CARBOHYDRATE POLYMERS
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2023
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IF 11.2
Guoguang Wei,Chi Zhang,Ning Zhou,Bin Wu,Hui Li,Alei Zhang,Pingkai Ouyang,Kequan Chen
In this study, co-immobilization of PLP and its dependent enzyme were investigated using a novel type of porous chitin bead (PCB). Crayfish shell was used to prepare PCB via dissolution of it to form beads, followed by the removal of CaCO 3 and protein in-situ. Scanning electron microscopy, Fourier transform infrared spectroscopy , and Brunauer-Emmett-Teller method showed that the PCB had abundant porous structures with deacetylation degree of 33 % and the specific surface area of 35.87 m 2 /g. Then, the beads are used to co-immobilize pyridoxal 5-phosphate (PLP) and l -lysine decarboxylase fused with chitin-binding protein ( Sp LDC-ChBD). Laser scanning confocal microscopy revealed that the beads could co-immobilize PLP and Sp LDC-ChBD successfully. In addition, a packed bed was also constructed using the PCB containing co-immobilized Sp LDC-ChBD and PLP. The substrate conversion remained at 91.09 % after 48 h with 50 g/L l -lysine, which showed good continuous catalysis ability. This study provides a novel method for co-immobilization of enzyme and PLP, as well as develops a new application of waste crustacean shells.
研究领域:
生物化学
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酶工程
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材料科学
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PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
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2023
·Vol. 89
·期 8
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IF 4.4
Ping Lu,Xiaoli Dong,Xuemeng Ji
Cronobacter sakazakii is an opportunistic pathogen capable of causing severe infections, particularly in neonates. Despite the bacterium’s strong pathogenicity, the pathogenicity of C. sakazakii is not yet well understood. Using a comparative proteomic profiling approach, we successfully identified pdxY, encoding a pyridoxal kinase involved in the recycling of pyridoxal 5′-phosphate (PLP), as a gene essential for the successful pathogenesis of C. sakazakii. Knocking out the pdxY gene resulted in slower growth and reduced virulence. Our study sheds light on the fundamental importance of pyridoxal kinase for the survival and virulence of C. sakazakii. The identification of pdxY as gene essential for successful pathogenesis provides a potential target for the development of new antibiotic treatments.IMPORTANCE The opportunistic pathogen Cronobacter sakazakii is known to cause severe infections, particularly in neonates, and can result in high mortality rates. In this study, we used a comparative proteomic profiling approach to identify genes essential for the successful pathogenesis of C. sakazakii. We successfully identified pdxY, encoding a pyridoxal kinase involved in the salvage pathway of pyridoxal 5′-phosphate (PLP), as a gene essential for the successful pathogenesis of C. sakazakii. Knocking out the pdxY gene resulted in impaired growth and reduced virulence. This study sheds light on the fundamental importance of pyridoxal kinase for the survival and virulence of C. sakazakii, which can be a potential target for the development of new antibiotic treatments. This study highlights the importance of comparative proteomic profiling in identifying virulence factors that can be targeted for the development of new antibiotics.
研究领域:
微生物学
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分子生物学
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病理学
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蛋白质组学
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传染病学
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高影响因子
ENZYME AND MICROBIAL TECHNOLOGY
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2020
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IF 3.448
Wenlong Zheng,Kaitong Chen,Sai Fang,Xiuli Cheng,Gang Xu,Lirong Yang,Jianping Wu
A number of organic synthesis involve threonine aldolase (TA), a pyridoxal phosphate (PLP)-dependent enzyme. Although the addition of exogenous PLP is necessary for the reactions, it increases the cost and complicates the purification of the product. This work constructed a PLP self-sufficient biocatalysis system for TA, which included an improvement of the intracellular PLP level and co-immobilization of TA with PLP. Engineered strain BL-ST was constructed by introducing PLP synthase PdxS/T to Escherichia coli BL21(ED3). The intracellular PLP concentration of the strain increased approximately fivefold to 48.5 μmol/g DCW . l -TA, from Bacillus nealsonii ( Bn LTA), was co-expressed in the strain BL-ST with PdxS/T, resulting in the engineered strain BL- Bn LTA-ST. Compared with the control strain BL- Bn LTA (254.1 U/L), the enzyme activity of the strain BL- Bn LTA-ST reached 1518.4 U/L without the addition of exogenous PLP. An efficient co-immobilization system was then designed. The epoxy resin LX-1000HFA wrapped by polyethyleneimine (PEI) acted as a carrier to immobilize the crude enzyme solution of the strain BL- Bn LTA-ST mixed with an extra 100 μM of exogenous PLP, resulting in the catalyst HFA PEI - Bn LTA-STPLP 100. HFA PEI - Bn LTA-STPLP 100 exhibited a half-life of approximately 450 h, and the application of the catalyst in the continuous biosynthesis of 3-[4-(methylsulfonyl) phenyl] serine had more than 180 batch reactions (>60% conv ) without the extra addition of exogenous PLP. The excellent compatibility and stability of the system were further confirmed by other TAs. This work introduced a PLP self-sufficient biocatalysis system that can reduce the cost of PLP and contribute to the industrial application of TA. In addition, the system may also be applied in other PLP-dependent enzymes.
