BIOTECHNOLOGY LETTERS
·
2017
·Vol. 39
·期 12
·1903-1910
·
IF 1.73
Liu Ke,Zhou Youzhi,Zhang Jinsong,Chu Jianlin,Zhang Yewei,He Bingfang
Objective To isolate a thermostable pyrimidine nucleoside phosphorylase (PyNP) from mesophilic bacteria by gene mining. Results
Bb PyNP from Brevibacillus borstelensis LK01 was isolated by gene mining. Bb PyNP had a highest 60% identity with that of reported PyNPs. Bb PyNP could catalyze the phosphorolysis of thymidine, 2′-deoxyuridine, uridine and 5-methyuridine. Bb PyNP had good thermostability and retained 73% of its original activity after 2 h incubation at 50 °C. Bb PyNP had the highest activity at an optimum alkaline pH of 8.5. Bb PyNP was stable from pH 7 to 9.8. Under preliminary optimized conditions, the biosynthesis of various 5-halogenated pyrimidine nucleosides by Bb PyNP reached the yield of 61–84%. Conclusion An efficient approach was estimated in isolating thermostable PyNP from mesophilic bacteria.
研究领域:
生物化学
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酶学
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生物技术
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产品关联
DOI
PubMed
高影响因子
Food Bioengineering
·
2024
·Vol. 3
·期 2
·151-159
·
IF 0
Ziye Zhao,Ning Nie,Xinwei Li,Ye Shi,Xudong Liu,Yifei Zhang
Nucleotides, such as uridine 5′-monophosphate (5′-UMP) and cytidine 5′-monophosphate (5′-CMP), are heavily demanded as food additives, dietary supplements, and medicinal intermediates. Although enzymatic phosphorylation is an effective approach to producing these nucleosides, its practical application is challenging due to the need for the expensive enzymes and coenzyme guanosine-5′-triphosphate (GTP). Here we prepared biocompatible calcium-based nanoflowers incorporated with cascade enzymes, uridine-cytidine kinase (UCK) and acetate kinase (ACK), for the production of nucleotides. The spatial distribution of these enzymes was optimized to maximize their catalytic performances. The most active nanoflowers (Ca 3 (PO 4 ) 2 & ACK)& UCK that display the rate-limiting enzyme UCK on the outer layer were used in a packed-bed reactor for continuous synthesis of 5′-UMP and 5′-CMP. The catalytic performance of the catalyst retained over 80% within 10 h, showing good operational stability.
研究领域:
生物催化
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纳米技术
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酶工程
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生物化学
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食品科学
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药物化学
✓ 已验证
DOI
ACS Synthetic Biology
·
2024
·
IF 3.7
Chi Zhang,Guoguang Wei,Ning Zhou,Yingying Wang,Jia Feng,Xin Wang,Alei Zhang,Kequan Chen
In this study, we proposed a biological approach to efficiently produce pseudouridine (Ψ) from glucose and uracil in vivo using engineered Escherichia coli. By screening host strains and core enzymes, E. coli MG1655 overexpressing Ψ monophosphate (ΨMP) glycosidase and ΨMP phosphatase was obtained, which displayed the highest Ψ concentration. Then, optimization of the RBS sequences, enhancement of ribose 5-phosphate supply in the cells, and overexpression of the membrane transport protein UraA were investigated. Finally, fed-batch fermentation of Ψ in a 5 L fermentor can reach 27.5 g/L with a yield of 89.2 mol % toward uracil and 25.6 mol % toward glucose within 48 h, both of which are the highest to date. In addition, the Ψ product with a high purity of 99.8% can be purified from the fermentation broth after crystallization. This work provides an efficient and environmentally friendly protocol for allowing for the possibility of Ψ bioproduction on an industrial scale.
