
生物化学
·
合成生物学
·
材料科学
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Small
·
2026
·
IF 11.8
Yan Li,Caiqin Zhang,Cuihong Chen,Yuan Li,Wanyu Jin,Xiangmei Liu,Chaofeng Wang,Yufeng Zheng,Talante Juma,Yongping Cao,Shuilin Wu,Congyang Mao
The rational design of bioactive molecules to precisely engineer the energy-conversion interfaces represents an unexplored frontier in advanced therapeutics. Herein, we report a curcumin (Cur)-adapted nano-composites (BiO 2-X /Cur), integrated into a dissolvable microneedles (MNs) patch, for synergistic piezo-sono-thermal therapy against drug-resistant skin infections. The natural compound Cur is engineered not merely as an anti-inflammatory agent but as a critical molecular adapter that precisely tunes the interfacial charge dynamics and energy landscape at the composites interface, which extends carrier lifetime to 0.63 ns and significantly enhances oxygen adsorption capacity ( E ads = -1.821 eV). Under ultrasound irradiation, the patch demonstrates superior piezo-sonocatalytic activity (≈0.6 V piezoelectric output and d 33 value of −2.9 × 10 −12 C/N) to release reactive oxygen species and simultaneously delivers a substantial sono-thermal effect (ΔT > 20 °C). The dissolvable MNs platform ensures precise and painless intradermal delivery of BiO 2-X /Cur, enabling in vitro and in vivo near-complete eradication (>99.97%) of Propionibacterium acnes and methicillin-resistant Staphylococcus aureus , downregulating pro-inflammatory cytokines (IL-6, TNF-α, HIF-1α) while upregulating reparative markers (IL-10, Nrf2). This work establishes an emerging paradigm of using natural molecules as precision adapters for engineering high-performance composites, offering an intelligent and versatile platform for treating complex infectious diseases.
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BMC Veterinary Research
·
2023
·Vol. 19
·期 1
·1-11
·
IF 2.6
Gao Yafan,Shen Yu,Fan Jiyuan,Ding Haojie,Zheng Bin,Yu Haijie,Huang Siyang,Kong Qingming,Lv Hangjun,Zhuo Xunhui,Lu Shaohong
Background Diseases caused by Toxoplasma gondii ( T. gondii ) have introduced serious threats to public health . There is an urgent need to develop a rapid detection method for T. gondii infection in cats, which are definitive hosts. Recombinant apical membrane antigen 1 (rAMA1) was produced in a prokaryotic expression system and used as the detection antigen. The aim of this study was to evaluate and optimize a reliable indirect enzyme-linked immunosorbent assay (iELISA) method based on rAMA1 for the detection of antibodies against T. gondii in cats. Results The rAMA1-iELISA method was developed and optimized by the chessboard titration method. There were no cross-reactions between T. gondii -positive cat serum and positive serum for other pathogens, indicating that rAMA1-iELISA could only detect T. gondii in most cases. The lowest detection limit of rAMA1-iELISA was 1:3200 (dilution of positive serum), and the CV of repeated tests within batches and between batches were confirmed to be less than 10%. The results of 247 cat serum samples detected by rAMA1-iELISA (kappa value = 0.622, p < 0.001) were in substantial agreement with commercial ELISA. The ROC curve analysis revealed the higher overall check accuracy of rAMA1-iELISA (sensitivity = 91.7%, specificity = 93.6%, AUC = 0.956, 95% CI 0.905 to 1.000) than GRA7-based iELISA (sensitivity = 91.7%, specificity = 85.5%, AUC = 0.936, 95% CI 0.892 to 0.980). Moreover, the positive rate of rAMA1-iELISA (6.5%, 16/247) was higher than that of GRA7-based iELISA (3.6%, 9/247) and that of commercial ELISA kit (4.9%, 12/247). Conclusion The iELISA method with good specificity, sensitivity, and reproducibility was established and can be used for large-scale detection of T. gondii infection in clinical cat samples.
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Foods
·
2023
·Vol. 12
·期 22
·4098
·
IF 5.2
Linlin Xu,Fei Pan,Yingnan Li,Huiqian Liu,Chengtao Wang
Lipase has great application potential in hydrolyzing residual yolk lipid in egg white liquid to restore its functional properties. In this study, a lipase gene fromBacillus subtiliswas expressed inE. coliBL21 (DE3) and named Lip-IM. Results showed that although Lip-IM has stronger specificity for medium- and short-chain substrates than long-chain substrates (C16, C18), due to its excellent enzyme activity, it also has strong hydrolysis activity for long-chain substrates and maintained over 80% activity at 4–20 °C, but significantly reduced when the temperature exceeds 40 °C. The addition of 0.5% Lip-IM enhanced foaming ability by 26% (from 475 to 501%) and reduced liquid precipitation rate by 9% (from 57 to 48%). Furthermore, molecular dynamics (MD) simulations were run to investigate the conformational stability of Lip-IM at different temperatures. Results showed that Lip-IM maintained a stable conformation within the temperature range of 277–303 K. Fluctuations in the flexible area and backbone movement of proteins were identified as the main reasons for its poor thermal stability.Keywords:lipase;foaming property;molecular dynamics simulation;thermal stability;dynamic cross-correlation analysis
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