研究领域:
生物化学
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酶工程
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工业微生物学
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高影响因子
Journal of Chemical Information and Modeling
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2025
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IF 5.3
Binbin Chen,Jiahui Huang,Yingchun Liu,Lirong Yang,Qi Wang,Haoran Yu
The catalytic mechanism of a pyridoxal 5′-phosphate (PLP)-dependent UstD was herein studied in atomic detail, employing the computational hybrid QM/MM methodology. UstD is a PLP-dependent enzyme that catalyzes the decarboxylative aldol reactions between l-aspartate and aldehyde or ketone derivatives to form γ-hydroxy-α-amino acids. In the reaction catalyzed by UstD, the loss of CO2 renders the C–C bond-forming reaction effectively irreversible, which makes UstD a special case among the enzymes catalyzing the C–C bond-forming reactions. This enzyme is currently seen as the optimal approach for the regioselective synthesis of γ-hydroxy-α-amino acids, which are very difficult to obtain by standard chemical methods. The results obtained herein showed that the catalytic mechanism of UstD might follow two paths to occur in three phases: (1) decarboxylation of substrate l-aspartate, (2) C–C bond formation by addition of aldehyde, and (3) the regeneration of catalytic sites. Although Path A and Path B showed a negligible difference in the energy barrier of the rate-determining step, Path A involves three additional steps in the overall pathway compared with Path B, which makes the reaction proceed more readily through Path B. According to the QM/MM energy profile of Path B, the rate-limiting step of the catalytic process is the decarboxylation of the side chain of l-aspartate, which has a calculated energy barrier of 19.19 kcal/mol. Two crucial residues, H263 and Y257, were identified to interact with the substrate aspartic acid. The knowledge about the transition states, intermediates, key residues, and protein conformational changes along the reaction path will be valuable for engineering UstD to improve the synthesis of γ-hydroxy-α-amino acids that serve as building blocks of various high-value chemicals such as antidiabetics and nutritional supplements.
研究领域:
生物化学
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计算生物学
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高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2026
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IF 8.7
Ping Liang,Bingke Li,Guorui Liu,Junran Xu,Qingyue Zhang,Yuchao Chen,Xinling Wu,Haoming Zhang,Zhongyu Li,Zhihui Li
Pyridoxal 5′-phosphate (PLP) serves as a dual crosslinker forming Schiff bases and electrostatic interactions. • Nanogels show pH-triggered release: 88.5% at pH 5.0 vs. 23.5% at pH 7.0. • TMX@CS-PLP NGs improve foliar wettability, rainfastness, and insecticidal efficacy. • The biocompatible nanogel platform enables intelligent, eco-friendly pesticide delivery. Conventional pesticide formulations suffer from poor targeting and low active ingredient utilization due to their inability to respond to microenvironmental signals at pest feeding sites. Here, we developed pH-responsive nanogels by crosslinking chitosan with PLP via Schiff base formation and electrostatic interactions using an inverse emulsion method, and loaded them with the neonicotinoid insecticide thiamethoxam (TMX). The resulting TMX@CS-PLP nanogels exhibited spherical morphology with a hydrodynamic diameter of 428.1 nm, a loading capacity of 9.2%, and an encapsulation efficiency of 79.4%. The Schiff base crosslinks imparted pH-dependent degradability: at acidic pH (5.0, simulating pest gut or infection sites), the nanogels released 88.5% of encapsulated TMX over 72 h, whereas only 23.5% was released at neutral pH (7.0, mimicking leaf surface conditions). The nanogel formulation also improved foliar wettability (contact angle reduced from 84.5o to 68.1o) and enhanced rainfastness compared with the technical suspension. In laboratory bioassays against Myzus persicae , TMX@CS-PLP nanogels caused 76.0% mortality after 24 h, significantly higher than the technical suspension (46.7%), while the blank nanogel showed no phytotoxicity or inherent insecticidal activity. These results demonstrate that PLP-crosslinked chitosan nanogels offer a biocompatible, pH-responsive platform for precision pesticide delivery, with enhanced foliar deposition and improved efficacy. Download: Download high-res image (124KB) Download: Download full-size image