研究领域:
合成生物学
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微生物工程
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DOI
PubMed
高影响因子
PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY
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2017
·
IF 1.361
Xiaoyan Li,Chen Qi,Peilian Wei,Lei Huang,Jin Cai,Zhinan Xu
Uridine 5′-diphosphate N-acetylglucosamine (UDP-GlcNAc) is a natural UDP-monosaccharide donor for bacterial glycosyltransferases, while uridine 5′-diphosphate N-trifluoacetyl glucosamine (UDP-GlcNTFA) is its synthetic mimic. The chemoenzymatic synthesis of UDP-GlcNAc and UDP-GlcNTFA was attempted by three recombinant enzymes. Recombinant N-acetylhexosamine 1-kinase was used to produce GlcNAc/GlcNTFA-1-phosphate from GlcNAc/GlcNTFA. N-acetylglucosamine-1-phosphate uridyltransferase from Escherichia coli K12 MG1655 was used to produce UDP-GlcNAc/GlcNTFA from GlcNAc/GlcNTFA-1-phosphate. Inorganic pyrophosphatase from E. coli K12 MG1655 was used to hydrolyze pyrophosphate to accelerate the reaction. The above enzymes were expressed in E. coli BL21 (DE3) and purified, respectively, and finally mixed in one-pot bioreactor. The effects of reaction conditions on the production of UDP-GlcNAc and UDP-GlcNTFA were characterized. To avoid the substrate inhibition effect on the production of UDP-GlcNAc and UDP-GlcNTFA, the reaction was performed with fed batch of substrate. Under the optimized conditions, high production of UDP-GlcNAc (59.51 g/L) and UDP-GlcNTFA (46.54 g/L) were achieved in this three-enzyme one-pot system. The present work is promising to develop an efficient scalable process for the supply of UDP-monosaccharide donors for oligosaccharide synthesis.
研究领域:
生物化学
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酶学
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合成生物学
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微生物学
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DOI
PubMed
高影响因子
Current Protocols
·
2021
·Vol. 1
·期 10
·e274
·
IF 0
Jingyi Wang,Jiachen Shang,Yu Xiang,Aijun Tong
Functionalized oligonucleotides (ONs) are widely applied as target recognition molecules for biosensing and gene regulation. Herein, we describe a general method for post-synthetic modification of ONs based on the oxidative amination of 4-thio-2′-deoxyuridine (4SdU) with sodium periodate and several amines. Alkyne-/azide-, biotin-, and fluorophore-modified ONs were prepared by modifying 4SdU-containing ONs with the corresponding amines and characterized for their bioorthogonal reactivity, streptavidin-binding affinity, and fluorescence properties, respectively. We synthesized three fluorophore-modified ONs with and without the aromatic fluorophores conjugated to modified nucleobases and investigated their emission properties. © 2021 Wiley Periodicals LLC. Basic Protocol 1 : Post-synthetic modification of ONs Supporting Protocol : Preparation of dansyl ethylenediamine Basic Protocol 2 : Quantification of ON reaction yields Basic Protocol 3 : Characterization of modified ONs
研究领域:
生物化学
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分子生物学
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化学生物学
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DOI
Nanomaterials
·
2022
·Vol. 12
·期 4
·696
·
IF 5.719
Chao Zhang,Shuangqing Fu,Fanghua Zhang,Mengnan Han,Xuming Wang,Jie Du,Honglei Zhang,Wei Li
Combination chemotherapy is emerging as an important strategy for cancer treatment with decreased side effects. However, chemotherapeutic drugs with different solubility are not easy to realize co-delivery in traditional nanocarriers. Herein, an affibody modified G-quadruplex DNA micellar prodrug (affi-F/GQs) of hydrophilic 5-fluorodeoxyuridine (FUdR) by integrating polymeric FUdRs into DNA strands is developed for the first time. To achieve synergistic efficacy with hydrophobic drugs, curcumin (Cur) is co-loaded into affi-F/GQs micelles to prepare the dual drug-loaded DNA micelles ([email protected]/GQs), in which affibody is employed as a targeting moiety to facilitate HER2 receptor-mediated uptake.[email protected]/GQs have a small size of approximately 130 nm and exhibit excellent stability. The system co-delivers FUdR and Cur in a ratiometric manner, and the drug loading rates are 21.1% and 5.6%, respectively. Compared with the physical combination of FUdR and Cur,[email protected]/GQs show higher cytotoxicity and greater synergistic effect on HER2 positive gastric cancer N87 cells. Surprisingly,[email protected]/GQs significantly enhance the expression and activity of apoptosis-associated proteins in Bcl-2/Bax-caspase 8, 9-caspase 3 apoptotic pathway, which is the main factor in the death of tumor cells induced by FUdR. Overall, this nanoencapsulation is a promising candidate for the targeted co-delivery of drugs with significant differences in solubility.Keywords:G-quadruplex;DNA micelles;affibody;5-fluorodeoxyuridine;curcumin;synergetic therapy
研究领域:
癌症治疗
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纳米技术
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药学
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分子生物学
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产品关联
DOI
PubMed
高影响因子
Chemical Biology & Drug Design
·
2016
·Vol. 88
·期 3
·329-340
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IF 2.802
Ke-Guang Cheng,Chun-Hua Su,Jia-Yan Huang,Jun Liu,Yuan-Ting Zheng,Zhen-Feng Chen
According to fused two bioactive moieties together by bonds covalently and available as a new single hybrid entity known as pharmacophore hybridization, a total of 10 targeted uridine–oleanolic acid hybrids were synthesized. Most of these hybrids showed excellent proliferation inhibition against tested Hep-G2, A549, BGC-823, MCF-7, and PC-3 tumor cell lines (IC 50 < 8 μ m ), even with some IC 50 values under 0.1 μ m . The detection of cytotoxicity selectivity revealed that hybrids 5 and 18 exhibited low cytotoxicity toward normal human liver cell HL-7702. Further studies revealed that selected hybrid 5 could induce apoptosis in Hep-G2 cells through the investigation of acridine orange/ethidium bromide, Hoechst 33258 fluorescence stainings, and annexin V/propidium iodide assay. It was also found that hybrid 5 could induce mitochondrial membrane potential disruption, arrest Hep-G2 cell line at G1 phase, and activate effector caspase-3/9 to trigger cell apoptosis.
