JOURNAL OF CHEMICAL TECHNOLOGY AND BIOTECHNOLOGY
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2018
·Vol. 93
·期 11
·3135-3142
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IF 2.587
Lingqia Su,Tian Wang,Lingang Yu,Jing Wu
BACKGROUND We previously showed that extracellular production of naturally cytoplasmic proteins could be realized through co-expression with phospholipase C (PLC), which enhances cell membrane permeability through limited hydrolysis of membrane phospholipids. Leuconostoc mesenteroides sucrose phosphorylase (SP Lc ), a naturally cytoplasmic enzyme, exhibits excellent properties for synthesis of α-arbutin, but no systemic investigation of its preparation has thus far been reported. RESULTS Three PLCs were selected and expressed, without signal peptide, in Escherichia coli . More than 90% of the PLCs from Listeria monocytogenes and Bacillus cereus were present in the culture medium. When SP Lc was co-expressed with these PLCs, and the location of the PLC and SP Lc genes in the co-expression vector was optimized, the highest production was obtained when SP Lc gene was inserted upstream of the B. cereus PLC gene, as in BL21(DE3)/pETDuet -sp - bcplc . SP Lc production was then scaled up to a 3 L fermenter and fermentation conditions were optimized. When the initial medium contained 0.6 mmol L −1 ZnCl 2 and protein expression was induced at a dry cell weight of 15 g L −1 by the addition of lactose at 0.2 g L −1 h −1 at 30 °C, the extracellular SP Lc activity reached its highest level of 1445.1 U mL −1 This constituted 81.6% of total activity; the remainder of the enzyme (18.4%) remained inside the cells. CONCLUSION This is the first report describing extracellular expression of sucrose phosphorylase, and the highest yield ever reported was obtained. This provides the basis for industrial-scale production and application of sucrose phosphorylase. It also provides a potential method for improving the production of other proteins and fine chemicals. © 2018 Society of Chemical Industry
研究领域:
生物化学工程
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微生物学
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酶学
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生物技术
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DOI
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2022
·
IF 5.895
Fenghua Wang,Zehui Guo,Zixuan Yang,Xueying Li,Xue Zhang,Xiangyang Ma,Zhuoxuan Han,Fuping Lu,Yihan Liu
Ceramide is a natural functional ingredient as food additive and medicine that has attracted extensive attention in the food, medical, and cosmetic industries. Here, we developed a biotechnological strategy based on a recombinant whole-cell biocatalyst for efficiently producing ceramide from crude soybean oil sediment (CSOS) waste. A novel phospholipase C (PLCac) from Acinetobacter calcoaceticus isolated from soil samples was identified and characterized. Furthermore, recombinant Komagataella phaffii displaying PLCac (dPLCac) on the cell surface was constructed as a whole-cell biocatalyst with better thermostability (30–60 °C) and pH stability (8.0–10.0) to successfully produce ceramide. After synergistical optimization of reaction time and dPLCac dose, the ceramide yield of hydrolyzing from CSOS using dPLCac was 51% (the theoretical maximum yield of converting sphingomyelin, ∼70%) and the relative yield was over 50% after seven consecutive 4 h batches under the optimized conditions. Our study provides a potentially promising strategy for the commercial production of ceramide.
研究领域:
生物技术
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生物化学
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环境科学
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食品科学
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制药科学
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化妆品科学
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DOI
PubMed
高影响因子
Frontiers in Pharmacology
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2024
·
IF 4.4
Wang Mengting,Mao Haiguang,Ke Zhijian,Wang Jinbo
Background: The hypoglycemic effects of Chinese bayberry leaves proanthocyanidins (BLPs) have been demonstrated. It is unclear, nevertheless, whether BLPs reduced postprandial blood glucose levels by regulating glucose uptake and glucose transport.
Method: This study investigated the effect of BLPs (25, 50 and 100 μg/mL) on glucose uptake and glucose transport in human intestinal epithelial cells (Caco-2 cells). The uptake of 2-Deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-D-glucose (2-NBDG) and disaccharidases activity in Caco-2 cells were measured. The glucose transport ability across the cell membrane was determined using the established Caco-2 monolayer model. The transcript and protein levels of key glucose transporters were analyzed using real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting, respectively.
