JOURNAL OF BIOTECHNOLOGY
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2018
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IF 2.533
Binglin Li,Dandan Duan,Jiao Wang,Huanyu Li,Xiaoli Zhang,Binxia Zhao
Phospholipase D (PLD) was firstly hyperactivated by the bio-imprinting and, then, these hyperactivated PLD molecules were immobilized by adsorption and precipitation, followed by cross-linking. The high degree of conformational rigidification provided by intra- and intermolecular cross-linking can make PLD “remember” imprint-induced characteristics even in aqueous solutions. The obtained immobilized PLD showed the excellent catalytic performance. The maximum activity of immobilized PLD reached 166953 U/g protein , which was approximately 14 times higher than that of free form (11922 U/g protein ). Moreover, the selectivity of PLD was significantly enhanced after immobilization. The yield of PG and PA reached 94.0% and 5.96%, respectively. Compared with the serious hydrolysis in the free PLD (35.3% yield of PA), the side reaction was minimized in this work. This may be the first description of the remarkably high improvement of the activity and selectivity of PLD through the immobilization technology.
研究领域:
生物化学
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酶工程
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产品关联
DOI
PubMed
高影响因子
Catalysts
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2020
·Vol. 10
·期 9
·1057
·
IF 3.52
Jing Wang,Sheng Xu,Yang Pang,Xin Wang,Kequan Chen,Pingkai Ouyang
To achieve efficient bio-production of phospholipase D (PLD), PLDs from different organisms were expressed inE.coli. An efficient secretory expression system was thereby developed for PLD. First, PLDs fromStreptomycesPMF andStreptomyces racemochromogeneswere separately over-expressed inE.colito compare their transphosphatidylation activity based on the synthesis of phosphatidylserine (PS), and PLDPMFwas determined to have higher activity. To further improve PLDPMFsynthesis, a secretory expression system suitable for PLDPMFwas constructed and optimized with different signal peptides. The highest secretory efficiency was observed when the PLD * (PLDPMFwith the native signal peptide Nat removed) was expressed fused with the fusion signal peptide PelB-Nat inE. coli. The fermentation conditions were also investigated to increase the production of recombinant PLD and 10.5 U/mL PLD was ultimately obtained under the optimized conditions. For the application of recombinant PLD to PS synthesis, the PLD properties were characterized and 30.2 g/L of PS was produced after 24 h of bioconversion when 50 g/L phosphatidylcholine (PC) was added.Keywords:phospholipase D;phosphatidylserine;secretory expression;enzymatic catalysis
研究领域:
生物化学
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分子生物学
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产品关联
DOI
高影响因子
APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
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2025
·
IF 3.3
He Ying,Huang Ao,Liu Yun
Phospholipase D (PLD) is a valuable enzyme in industrial processes for converting phosphatidylcholine (PC) to phosphatidylserine (PS). In this study, a strain of Bacillus cereus was isolated from soil and identified through 16S rRNA sequencing. To enhance PLD activity, various mutagenesis strategies—including chemical treatment, irradiation, and their combinations—were employed, resulting in four high-activity positive mutants (C-7, I-12, CI 7–12, and IC 13–14). Among these, the CI 7–12 mutant exhibited a significantly higher enzymatic activity, showing a 3.12-fold increase (312.2%) compared to the wild-type strain. Fermentation conditions were optimied using response surface methodology (RSM), achieving a PLD activity of 35 U/mL. The enzyme demonstrated stability over a wide temperature range (30–60 °C) and pH range (6–10), with a half-life of 128 days. Kinetic analysis revealed a V max of 20.04 μmol/h and a K m of 7.13 μmol/mL, indicating efficient activity. In bioconversion experiments, the PLD-enriched fermentation broth catalyzed the conversion of PC to PS, achieving a 53.0% conversion rate and a 92.3% selectivity for PS in a two-phase system. These findings expand the potential sources of PLD and underscore its applicability for PS production in biotechnological applications.
