Catalysis Science & Technology
·
2018
·Vol. 8
·期 18
·4718-4725
·
IF 5.365
Jiang-Wei Shen,Jia-Mei Qi,Xiao-Jian Zhang,Zhi-Qiang Liu,Yu-Guo Zheng
Candida antarctica lipase B (CALB) is a versatile and robust lipase with high activity and enantioselectivity in the resolution of alcohols and amines. Poor specific activity toward carboxylic esters that are branched or substituted in close proximity to the carboxyl group limits the application of CALB. In this study, a semi-rational engineering approach was successfully applied to tailor CALB for efficient synthesis of the moxifloxacin chiral intermediate through enzymatic resolution of cis-(±)-dimethyl 1-acetylpiperidine-2,3-dicarboxylate. The combinatorial active-site saturation test was performed on CALB and two key residue sites, 189 and 190, were identified in the determination of hydrolytic activity. The specific activity and catalytic efficiency (kcat/Km) of the best variant mut-I189K were increased by 286- and 193-fold, respectively, without impacting its enantioselectivity. With 0.1 g L−1 of purified mut-I189K loading, 1 M substrate was asymmetrically hydrolyzed with 49.9% conversion within 5 h, resulting in a space–time yield of 583.8 g L−1 d−1. These results demonstrated that mut-I189K is a competitive biocatalyst for large-scale preparation of the moxifloxacin chiral intermediate.
研究领域:
生物催化
·
酶工程
·
药物化学
·
有机合成
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产品关联
DOI
高影响因子
BIOCONJUGATE CHEMISTRY
·
2019
·
IF 4.349
Qiang Zhang,Dan Xu,Qiuyan Guo,Wenjun Shan,Jun Yang,Tongtong Lin,Shefang Ye,Xi Zhou,Yunlong Ge,Shengli Bi,Lei Ren
The progression of hepatic fibrosis can lead to cirrhosis and hepatic failure, but the development of antifibrotic drugs have faced the challenges of poor effectiveness and targeted specificity. Herein, a theranostic strategy was carried to encapsulate a natural medicine (Quercetin, QR) into hepatitis B core (HBc) protein nanocages (NCs) for imaging and targeted treatment of hepatic fibrosis. It was noted that nanoparticles (RGD-HBc/QR) with surface-displayed RGD targeting ligand exhibit a rather high selectivity toward activated HSCs via the binding affinity with integrin αvβ3, and an efficient inhibition of proliferation and activation of hepatic stellate cells (HSCs) in vitro and in vivo. Once encapsulated in quercetin–gadolinium complex and/or labeled with the NIR fluorescent probes (Cy5.5), the resulting nanoparticles (RGD-HBc/QGd) show great potential as NIR fluorescent and magnetic resonance imaging contrast agents for hepatic fibrosis in vivo. Therefore, the multifunctional integrin-targeted nanoparticles could selectively deliver QR to the activated HSCs, and may provide an effective antifibrotic theranostic strategy.