Results: The results showed that BLPs significantly decreased glucose uptake and disaccharidases activity (p<0.05). Otherwise, BLPs treatment obviously inhibited glucose transport across the Caco-2 monolayer in both simulated-fast (5 mM glucose) and simulated-fed (25 mM glucose) conditions. It was attributed to the suppression of glucose transporter2 (GLUT2) and sodium-dependent glucose cotransporter 1 (SGLT1) by BLPs. BLPs were found to significantly downregulated the transcript level and protein expression of glucose transporters (p<0.05). Meanwhile, the mRNA expression of phospholipase C (PLC) and protein kinase C (PKC) involved in the signaling pathway associated with glucose transport were decreased by BLPs.
Conclusion: These results suggested that BLPs inhibited intestinal glucose transport via inhibiting the expression of glucose transporters. It indicated that BLPs could be potentially used as a functional food in the diet to modulate postprandial hyperglycemia.
研究领域:
药理学
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营养学
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细胞生物学
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DOI
PubMed
高影响因子
ANALYST
·
2023
·Vol. 148
·期 10
·2352-2361
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IF 4.2
Xiwen Li,Peiran Meng,Mingyang Sun,Yue Chen,Zhiyi Song,Xinyao Wang,Na Li,Yue Sun
Phospholipase C (PLC) has important biological functions in specific cancer types, immune disorders and neurodegeneration. Here, an ultrasensitive electrochemical sensor for PLC was developed via signal amplification based on breathing atom transfer radical polymerization (ATRP). First, phosphatidylethanolamine (PE) was immobilized on the surface of a gold electrode by L-cysteine and cross-linker. Then, PE was specially hydrolyzed by PLC to obtain the phosphate groups and tethered with the ATRP initiator α-bromophenacetic acid (BPAA) by the coordination action of Zr4+. After the breathing ATRP, a large number of electroactive monomers (ferrocenylmethyl methacrylate, FcMMA) were successfully grafted from BPAA. The experimental results indicated that the detection signal of the obtained electrode (sensor) was proportional to the concentration of PLC. The sensor showed a low detection limit of 0.270 U L−1 and a wide linear range of 1–40 U L−1 (R2 = 0.997). Most importantly, the sensor was successfully applied to detect PLC in breast cancer cells (MCF-7, MDA-MB-231) and nontumor cells (MCF-10A). The value obtained by our electrochemical sensor had no obvious difference from that determined by the commercial ELISA kit. These results showed that the fabricated PLC sensor had acceptable potential in clinical applications.
研究领域:
生物化学
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生物技术
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分析化学
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癌症研究
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免疫学
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神经科学
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DOI
PubMed
高影响因子
STAR Protocols
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2023
·
IF 0
Huan Li,Jie Yang,Jingfeng Li,Zhenxing Gao,Jiang Xu,Chaoyang Li
Summary Defects of glycosylphosphatidylinositol (GPI)-anchor synthesis lead to the production of pro-proteins with altered functions. However, pro-protein-specific antibodies for functional analysis are lacking. Here, we present a protocol to differentiate GPI-anchored prion protein (PrP) from pro-PrP in cancer cells using a complementary approach applicable to other GPI-anchored proteins. We first describe steps for phosphatidylinositol-specific phospholipase C treatment and flow-cytometry-based detection. We then detail the carboxypeptidase Y (CPDY) assay including antibody immobilization, affinity purification, CPDY treatment, and western-blot-based detection. For complete details on the use and execution of this protocol, please refer to Li et al. (2022). 1