研究领域:
微生物学
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生物化学
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工业生物技术
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DOI
PubMed
高影响因子
3 Biotech
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2021
·Vol. 11
·期 4
·1-9
·
IF 2.406
Ren Runxian,Zhai Lixin,Tian Qiaopeng,Meng Di,Guan Zhengbin,Cai Yujie,Liao Xiangru
Diphenyl phosphate (DPHP) has been increasingly detected in environmental samples, posing a potential hazard to humans and other organisms and arousing concern regarding its adverse effects. Biological degradation of DPHP is considered a promising and environmentally friendly method for its removal. In this study, the bagdpd gene was mined from the Bacillus altitudinis W3 genome and identified as a glycerophosphodiester phosphodiesterase by bioinformatics analysis. The enzyme was expressed and its biochemical properties were studied. When using bis(4-nitrophenyl) phosphate as substrate, enzyme activity was optimal at 55 °C and a pH of 8.5. The enzyme remained stable in the pH range of 8.0 − 10.0. The rBaGDPD enzyme degraded DPHP and the reaction product was identified as phenyl phosphate by LC–MS. This is the first report of a glycerophosphodiester phosphodiesterase exhibiting hydrolytic activity against DPHP. This study demonstrated that rBaGDPD could have the potential for bioremediation and industrial applications.
研究领域:
环境科学
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微生物学
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生物化学
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DOI
PubMed
高影响因子
INDUSTRIAL & ENGINEERING CHEMISTRY RESEARCH
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2016
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IF 2.567
Binglin Li,Jiao Wang,Xiaoli Zhang,Binxia Zhao
Phospholipase D (PLD) was immobilized in a simple and effective way by adsorption and precipitation of the enzyme, followed by chemical cross-linking to form an “enzyme net” covering the surface of nonporous silicon dioxide nanoparticles. For catalyzing transphosphatidylation to produce phosphatidylethanolamine (PE), at pH 6.0 and 35 °C (the optimum operational conditions), the specific activity of immobilized PLD reached 15872 U/gprotein, which was approximately 1.15 times higher than the maximum value of specific activity of free PLD (13813 U/gprotein). A kinetic study demonstrates immobilized PLD had increases in catalytic activity and enzyme–substrate affinity. In addition, the thermostability and pH tolerance were significantly enhanced compared with free PLD. The half-life of immobilized PLD was significantly increased from 30 to 70 days at 4 °C (approximately 2.3 times). This novel method has been proven to be suitable for the production of robust biocatalysts.
研究领域:
生物化学
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纳米生物学
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产品关联
DOI
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2019
·
IF 4.784
Yan Li,Jia-Qin Wu,Neng-Bing Long,Rui-Feng Zhang
In this article, novel epoxy resin-based hierarchical porous polymers (HPSs) have been prepared through a non-sol-gel and template-free approach using crystalline trimethylolpropane (TMP) as porogen. The polymers exhibit dimensional stability and possess 3-dimentional interconnected multi-scale pores. In range of 50 μm~10 nm are ultra-macro-pore in between skeleton, macro-pore on skeleton and meso-pore in network, respectively. The porosity and specific surface area can be adjusted in range of 91.2–82.5% and 225–156 m 2 /g, respectively. Using three kinds of hierarchical porous polymers as supports phospholipase D (PLD) was effectively immobilized through physical adsorption. Owing to high porosity of the support and improvement of mass transfer the loading amount of PLD reached as high as 223 mg/g support and the corresponding specific activity achieved up to 3.75 × 10 3 U/g support . Under optimized conditions and the phosphatidylserine (PS) yield reached 95.5% within 40 min at 45 °C. The immobilized PLD exhibited not only better storage stability and but also resistance to pH and thermal inactivation than free PLD. It was found that 73.5% of PS yield retained after 12 cycling reuses.