研究领域:
医学
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药学
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生物技术
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产品关联
DOI
PubMed
高影响因子
PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY
·
2019
·
IF 2.87
Kai Tian,Jiang Zhu,Mei Li,Xinghui Qiu
Capsaicin (8-methyl- N -vanillyl-6-nonenamide) is the most abundant capsaicinoids found in hot peppers ( Capsicum annum and Capsicum frutescens ). It has been well documented that capsaicin plays an important role in the defense against the attack of herbivores or pathogens on Capsicum plants. A few insect herbivores such as Helicoverpa armigera and Helicoverpa assulta have been recorded to be capable of feeding on hot pepper fruits, suggesting that these insects evolve mechanisms against the toxicity of capsaicin. Although cytochrome P450-meidated detoxification is considered to be an important mechanism by which cotton bollworms cope with capsaicin, experimental evidence is lacking. In this study, we compared the capacity of four H. armigera P450s (CYP6B6, CYP9A12, CYP9A14 and CYP9A17) in capsaicin metabolism, and the capsaicin metabolites were screened and tentatively identified by liquid chromatography–tandem mass spectrometry (LC–MS/MS). HPLC analyses showed that depletion rates of capsaicin were 21.9 ± 0.1, 11.9 ± 1.5, 16.3 ± 1.4 and 14.8 ± 0.2 min −1 for CYP6B6, CYP9A12, CYP9A14 and CYP9A17 respectively. The transformation of capsaicin was inhibited by the P450 inhibitor piperonyl butoxide . A total of seven products were detected, and hydroxylation (aromatic and aliphatic) and dehydrogenation were found to be two main pathways in capsaicin metabolism. In addition, capsaicin metabolism was enzyme selective: M1 (ω-hydroxylated N-macrocyclic metabolite) and M3 (ω-hydroxylated metabolite) were uniquely detected in the CYP6B6 catalytic reaction, while M4 (ω-n hydroxylated capsaicin), M5 (diene of capsaicin) and M6 (doubly oxidized metabolite of dehydrogenated capsaicin) were only detectable in CYP9A metabolisms. A capsaicin dimer (5, 5′-dicapsaicin) was found to be the major metabolite of CYP9A reactions, but the minor product produced by CYP6B6. An overall more similar behavior in capsaicin metabolism was observed among CYP9As than between CYP6B6 and CYP9As. Our data demonstrate that CYP6B6 and CYP9As have a potent capability to transform capsaicin, and individual P450 produce unique metabolite profile. These findings help us to understand the molecular basis of capsaicin adaptation in H. armigera .
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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产品关联
DOI
PubMed
高影响因子
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
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合成生物学
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产品关联
DOI
PubMed
高影响因子
Food Science and Human Wellness
·
2023
·
IF 7
Weijun Leng,Xiaoyun Wu,Xianghui Qi,Hongying Liu,Li Yuan,Ruichang Gao
In this study, a gene encoding serine protease ( PmSpr288 ) from cold-adapted bacterium, namely Planococcus maritimus XJ11, was cloned and overexpressed in Escherichia coli BL21 (DE3). Bioinformatics analysis revealed that PmSpr288 belongs to serine protease S8 superfamily with a classical catalytic triad comprised by the Asp49, His86 and Ser251. Moreover, PmSpr288 was found to be active over broad alkaline pH and low-moderate temperature, and exhibited wide range of protein substrate specificity. In addition, PmSpr288 was able to hydrolyze the meat proteins actin and myosin, and molecular docking results suggested that the crucial interaction between PmSpr288 and actin/myosin complexes was mainly occupied by hydrogen bonds. The muscle protein hydrolysates of silver carp prepared by PmSpr288 was shown to have antioxidant activity via DPPH radical scavenging assay, which presented an IC 50 valve of 1.309 mg/mL. In conclusion, these characteristics imply that PmSpr288 has potential biotechnological application prospect for the production of bioactive peptides.
研究领域:
微生物学
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生物化学
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生物技术
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食品科学
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产品关联
DOI
高影响因子
Nature Communications
·
2023
·Vol. 14
·期 1
·1-14
·
IF 16.6
Pan Yunjun,Li Guobang,Liu Ruxin,Guo Jiawei,Liu Yunjie,Liu Mingyu,Zhang Xingwang,Chi Luping,Xu Kangwei,Wu Ruibo,Zhang Yuzhong,Li Yuezhong,Gao Xiang,Li Shengying
Cytochrome P450 enzymes play important roles in the biosynthesis of macrolide antibiotics by mediating a vast variety of regio- and stereoselective oxidative modifications, thus improving their chemical diversity, biological activities, and pharmaceutical properties. Tremendous efforts have been made on engineering the reactivity and selectivity of these useful biocatalysts. However, the 20 proteinogenic amino acids cannot always satisfy the requirement of site-directed/random mutagenesis and rational protein design of P450 enzymes. To address this issue, herein, we practice the semi-rational non-canonical amino acid mutagenesis for the pikromycin biosynthetic P450 enzyme PikC, which recognizes its native macrolide substrates with a 12- or 14-membered ring macrolactone linked to a deoxyamino sugar through a unique sugar-anchoring mechanism. Based on a semi-rationally designed substrate binding strategy, non-canonical amino acid mutagenesis at the His238 position enables the unnatural activities of several PikC mutants towards the macrolactone precursors without any sugar appendix. With the aglycone hydroxylating activities, the pikromycin biosynthetic pathway is rewired by the representative mutant PikC H238 p AcF carrying a p- acetylphenylalanine residue at the His238 position and a promiscuous glycosyltransferase. Moreover, structural analysis of substrate-free and three different enzyme-substrate complexes of PikC H238 p AcF provides significant mechanistic insights into the substrate binding and catalytic selectivity of this paradigm biosynthetic P450 enzyme.