研究领域:
药理学
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肿瘤学
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有机化学
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DOI
PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2025
·
IF 6.2
Jihang Zhang,Xiao Zhang,Jinming Zhang,Jinglan Wu,Pengpeng Yang,Chenglun Tang,Fengxia Zou,Hanjie Ying,Wei Zhuang
Interfacial modulation of protein microenvironments plays a pivotal role in enhancing enzyme immobilization and catalysis. In this study, we proposed a polyethylenimine (PEI)-assisted strategy that combines electrostatic and affinity interactions to improve the performance of Cytidine 5′-Monophosphate (CMP) conversion by uridine-cytidine kinase (UCK). The PEI-modified interface creates an optimal local microenvironment that maintains a balanced charge distribution, stabilizes UCK’s conformation, and prevents denaturation. Electrostatic interactions promote product adsorption, enhance diffusion, and reduce substrate accumulation, boosting reaction efficiency. The kinetic assays revealed an increase in the maximum reaction rate from 16.8 to 113.2 μM·min–1 with a remarkable increase in substrate affinity and enzyme activity. The relative enzyme activity at the optimal substrate concentration increased from 70.4 to 106.9%, and by 113.3% under conditions of substrate inhibition. This study provides theoretical and technical support for the efficient production of CMP with promising applications in food, feed, and medical fields.
研究领域:
生物化学
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酶工程
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生物催化
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DOI
PubMed
高影响因子
FOOD CHEMISTRY
·
2022
·
IF 9.231
Xiaoxiang Gao,Chenbo Yue,Ruocen Tian,Leilei Yu,Fengwei Tian,Jianxin Zhao,Wei Chen,Qixiao Zhai
We examined the microbial regulatory capacity of four polyphenols with different structure in healthy mice and explore the mechanism according to exogenous metabolites and microbial metabolites. Oral administration of four polyphenols, including caffeic acid (CA), procyanidin (PA), puerarin (Pue), and resveratrol (Res), did not lead to metabolic disorder in healthy mice. Gut microbiota analysis revealed that CA, PA, and Pue administration significantly enhanced the abundance of Akkermansia and Ruminococcaceae UCG-014 while Res supplement mainly promoted the growth of Lactobacillus and Bacteroides . Furthermore, correlation analysis and exogenous metabolite prediction revealed that the effects of polyphenols, including CA, PA, and Pue, on Akkermansia have strong relationship with uridine while the regulation of Res on microbiota might be dependent on the decrease on petroselinic acid. These investigations considerably suggest the importance of exploration of exogenous metabolites and reveal the similarity of effects of polyphenols on microbiota and metabolites.