研究领域:
生物化学
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材料科学
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产品关联
DOI
PubMed
高影响因子
APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
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2023
·
IF 3
Zhao Xia,Guo Meijing,Li Xian,Liu Bo,Li Binglin,Wang Jiao
Phospholipase D (PLD) with the higher transphosphatidylation activity was screened from Streptomyces sp. LD0501 basing on the protoplast mutagenesis technology. Then, it was successfully bio-imprinted to form a hyperactivated structure and rigidified by the intramolecular cross-linking, which was immobilized on the nonporous nanoscale silica. Characterization techniques were employed to investigate the structure and physicochemical properties of the catalysts, including Fourier transform infrared (FTIR) spectra and scanning electron microscopy (SEM) analysis. Transphosphatidylation activity and selectivity were improved significantly when immobilized PLD was used. The maximum yield for the production of phosphatidylserine (PS) reached 97% and the side reaction, the hydrolysis, was minimized. These results were further confirmed by the nuclear magnetic resonance (NMR) and mass spectrometry (MS) analysis. The imprint-induced characteristics of PLD was successfully “remembered” even in the present of much water. In addition, this immobilized hyperactivated PLD showed the excellent operational stabilities and environmental tolerances. Graphical abstract
研究领域:
生物化学
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酶工程
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产品关联
DOI
PubMed
高影响因子
ENZYME AND MICROBIAL TECHNOLOGY
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2020
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IF 3.448
Jia-Qin Wu,Xiao-Mei Xu,Ding-Lin Wang,Neng-Bing Long,Rui-Feng Zhang
Novel nano-composites were prepared by coating epoxy resin-based cationic polymer in nano-thickness via in-situ curing on the nano-wall of macroporous SiO 2 with pore size of 0.5∼1 μm. By changing the thickness of polymer coating the specific surface area and porosity varied in range of 115∼74 m 2 /g and 90.4∼83.9 %, respectively. Through ion exchange phospholipase D (PLD, from Streptomyces sp ) was efficiently immobilized on the nano-composites as support and the immobilized PLD was applied for the highly efficient synthesis of phosphatidylserine (PS). The loading amount of PLD on the nano-composited support reached to a maximum of 90.2 mg/g support , 4 times as high as that on the pure macroporous silica. The specific activity of the immobilized PLD reached as high as 16,230 U/g protein , while that of free PLD was 18,780 U/g protein . Under a wide range of temperature and pH the stability and activity of the immobilized PLD were greatly improved as compared with the free ones. Under optimized conditions at 45 °C and pH 7.0, the PS yield reached as high as 96.2 % within 40 min. After 28 days storage the immobilized PLD retained 82.2 % of original activity, and after 12 cycling reuses it retained 79.3 % of PS yield, which indicated that the immobilized PLD exhibited good stability.
研究领域:
生物化学
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酶工程
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DOI
PubMed
高影响因子
PROCESS BIOCHEMISTRY
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2017
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IF 2.497
Wen Qin,Chengjin Wu,Wei Song,Xiulai Chen,Jia Liu,Qiuling Luo,Liming Liu
In this study, an eco-friendly and high-efficient method for the bioconversion of phosphatidylserine (PS) from phosphatidylcholine (PC) and l -serine was developed. After the strain screening, the Streptomyces chromofuscus phospholipase D ( Sc PLD) was chosen and expressed in Escherichia coli BL21 with pET28a (+). After optimization, the maximal specific productivity and Sc PLD hydrolysis activity reached 9049.6 U/g and 10.6 U/mL, respectively, while induction with 2 g/L lactose, at 25 °C for 12 h. The maximal conversions of PC to PS was 93.4% with a final PS concentration of 106.2 g/L (53.1 g/L/h) under theoptimal conditions: 0.153 M PC in CPME, 2.3 M l-ser in sodium acetate buffer, 2.0 g lyophilized cells/mol PC, 100 mM Ca 2+ , 40 °C, and 2 h of incubation. After scale-up to 1 L, the conversions and yield maintained at 85.4% and 97.2 g/L. Therefore, the enzymatic process showed great potential for industrialization of PS.