研究领域:
分子生物学
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细胞生物学
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癌症研究
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生物化学
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DOI
PubMed
Analytical Methods
·
2019
·Vol. 11
·期 42
·5398-5404
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IF 2.378
Shengting Zhang,Mingrui Li,Dongbo Liu,Kunhao Qin,Yingying Xiang,Xiuling Ji,Congwen Li,Yunlin Wei
The human purinergic P2Y1 receptor (P2Y1R) belongs to the family of G-protein coupled receptors (GPCRs). Two stable cell lines, hP2Y1R-tGFP/U2OS and hP2Y1R-β-AR2-tGFP/U2OS, were generated with steady expression of fluorescent tGFP tagged P2Y1R or tGFP tagged β-arrestin2. The High Content Screening (HCS) technique was used to investigate P2Y1R mediated signal transduction processes based on the distribution of fluorescent receptors or the β-arrestin2/receptor complex. All procedures including aggregation, endolysis and resensitization that were involved in the interaction between the P2Y1 receptor and its ligand can be visualized using the tGFP labeled receptor combined with HCS technology in this study. ADP activated P2Y1R to induce a distinguished pattern of fluorescent vesicle formation inside both cell types. These two cell lines produced different peak fluorescence signals when activated by ADP at 100 μM, and the time to form the maximal vesicles was detected as 15 and 20 min respectively. The EC50 values of the concentration–response curves of ADP induced formation of fluorescent vesicles were 35.7 nM and 71.8 nM respectively. The ADP effects could be blocked by a P2Y1R specific antagonist MRS2179. The P2Y1R are coupled to Gq protein to activate PLC first, PLC cleave PIP2 into IP3 and DAG. And then DAG and Ca2+ activate PKC pathway, and finally the phospholipase D initiates β-arrestin2 mediated signal pathway. It took 30 and 45 min to complete P2Y1R re-sensitization in these two constructs respectively. Our results demonstrated that P2Y1R triggered downstream responses not only via heterotrimeric Gq proteins but also through β-arrestin2.
研究领域:
细胞生物学
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药理学
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分子生物学
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信号转导
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DOI
高影响因子
MICROCHEMICAL JOURNAL
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2023
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IF 4.8
Xiwen Li,Zuan Yang,Peiran Meng,Shipeng Jiang,Mingyang Sun,Xiaoyu Zhang,Yue Sun
An ultrasensitive electrochemical sensor for phospholipase C (PLC) was developed via dual signal amplification based on metal-free visible-light induced atom transfer radical polymerization (MVL ATRP) and Ag nanoparticles (AgNPs). The environment-friendly MVL ATRP method was used to form aldehyde based polymers on the electrode surface, providing more reaction sites for further silver mirror reaction. In addition, a large number of electrically active AgNPs were obtained through efficient and low-cost silver mirror reaction, which improved the detection signal. Under the optimal conditions, the proposed sensor showed a linear response from 1 mU/L to 10 6 mU/L for PLC with a detection limit of 0.1032 mU/L (S/N = 3). Most importantly, the sensor was successfully applied to detect PLC in non-tumor cells (MCF-10A) and tumor cells (MCF-7, MDA-MB-231). The obtained values had no obvious difference with that determined by commercial ELISA kit. When 0.5–10 U/L standard PLC were added into the above cells extracts, the recovery rate was between 96.1% and 102.9%, and the RSD was less than 4.02%. The results showed that the fabricated PLC sensor had acceptable potential in clinical applications.