研究领域:
生物化学
·
分子生物学
·
药学
·
酶学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ENZYME AND MICROBIAL TECHNOLOGY
·
2022
·
IF 3.705
Hong-Ru Zhao,Bing-Mei Su,Yi-Bing Shi,Juan Lin
Ethyl 3-hydroxy-3-phenylpropionate (EHPP), ( R )-EHPP or ( S )-EHPP, is an important chiral intermediate for pharmaceuticals. Its synthesis from ethyl benzoyl acetate (EBA) by alcohol dehydrogenase is regarded as a green method. However, scarcely any alcohol dehydrogenase has been reported competent in asymmetric synthesis of chiral EHPP at high EBA loading. Present study developed two robust and efficient bio-catalysts Mu-S2 and Mu-R4 for preparation of ( S )-EHPP and ( R )-EHPP respectively by rational design of alcohol dehydrogenase PcSDR from Pedobacter chitinilyticus based on molecular dynamics (MD) simulation analysis. BtGDH, a glucose dehydrogenase from Bacillus toyonensis catalyzing the oxidation of glucose for cofactor regeneration, was co-expressed with the screened mutants to form enzyme systems Mu-S2-BtGDH and Mu-R4-BtGDH. After reaction condition optimization, Mu-S2-BtGDH and Mu-R4-BtGDH were efficient in the synthesis of ( S )-EHPP (94% conv. and 99% e.e.) and ( R )-EHPP (99% conv. and 98% e.e. ) respectively in 100 mL scale under 500 mM of EBA loading in 10 h following a substrate continuous feeding mode. After purifying, the isolated yield for each EHPP enantiomer is > 93%. This work not only provides potential biocatalysts for the industrial production of ( R )-EHPP and ( S )-EHPP, but also enriches the constructure-function relationship of alcohol dehydrogenases.
研究领域:
生物化学
·
酶工程
·
药物化学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
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.
研究领域:
生物化学
·
酶学
·
生物技术
·
药理学
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产品关联
DOI
PubMed
高影响因子
Advanced Healthcare Materials
·
2019
·Vol. 8
·期 23
·1900911
·
IF 6.27
Peng Ji,Haiqin Huang,Shirui Yuan,Le Wang,Siqi Wang,Yiwei Chen,Na Feng,Hanitrarimalala Veroniaina,Ziheng Wu,Zhenghong Wu,Xiaole Qi
Reactive oxygen species (ROS)-mediated apoptosis is considered a crucial therapeutic mechanisms for artesunate (AS). As an Fe(II)-dependent drug, the anticancer effect of AS is often limited due to insufficient Fe(II) concentration in targeted cells. To overcome this problem, a recombinant apoferritin nanocarrier containing ferriferous oxide (M-HFn) is constructed to produce auxiliary exogenous Fe(II) when delivering AS to cancer cells. Here, the newly fabricated AS-loaded M-HFn nanoparticles (M-HFn@AS NPs) can significantly improve the tumor-specific targeting and intracellular uptake efficiency of AS in human cervical carcinoma cells. After being captured in the acidic cavity of endosomes, M-HFn@AS NPs can simultaneously release Fe(II) and allow AS to activate satisfactory ROS-mediated apoptosis. Furthermore, in vivo studies demonstrate that M-HFn@AS NPs can selectively accumulate in tumors to efficiently inhibit tumor growth. Thus, M-HFn@AS NPs are a promising system to enhance the therapeutic effect of Fe(II)-dependent drugs.
研究领域:
药学
·
癌症研究
·
纳米技术
·
分子生物学
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产品关联
DOI
PubMed
高影响因子
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
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药学
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产品关联
DOI
PubMed
高影响因子
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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产品关联
DOI
高影响因子
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
✓ 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）