Whole-cell catalytic synthesis of 2-O-α-glucopyranosyl-l-ascorbic acid by sucrose phosphorylase from Bifidobacterium breve via a batch-feeding strategy

2022 ◽  
Vol 112 ◽  
pp. 27-34
Author(s):  
Yaoyao Zhou ◽  
Tian Gan ◽  
Ruini Jiang ◽  
Hanchi Chen ◽  
Zhi Ma ◽  
...  
Keyword(s):  
Ascorbic Acid ◽  
Feeding Strategy ◽  
Catalytic Synthesis ◽  
Sucrose Phosphorylase ◽  
Whole Cell ◽  
Bifidobacterium Breve

scholarly journals Whole cell-based catalyst for enzymatic production of the osmolyte 2-O-α-glucosylglycerol

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Katharina N. Schwaiger ◽  
Monika Cserjan-Puschmann ◽  
Gerald Striedner ◽  
Bernd Nidetzky
Keyword(s):  
Specific Activity ◽  
Dry Mass ◽  
High Yield ◽  
Sucrose Phosphorylase ◽  
Bifidobacterium Adolescentis ◽  
Enzymatic Production ◽  
Intracellular Enzyme ◽  
Whole Cell ◽  
Freeze Thaw ◽  
Thaw Cycle

Abstract Background Glucosylglycerol (2-O-α-d-glucosyl-sn-glycerol; GG) is a natural osmolyte from bacteria and plants. It has promising applications as cosmetic and food-and-feed ingredient. Due to its natural scarcity, GG must be prepared through dedicated synthesis, and an industrial bioprocess for GG production has been implemented. This process uses sucrose phosphorylase (SucP)-catalyzed glycosylation of glycerol from sucrose, applying the isolated enzyme in immobilized form. A whole cell-based enzyme formulation might constitute an advanced catalyst for GG production. Here, recombinant production in Escherichia coli BL21(DE3) was compared systematically for the SucPs from Leuconostoc mesenteroides (LmSucP) and Bifidobacterium adolescentis (BaSucP) with the purpose of whole cell catalyst development. Results Expression from pQE30 and pET21 plasmids in E. coli BL21(DE3) gave recombinant protein at 40–50% share of total intracellular protein, with the monomeric LmSucP mostly soluble (≥ 80%) and the homodimeric BaSucP more prominently insoluble (~ 40%). The cell lysate specific activity of LmSucP was 2.8-fold (pET21; 70 ± 24 U/mg; N = 5) and 1.4-fold (pQE30; 54 ± 9 U/mg, N = 5) higher than that of BaSucP. Synthesis reactions revealed LmSucP was more regio-selective for glycerol glycosylation (~ 88%; position O2 compared to O1) than BaSucP (~ 66%), thus identifying LmSucP as the enzyme of choice for GG production. Fed-batch bioreactor cultivations at controlled low specific growth rate (µ = 0.05 h−1; 28 °C) for LmSucP production (pET21) yielded ~ 40 g cell dry mass (CDM)/L with an activity of 2.0 × 104 U/g CDM, corresponding to 39 U/mg protein. The same production from the pQE30 plasmid gave a lower yield of 6.5 × 103 U/g CDM, equivalent to 13 U/mg. A single freeze–thaw cycle exposed ~ 70% of the intracellular enzyme activity for GG production (~ 65 g/L, ~ 90% yield from sucrose), without releasing it from the cells during the reaction. Conclusions Compared to BaSucP, LmSucP is preferred for regio-selective GG production. Expression from pET21 and pQE30 plasmids enables high-yield bioreactor production of the enzyme as a whole cell catalyst. The freeze–thaw treated cells represent a highly active, solid formulation of the LmSucP for practical synthesis.

Characterisation of a Thermobacillus sucrose phosphorylase and its utility in enzymatic synthesis of 2-O-α-d-glucopyranosyl-l- ascorbic acid

2019 ◽  
Vol 305 ◽  
pp. 27-34
Author(s):  
Yan Li ◽  
Zheng Li ◽  
Xiaoying He ◽  
Liangliang Chen ◽  
Yinchu Cheng ◽  
...  
Keyword(s):  
Ascorbic Acid ◽  
Enzymatic Synthesis ◽  
Sucrose Phosphorylase

Oxidative Downmodulation of the Transient K-CurrentI A by Intracellular Arachidonic Acid in Rat Hippocampal Neurons

1999 ◽  
Vol 82 (1) ◽  
pp. 508-511 ◽  
Author(s):  
Katrin Bittner ◽  
Wolfgang Müller
Keyword(s):  
Oxidative Stress ◽  
Ascorbic Acid ◽  
Arachidonic Acid ◽  
Patch Clamp ◽  
Hippocampal Neurons ◽  
Whole Cell ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp ◽  
Intracellular Glutathione

Membrane-permeable arachidonic acid (AA) is liberated in a Ca2+-dependent way inside cells. By using whole cell patch clamp we show that intracellular AA (1 pM) selectively reduces I A in rat hippocampal neurons, whereas extracellular application requires a 106-fold concentration. The nonmetabolized AA analogue ETYA mimics the effect of AA that is blocked by ascorbic acid or intracellular glutathione, suggesting an intracellular oxidative mechanism. We conclude that intracellular AA is extremely potent in reducing I A by an oxidative mechanism, particularly during oxidative stress.

Batch-feeding whole-cell catalytic synthesis of α-arbutin by amylosucrase from Xanthomonas campestris

2019 ◽  
Vol 46 (6) ◽  
pp. 759-767 ◽  
Author(s):  
Linjiang Zhu ◽  
Dan Jiang ◽  
Yaoyao Zhou ◽  
Yuele Lu ◽  
Yongxian Fan ◽  
...  
Keyword(s):  
Xanthomonas Campestris ◽  
Catalytic Synthesis ◽  
Whole Cell

scholarly journals Designing a whole cell bioreporter to show antioxidant activities of agents that work by promotion of the KEAP1–NRF2 signaling pathway

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Negar Mozaheb ◽  
Ehsan Arefian ◽  
Mohammad Ali Amoozegar
Keyword(s):  
Ascorbic Acid ◽  
Signaling Pathway ◽  
Antioxidant Activities ◽  
Detection System ◽  
Regulatory Region ◽  
Related Factor ◽  
Coding Region ◽  
Whole Cell ◽  
Antioxidant Agents ◽  
Nrf2 Signaling Pathway

Abstract The major signaling pathway in human cells is related to the antioxidant defense system. The main component of this system is a transcription factor, Nuclear Factor Erythroid 2-Related Factor 2 (NRF2). It regulates this system in different cellular situations under stimulation by oxidative stress or antioxidants. Thus, detecting the stimulation of NRF2 via a screening strategy may enable us to discover stimulating agents of NRF2-related signaling pathway. With this in mind, we designed a whole cell bioreporter containing the NRF2 response elements that are inserted in a luciferase vector, immediately upstream of a luciferase gene whose promoter has been removed. This bioreporter is activated by stimulators such as 3H-1,2-dithiole-3-thione (D3T), butyl hydroxyanisole (BHA) and ascorbic acid reacting as antioxidant agents. It was observed that the regulatory region of the NRF2 gene, which is identified by NRF2 protein, is located inside its coding region. This designed bioreporter can detect the presence of antioxidant agents. It also exhibits a significant linear correlation over different doses of these agents ranging from 0.8 to 80 μM for ascorbic acid, 0.1 to 100 μM for D3T, and 0.1 to 100 μM for BHA. This detection system is proven to be more sensitive than Real-time PCR, suggesting it to be a highly sensitive system among the available methods.

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