研究领域:
分析化学
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生物化学
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纳米技术
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生物医学工程
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产品关联
DOI
高影响因子
FOOD CONTROL
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2023
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IF 6
Yuge Guan,Xinghua Lu,Jiyu Cheng,Sainan Lu,Liqing Yin,Junfeng Cheng,Mingyi Yang,Yunjie Chen,Jian Sun,Guoquan Lu,Linjiang Pang
The quality deterioration of sweetpotatoes easily during postharvest storage owing to their high respiration rate and accumulation of reactive oxygen species (ROS). In this study, an edible coating (MCW) assembled using montmorillonite, chitosan, and whey protein isolate was fabricated to improve the postharvest quality of sweetpotatoes, and the regulatory mechanism of the edible coating treatment on sweetpotato quality has investigated. The results confirmed that edible coating treatment had a positive effect on maintaining the appearance, firmness, colour, and sensory quality of sweetpotatoes, especially in maintaining free water, starch and soluble protein content, inhibiting respiration rate and delaying weight loss of sweetpotatoes stored at 25 °C for 30 d. Meanwhile, this study illuminated the MCW coating regulates ROS metabolism by upregulating superoxide dismutase, ascorbate peroxidase, and glutathione reductase gene expression, resulting in lower O 2 −. content in postharvest sweetpotato. Furthermore, the relative expression of the genes encoding lipoxygenase, phospholipase C, and phospholipase D was downregulated by the edible coating treatment and contributed to lower enzyme activity, electrolyte leakage and malondialdehyde content, which further reduced the degree of membrane lipid peroxidation and effectively maintained the structure of plasma membrane and then protected the cell integrity of sweetpotato. Collectively, this study provides useful information at the transcriptional level that MCW coating can enhance the post-harvest quality of sweetpotatoes by regulating ROS and membrane lipid metabolism.
研究领域:
农业科学
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食品科学
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植物生理学
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采后技术
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产品关联
DOI
高影响因子
MOLECULES
·
2026
·Vol. 31
·期 5
·816
·
IF 5.1
Xuefeng Li,Wenlong Yuan,Lin Mei,Yufei Zhang,Lei Ping,Mingming Zheng,Shengjiao Yu,Xi Du,Jun Wei
This study explores the application of immobilized phospholipase A1 (PLA1) on hollow double-layer mesoporous silica nanoparticles (PLA1@NH2/C8-HdlMS) for the degumming of crude arachidonic acid (ARA) oil for the first time. The immobilized enzyme was comprehensively characterized, and the reaction conditions were optimized via single-factor experiments. Under the optimized conditions (enzyme dosage 0.3%w/w, 35 °C, water addition 3%, and reaction time 90 min), PLA1@NH2/C8-HdlMS achieved a remarkable phosphorus removal rate of 97.9%, reducing the phosphorus content from 441.21 mg/kg to 9.29 mg/kg in 90 min (well below the food-grade standard of <10 mg/kg). The fatty acid composition of the oil remained almost unchanged, while the oxidative induction time of the degummed oil significantly improved by 42%. Notably, PLA1@NH2/C8-HdlMS demonstrated broad applicability across crude oils, with initial phosphorus contents ranging from 294.98 mg/kg to 537.44 mg/kg, and it maintained ~93% of its initial activity after 11 reuse cycles. Compared to traditional hydration degumming (with a phosphorus removal rate of 56.3%), this enzymatic method offers superior efficiency at lower temperatures, minimizing energy consumption and the thermal degradation of ARA. This green, efficient, and sustainable method for degumming heat-sensitive oils offers significant potential for the industrial application of high-quality functional oils by preserving PUFA integrity and reducing environmental impact.
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产品关联
DOI
PubMed
高影响因子
Journal of Nanophotonics
·
2026
·
IF 1
Zhi Sun,Yu Zhao,Tao Chen,Shuwen Zeng
In this study, we present a biosensor that employs a microfluidic approach to fabricate nematic liquid crystal (NLC) microspheres for the detection of phospholipase A1 (PLA1), a biomarker linked to Alzheimer’s disease (AD). We created uniform-diameter NLC microspheres utilizing a custom-designed microfluidic chip and functionalized them with phosphatidylcholine, which facilitates a radial arrangement. Following this, the NLC microspheres underwent a transition from a radial to a bipolar arrangement due to the hydrolysis of PC by PLA1. This method allows for direct observation of the detection process using a polarizing microscope. Image binarization is subsequently applied to enhance detection sensitivity, achieving a detection limit for PLA1 of 400 pg/μL. Furthermore, the detection time is reduced by more than twelve-fold compared with traditional methods. The rapid response capability of NLC microspheres demonstrates significant potential for the detection of various diseases and biomarkers. Our research establishes a foundation for the further development of a detection platform for the AD marker PLA1, paving the way for the creation of a low-cost, rapid, and portable diagnostic tool.
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DOI
高影响因子
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