PROTEIN EXPRESSION AND PURIFICATION
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2024
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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
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生物技术
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化学工程
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DOI
PubMed
高影响因子
MICROCHIMICA ACTA
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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
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肿瘤学
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分析化学
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DOI
PubMed
高影响因子
JOURNAL OF ORGANIC CHEMISTRY
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2021
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IF 4.354
Jiawei Guo,Fengwei Li,Fangyuan Cheng,Li Ma,Xiaohui Liu,Pradeepraj Durairaj,Gang Zhang,Dandan Tang,Xiangtian Long,Wei Zhang,Lei Du,Xingwang Zhang,Shengying Li
Human drug metabolites (HDMs) are important chemicals widely used in drug-related studies. However, acquiring these enzyme-derived and regio-/stereo-selectively modified compounds through chemical approaches is complicated. PikC is a biosynthetic P450 enzyme involved in pikromycin biosynthesis from the bacterium Streptomyces venezuelae. Here, we identify the mutant PikCD50N as a potential biocatalyst, with a broad substrate scope, diversified product profile, and high catalytic efficiency, for preparation of HDMs. Remarkably, PikCD50N can mediate the drug-metabolizing reactions using the low-cost H2O2 as a direct electron and oxygen donor.
研究领域:
生物化学
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药理学
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酶学
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产品关联
DOI
PubMed
高影响因子
Journal of Cleaner Production
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2021
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IF 9.297
Guotao Mao,Fangyuan Wang,Jie Wang,Pengyu Chen,Xiaodong Zhang,Hongsen Zhang,Zhimin Wang,Andong Song
Laccases are green biocatalysts that are advantageous in the bioremediation of the triphenylmethane dye malachite green (MG). Here, Cblac from Caldicellulosiruptor bescii was characterized to be a novel DUF152 laccase. Cblac mutant Mut8 was designed with enhanced thermostability, which was demonstrated by the Thermofluor assay, circular dichroism spectrum, and structural analyses. MG bioremediation by Mut8 at high temperatures was performed and examined for its potential to degrade and detoxify the high-temperature MG effluent. Approximately 100% of 100 mg/L of MG was degraded by 40 U/L of Mut8 within 2 h at 75 °C. Mut8 initiated the degradation via oxidative cleavage of the central C–C bond and N-demethylation of MG, and the intermediates were further demethylated, hydroxylated and oxidized. Moreover, the systematic toxicity assessment of the MG degradation products using bacteria, Zea mays , and zebrafish demonstrated that Mut8 thoroughly eliminated MG toxicity. Taken together, Mut8 could effectively decolorize and detoxify MG at high temperatures, showing great promise as an environmental friendly and effective approach to remediate MG effluent immediately released from the dyeing process, thus avoiding the cooling process and lowering the cost simultaneously.
研究领域:
环境生物技术
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生物催化
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生物修复
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酶工程
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产品关联
DOI
高影响因子
BIOPROCESS AND BIOSYSTEMS ENGINEERING
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2021
·Vol. 45
·期 2
·353-364
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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
研究领域:
生物技术
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酶学
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化学工程
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产品关联
DOI
PubMed
高影响因子
BIORESOURCE TECHNOLOGY
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2020
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IF 7.539
Guoyan Zhang,Hossain M. Zabed,Junhua Yun,Jiao Yuan,Yufei Zhang,Yang Wang,Xianghui Qi
In this study, a new strain of Lactobacillus plantarum (CY.6) was identified and its L-arabinose isomerase (L-AI) encoding gene ( ara A) was overexpressed in Escherichia coli BL21 for the biosynthesis of D-tagatose from milk whey powders (WP). Whole-cell biotransformation of lactose in WP into D-tagatose was done by three technological approaches, including 100%, 50% and 0% hydrolysis of lactose in WP before biotransformation, where simultaneous saccharification and biotransformation (SSB, 0% prior hydrolysis of lactose) produced maximum amounts of D-tagatose. Two-stage SSB provided 73.6% conversion efficiency (based on D-galactose) and 36.8% (in term of lactose), with 51.5 g/L of D-tagatose after 96 h, while concentration of D-tagatose produced after first stage was 34.4 g/L. Yield and volumetric productivity of D-tagatose after two-stage SSB were found to be 0.26 g/g of WP (0.37 g/g of lactose, 0.74 g/g of D-galactose produced from lactose) and 0.54 g/L/h, respectively.