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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产品关联
DOI
高影响因子
JOURNAL OF THE AMERICAN OIL CHEMISTS SOCIETY
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2018
·Vol. 96
·期 1
·15-23
·
IF 1.601
Saisai Li,Yan Li,Nengbing Long,Ruifeng Zhang
Phospholipase D (PLD) was effectively immobilized on a nanocomposite support composed of ZnO nanowires and SiO 2 through in-situ co-cross-linking with poly-L-lysine (PLL). An anionic and long-chained bisepoxide cross-linker was electrostatically adsorbed on the surface of ZnO nanowires prior to co-cross-linking. Under the optimal pH, 6.5, the loading amount of PLD reached an average of 114 mg per g support for PLL concentrations in the range of 0–0.6 mg mL −1 . Co-cross-linking of PLD with PLL enhanced the activity of PLD remarkably. The highest specific activity of co-cross-linked PLD reached 25.8 U per mg protein , which was 1.62 times as high as that of the cross-linked PLD (15.9 U per mg protein ). (One unit of PLD activity [U] refers to the amount of PLD, which liberates 1 μmol of p-nitrophenol per min via hydrolysis of phosphatidyl-p-nitrophenol at pH 8.0 and 30 °C). Under the optimized conditions, the yield of phosphatidylglycerol (PtdGro) reached 98.6% within 80 min at 45 °C and pH 6.5. The co-cross-linked PLD not only exhibited good resistance to pH and heat inactivation but also improved storage stability and reusability. The yield of PtdGro remained ≥85.4% after 13 repeat uses.
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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DOI
高影响因子
Bioengineered
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2017
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IF 1.691
Wen-Bin Zhou,Jin-Song Gong,Hai-Juan Hou,Heng Li,Zhen-Ming Lu,Hong-Yu Xu,Zheng-Hong Xu,Jin-Song Shi
Phosphatidylserine (PS) is useful as the additive in industries for memory improvement, mood enhancement and drug delivery. Conventionally, PS was extracted from soybeans, vegetable oils, egg yolk, and biomass; however, their low availability and high extraction cost were limiting factors. Phospholipase D (PLD) is a promising tool for enzymatic synthesis of PS due to its transphosphatidylation activity. In this contribution, a new and uncharacterized PLD was first obtained from GenBank database via genome mining strategy. The open reading frame consisted of 1614 bp and potentially encoded a protein of 538-amino-acid with a theoretical molecular mass of 60 kDa. The gene was successfully cloned and expressed in Escherichia coli. Its enzymatic properties were experimentally characterized. The temperature and pH optima of PLD were determined to be 60°C and 7.5, respectively. Its hydrolytic activity was improved by addition of Ca2+ at 5 mM as compared with the control. The enzyme displayed suitable transphosphatidylation activity and PS could be synthesized with L-serine and soybean lecithin as substrates under the catalysis of PLD. This PLD enzyme might be a potential candidate for industrial applications in PS production. To the best of our knowledge, this is the first report on genome mining of PLDs from GenBank database.
研究领域:
生物化学
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分子生物学
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工业酶学
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产品关联
DOI
PubMed
高影响因子
BIOCHEMICAL ENGINEERING JOURNAL
·
2024
·
IF 3.7
Yun Liu,Ao Huang,Xiaowei Wen
Phospholipase D (PLD) is essential for the bioconversion of phosphatidylcholine (PC) to phosphatidylserine (PS), a process valuable in functional food and medicine. This study explores the stability and catalytic properties of PLD immobilized on chitosan-encapsulated magnetic nanoparticles (CMNPs), utilizing oxidized dextran (DX) and glutaraldehyde (Glu) as cross-linkers. The cross-linker concentration and immobilization time were optimized to assess their effects on PLD catalytic performance. PLD immobilized on CMNPs with DX (DX-CMNPs-PLD) exhibited optimal activity at pH 8.0 and 30 °C, retaining over 40 % activity after 14 cycles, while Glu-cross-linked PLD (Glu-CMNPs-PLD) retained approximately 65 %. DX-CMNPs-PLD demonstrated superior pH, temperature, and operational stability compared to free PLD. Additionally, the immobilized PLD was characterized using transmission electron microscopy, X-ray diffraction, and Fourier-transform infrared spectroscopy. Kinetics parameters (V max and K m ) of the immobilized PLD were also studied with free PLD serving as a control. Conformational analyses indicated a significant change in PLD's secondary structure, particularly in β-sheet content, which likely contributed to the enhanced stability and activity. These findings suggest a promising approach for PLD immobilization on CMNPs, with notable implications for biotechnological applications.
研究领域:
生物技术
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食品科学
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医学
✓ 已验证
DOI
高影响因子
危险品化学品经营许可证（带存储）