研究领域:
微生物学
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营养学
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代谢组学
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产品关联
DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
·
2023
·Vol. 24
·期 18
·14211
·
IF 5.6
Jie Li,Ning Han,Yangyang Li,Feifei Zhao,Wenguang Xiong,Zhenling Zeng
Streptococcus suisis an emerging zoonotic pathogen that can cause fatal diseases such as meningitis and sepsis in pigs and human beings. The overuse of antibiotics is leading to an increased level of resistance inS. suis, and novel antimicrobial agents or anti-virulence agents for the treatment of infections caused byS. suisare urgently needed. In the present study, we investigated the antibacterial activity, mode of action and anti-virulence effects of floxuridine againstS. suis. Floxuridine showed excessive antibacterial activity againstS. suisboth in vivo and in vitro; 4 × MIC of floxuridine could killS. suiswithin 8 h in a time–kill assay. Meanwhile, floxuridine disrupted the membrane structure and permeability of the cytoplasmic membrane. Molecular docking revealed that floxuridine and SLY can be directly bind to each other. Moreover, floxuridine effectively inhibited the hemolytic capacity and expression levels of the virulence-related genes ofS. suis. Collectively, these results indicate that the FDA-approved anticancer drug floxuridine is a promising agent and a potential virulence inhibitor againstS. suis.Keywords:floxuridine;Streptococcus suis;mode of action;anti-virulence
研究领域:
微生物学
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药理学
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传染病学
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产品关联
DOI
PubMed
高影响因子
Rice Science
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2023
·
IF 4.8
Yang Xiaozheng,Liu Yusong,Huang Jing,Tao Ye,Wang Yifeng,Shen Renfang,Zhu Xiaofang
Phosphorus (P) starvation in rice facilitates the reutilization of root cell wall P by enhancing the pectin content. NaCl modulates pectin content, however, it is still unknown whether NaCl is also involved in the process of pectin regulated cell wall P remobilization in rice under P starved conditions. In this study, we found that 10 mmol/L NaCl increased the shoot and root biomasses under P deficiency to a remarkable extent, in company with the elevated shoot and root soluble P contents in rice. Further analysis indicated that exogenous NaCl enhanced the root cell wall P mobilization by increasing the pectin methylesterase activity and uronic acid content in pectin suggesting the involvement of NaCl in the process of cell wall P reutilization in P starved rice roots. Additionally, exogenous NaCl up-regulated the expression of P transporter OsPT6, which was induced by P deficiency, suggesting that NaCl also facilitated the P translocation prominently from root to shoot in P starved rice. Moreover, exogenous abscisic acid (ABA) can reverse the NaCl-mediated mitigation under P deficiency, indicating the involvement of ABA in the NaCl regulated root cell wall P reutilization. Taken together, our results demonstrated that NaCl can activate the reutilization of root cell wall P in P starved rice, which is dependent on the ABA accumulation pathway.
研究领域:
植物生理学
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分子生物学
·
农学
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产品关联
DOI
高影响因子
Microbial Cell Factories
·
2022
·Vol. 21
·期 1
·1-13
·
IF 6.352
Xin Qinglong,Chen Yudan,Chen Qianlin,Wang Bin,Pan Li
Background Bacillus amyloliquefaciens is generally recognized as food safe (GRAS) microbial host and important enzyme-producing strain in the industry. B.amyloliquefaciens LB1ba02 is a production strain suitable for secreting mesophilic α-amylase in the industry. Nevertheless, due to the low transformation efficiency and restriction-modification system, the development of its CRISPR tool lags far behind other species and strains from the genus Bacillus . This work was undertaken to develop a fast and efficient gene-editing tool in B.amyloliquefaciens LB1ba02. Results In this study, we fused the nuclease-deficient mutant Cas9n (D10A) of Cas9 with activation-induced cytidine deaminase (AID) and developed a fast and efficient base editing system for the first time in B. amyloliquefaciens LB1ba02. The system was verified by inactivating the pyrF gene coding orotidine 5'-phosphate decarboxylase and the mutant could grow normally on M9 medium supplemented with 5-fluoroorotic acid (5-FOA) and uridine (U). Our base editing system has a 6nt editing window consisting of an all-in-one temperature-sensitive plasmid that facilitates multiple rounds of genome engineering in B. amyloliquefaciens LB1ba02. The total editing efficiency of this method reached 100% and it achieved simultaneous editing of three loci with an efficiency of 53.3%. In addition, based on the base editing CRISPR/Cas9n-AID system, we also developed a single plasmid CRISPR/Cas9n system suitable for rapid gene knockout and integration. The knockout efficiency for a single gene reached 93%. Finally, we generated 4 genes ( aprE , nprE , wprA , and bamHIR ) mutant strain, LB1ba02△4. The mutant strain secreted 1.25-fold more α-amylase into the medium than the wild-type strain. Conclusions The CRISPR/Cas9n-AID and CRISPR/Cas9n systems developed in this work proved to be a fast and efficient genetic manipulation tool in a restriction-modification system and poorly transformable strain.
研究领域:
微生物学
·
基因工程
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生物技术
✓ 已验证
DOI
PubMed
高影响因子
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