研究领域:
生物技术
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微生物学
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食品工程
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酶学
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产品关联
DOI
PubMed
高影响因子
Biomass Conversion and Biorefinery
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2022
·Vol. 13
·期 17
·15837-15844
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IF 4.05
Di Junhua,Qin Lizhen,He Yu-Cai
A new type of metal-free solid acid catalyst ChCl:OA-SG(SiO 2 ) was firstly synthesized by loading deep eutectic solvent ChCl:oxalic acid on silica with sol–gel process using tetraethyl orthosilicate as silicon source. The properties of the catalyst were characterized by Fourier transform infrared spectrum, X-ray diffraction, scanning electron microscope, and Brunauer-Emmet-Teller. Highest furfural (FF) yield reached 41.2% from corncob with ChCl:OA-SG(SiO 2 ) catalyst (2.0 wt%) in water at 170 °C for 0.5 h. The catalytic mechanism of ChCl:OA-SG(SiO 2 )-catalyzed biomass into furfural was interpreted in the aqueous system. Corncob-valorized FF was wholly transformed to furfuryl alcohol with recombinant Escherichia coli CG-19 cells after bioreduction for 3 h of at 30 °C and pH 7.0. Yield of 0.416 g furfuryl alcohol/g xylan in corncob was obtained. This established hybrid catalysis could efficiently valorize biomass into value-added furfuryl alcohol.
研究领域:
生物化学
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催化化学
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可再生资源工程
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产品关联
DOI
高影响因子
Biomaterials Advances
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2022
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IF 0
Lichao Chen,Bo Wang,Haohao Ren,Yanan Wu,Defu Lyu,Yanan Ouyang,Qiyi Zhang,Yonggang Yan
Poly-amino acid (PAA) is a promising biomaterial in biomedical engineering due to its similar amide bond structure to collagen and excellent biocompatibility, but the lack of osteogenic activity and inferior mechanical strength limit its long-term application in orthopedics. In this study, a poly-amino acid/poly (p-benzamide) (PAA-PBA) copolymer with high mechanical strength was designed and fabricated by the method of solution polymerization. The chain structures, thermal properties and mechanical properties of these polymers were evaluated and results showed that PBA greatly promoted the mechanical properties of PAA, and the copolymer performed the maximum mechanical strengths with compressive strength, bending strength and tensile strength of 123 MPa, 107 MPa and, 95 MPa, respectively. To increase the bioactivity of surface, a bioactive coating that consists of poly-(dopamine) (PDA) nanolayers and tripeptide Arginine-Glycine-Aspartic acid (RGD) on sulfonated PAA-PBA copolymer was created. A porous structure appeared on the surface after modification, the surface roughness and hydrophilicity of copolymer has been improved obviously after introducing PDA and RGD peptide coating. The in vitro bioactivity evaluation demonstrated that the RGD-functionalized sample showed a significantly improved ability to promote bone apatite mineralization, cell adhesion, proliferation and osteogenic differentiation. In a word, such a strategy of material synthesis and surface modification method shows a great potential for broadening the use of PAA in the application of load-bearing bone substitute biomaterials.
研究领域:
生物医学工程
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材料科学
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组织工程
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产品关联
DOI
PubMed
PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY
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2019
·
IF 2.87
Jiang Zhu,Kai Tian,Christopher A. Reilly,Xinghui Qiu
Helicoverpa armigera and H. assulta are two of the few insects that can feed on hot pepper fruits. Capsaicin and dihydrocapsaicin ( i.e. , capsaicinoids) are the principal pungent compounds in hot peppers. To explore possible molecular mechanisms of adaptation that allow these two species to consume capsaicinoids, the capacity of the three detoxification tissues (fat body, midgut, and Malpighian tubule) of the two pests, to metabolically degrade capsaicin and dihydrocapsaicin, was compared. The results showed that capsaicin and dihydrocapsaicin were metabolized by crude enzyme preparations from all three tissues of the two pests. Five metabolites of capsaicin, and five metabolites of dihydrocapsaicin were identified. Tissue and species differences in the degree of capsaicin and dihydrocapsaicin metabolism were observed. The specialist H. assulta had an overall greater capacity to degrade the capsaicinoids compared to the generalist H. armigera. Further, the midgut was the most significant contributor to capsaicinoid metabolism. The notably high specific activity in Malpighian tubules of H. armigera also further highlights the significance of this organ in xenobiotic detoxification. Alkyl hydroxylation and dehydrogenation were the main pathways for the oxidative biotransformation of both capsaicin and dihydrocapsaicin by cytochrome P450s. This study provides evidence that enhanced metabolic decomposition of capsaicinoids may be an adaptation explaining dietary preferences for Capsicum fruits by these two pests.