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高影响因子
Synthetic and Systems Biotechnology
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2025
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IF 4.4
Yanghao Liu,Changgeng Li,Yu Miao,Zhichao Chen,Tangen Shi,Ling Ma,Siyu Zhao,Zichen Yu,Lanxiao Li,Qingyang Xu
5-Aminolevulinic acid (5-ALA) is an important non-proteinogenic amino acid with wide applications in agriculture and medicine. To achieve high-level microbial production, this study established a dual-pathway reconstruction strategy in Escherichia coli by integrating the endogenous C5 pathway with an inducible exogenous C4 pathway. Multi-copy overexpression of gltX , hemA , and hemL , combined with enhanced glutamate supply and the introduction of a non-oxidative glycolysis pathway, effectively increased the C5 pathway flux and carbon efficiency, while product toxicity was significantly alleviated by strengthening efflux mechanisms and oxidative stress tolerance. A quorum sensing–based regulatory system was employed to dynamically regulate hemB expression, thereby balancing cell growth and 5-ALA biosynthesis, and a controlled glycine feeding strategy specifically activated the C4 pathway during the later fermentation stage, forming a stage-specific dynamic activation mechanism. Promoter regulation of sucC/sucD expression and enhanced endogenous PLP biosynthesis further stabilized the C4 flux. Fed-batch fermentation in a 5 L bioreactor resulted in a final 5-ALA titer of 37.34 g/L, demonstrating the industrial potential of this systems metabolic engineering strategy combining dual pathways and stage-specific activation control.
研究领域:
合成生物学
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代谢工程
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生物技术
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微生物学
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高影响因子
ENZYME AND MICROBIAL TECHNOLOGY
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2023
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IF 3.4
Fangfang Chang,Yuheng Wang,Jie Zhang,Tao Tu,Huiying Luo,Huoqing Huang,Yingguo Bai,Xing Qin,Yaru Wang,Bin Yao,Yuan Wang,Xiaolu Wang
γ-Aminobutyric acid (GABA) has been widely used in the food, feed, pharmaceutical, and chemical industry fields. Previously, we developed a whole-cell catalyst capable of converting L-glutamate (L-Glu) into GABA by overexpressing the glutamate decarboxylase gene ( gadz11 ) from Bacillus sp. Z11 in Escherichia coli BL21(DE3). However, to enhance cell permeability , a freeze-thaw treatment is required, and to enhance GADZ11 activity, pyridoxal 5′-phosphate (PLP) must be added to the reaction system. The aim of this study is to provide a more efficient approach for GABA production by engineering the recombinant E. coli above. First, the inducible expression conditions of the gadz11 in E. coli were optimized to 37 °C for 6 h. Next, an ideal engineered strain was produced via increasing cell permeability by overexpressing sulA and eliminating PLP dependence by constructing a self-sufficient system. Furthermore, an efficient whole-cell biocatalytic process was optimized. The optimal substrate concentration , cell density, and reaction temperature were 1.0 mol/L (the molecular ratio of L-Glu to L-monosodium glutamate (L-MSG) was 4:1), 15 and 37 °C, respectively. Finally, a whole-cell bioconversion procedure was performed in a 3-L bioreactor under optimal conditions. The strain could be reused for at least two cycles with GABA yield, productivity and conversion ratio of 206.2 g/L, 117.8 g/L/h and 100.0%, respectively. This is currently the highest GABA productivity from a mixture of L-Glu and L-MSG reported without the addition of cofactors or additional treatment of cells. This work demonstrates that the novel engineered E. coli strain has the potential for application in large-scale industrial GABA production.
研究领域:
生物技术
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微生物学
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生物化工
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代谢工程
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DOI
PubMed
高影响因子
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