研究领域:
昆虫学
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植物化学
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分子生物学
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DOI
PubMed
高影响因子
ACS Catalysis
·
2019
·
IF 12.221
Bing-Mei Su,Ze-Hui Shao,Ai-Peng Li,Muhammad Naeem,Juan Lin,Li-Dan Ye,Hong-Wei Yu
Inspired by the conformational change of the enzyme–substrate complex in molecular dynamics (MD) simulation with distance restriction, we propose a strategy for identifying the engineering targets based on the comparative analysis of enzyme-/substrate- binding modes in MD simulations with and without distance restriction (prereaction-state simulation and free-state simulation). Taking the short-chain dehydrogenase/reductase (SDR) mutant EbSDR8-G94A/S153L (Mu0) with poor activity toward bulky aryl ketones as an example, H145 and Y188 were identified as the engineering targets due to the distinct conformation difference in the two simulation modes. To break the “beam” structure formed by these residues at the entry of cavity C2 in free-state simulation, the substrate-binding pocket was reconstructed, and meanwhile the relative size of cavities C1 and C2 was modulated to improve the enantioselectivity. In this way, mutants for efficient asymmetric reduction of o-halogenated acetophenones, propiophenones, aromatic ketoesters, and diaryl ketones were designed, delivering chiral alcohols with >99% conversion and >98% ee. The effectiveness of this design strategy was also validated by the successful redesign of PpYSDR, generating a variant for efficient reduction of (4-chlorophenyl) 2-pyridyl ketone into the S-product with >99% conversion and 96% ee. MD simulations suggested a suitable binding pocket with proper energy contribution as the ubiquitous mechanism for the improvement of activity and enantioselectivity toward substrates with varied structures. The success in this study provides hints for the rational design of alcohol dehydrogenases/reductases with both a broad substrate spectrum and high enantioselectivity.
研究领域:
生物化学
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计算生物学
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酶工程
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产品关联
DOI
高影响因子
PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY
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2020
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IF 1.415
Cai-Yun Liu,Wen-Bin Su,Li-Bin Guo,Ye-Wang Zhang
Heparinase I (Hep I) specifically degrades heparin to oligosaccharide or unsaturated disaccharide and has been widely used in preparation of low molecular weight heparin (LMWH). In this work, a novel Hep I from Bacteroides eggerthii VPI T5-42B-1 was cloned and overexpressed in Escherichia coli BL21 (DE3). The enzyme has specific activity of 480 IU·mg−1 at the optimal temperature and pH of 30 °C and pH 7.5, and the Km and Vmax were 3.6 mg·mL−1 and 647.93 U·mg−1, respectively. The Hep I has good stability with t1/2 values of 350 and 60 min at 30 and 37 °C, respectively. And it showed a residual relative activity of 70.8% after 21 days incubation at 4 °C. Substrate docking study revealed that Lys99, Arg101, Gln241, Lys270, Asn275, and Lys292 were mainly involved in the substrate binding of Hep I. The shorter hydrogen bonds formed between heparin and these residues suggested the higher specific activity of BeHep I. And the minimum conformational entropy value of 756 J·K−1 provides an evidence for the improved stability of this enzyme. This Hep I could be of interest in the industrial preparation of LMWH for its high specific activity and good stability.
研究领域:
生物化学
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酶学
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药学
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产品关联
DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2022
·
IF 8.025
Ling Zheng,Zilong Guo,Yinxiao Xu,Benwei Zhu,Zhong Yao
Pectate lyase (ErPL2) from Echinicola rosea JL3085 showed maximal activity at 45 °C and pH 9.0 with 0.6 mM CaCl 2 . The K m and V max values of ErPL2 for polygalacturonic sodium were 2.098 mmol/L and 0.955 mmol/s, respectively. ErPL2 endolytically degraded pectic substances into oligosaccharides with degree of polymerization (DP) 1–5. To improve the thermostability and pH operation range, recombinant ErPL2 was immobilized onto mesoporous titanium oxide particles (MTOPs). MTOPs have abundant hydroxyl groups on the surface, which is a non-toxicity and good biocompatibility carrier. The residual enzyme activity of immobilized ErPL2 at 40 °C increased remarkably from 11% to 91% compared with free enzyme. The operable pH range was extended from 8–9 to 9–11. Surprisingly, the catalytic efficiency of immobilized ErPL2 was about 19 times higher than free enzyme. To our knowledge this is the first attempt of pectate lyase immobilized on MTOPs and it provides a new option for improving the catalytic performance.
研究领域:
酶学
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生物技术
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食品科学
·
化学工程
✓ 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）