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1.
Plant Physiol Biochem ; 211: 108716, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38744087

RESUMEN

In the context of global climate change, recurrent freeze-thaw cycles (FTC) and concurrent exposure to polystyrene nanoplastics (PSNPs) directly impact crop growth and indirectly affect resilience to abiotic stress. In January 2023, experiments at the Environmental Biology Laboratory, Jilin University, Changchun, China, exposed rye seedlings to 100 nm PSNPs at concentrations of 0, 10, 50, and 100 mg/L for seven days, followed by three FTC. Scanning electron microscopy (SEM) demonstrated that PSNPs migrated from the roots to the leaves, with FTC significantly exacerbating their accumulation within plant tissues. Transmission electron microscopy (TEM) observations showed that FTC disrupted normal cell division, and combined stress from NPs damaged plant organs, particularly chloroplasts, thereby substantially inhibiting photosynthesis. FTC delayed plant phenological stages. Under combined stress, malondialdehyde (MDA) accumulation in plant tissues increased by 15.6%, while hydrogen peroxide (H2O2) content decreased. Simultaneously, the activities of peroxidase (POD) and catalase (CAT) increased by 34.2% and 38.6%, respectively. Molecular docking unveiled that PSNPs could bind to the active center of POD/CAT through hydrogen bonding or hydrophobic interactions. The Integrated Biomarker Response (IBR) index highlighted FTC as a crucial determinant for pronounced effects. Moreover, an apparent dose-dependent effect was observed, with antioxidant enzyme activities in rye seedlings induced by low pollutant concentrations and inhibited by high concentrations. These results indicate that FTC and PSNPs can disrupt plant membrane systems and cause severe oxidative damage. Overall, this study provides compelling scientific evidence of the risks associated with NPs exposure in plants subjected to abiotic stress.


Asunto(s)
Congelación , Poliestirenos , Secale , Plantones , Plantones/efectos de los fármacos , Plantones/metabolismo , Poliestirenos/toxicidad , Secale/efectos de los fármacos , Secale/metabolismo , Peroxidasa/metabolismo , Catalasa/metabolismo , Nanopartículas/toxicidad , Simulación del Acoplamiento Molecular , Malondialdehído/metabolismo
2.
Bioresour Technol ; 341: 125799, 2021 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-34425465

RESUMEN

l-glutamine is a semi-essential amino acid widely used in the food and pharmaceutical industries. The microbial synthesis of l-glutamine is limited by lack of effective strains with high titer and safety. First, ARTP mutagenesis combined with high-throughput screening generated an l-glutamine-producing strain of Corynebacterium glutamicum with titer of 25.7 ± 2.7 g/L. Subsequently, a series of rational metabolic approaches were used to further improve l-glutamine production, which included increasing the carbon flow to l-glutamine (proB and NCgl1221 knockout), enhancing the catalytic efficiency of the key enzyme (glnE knockout and glnA screening and overexpression) and reinforcement of ATP regeneration (ppk overexpression and RBS optimization). Finally, we proposed a two-stage pH control strategy to address the inconsistent effect of pH on cell growth and l-glutamine production. These combined strategies led to a 186.0% increase of l-glutamine titer compared to that of the initial strain, reaching 73.5 ± 3.1 g/L with a yield of 0.368 ± 0.034 g/g glucose.


Asunto(s)
Corynebacterium glutamicum , Corynebacterium glutamicum/genética , Fermentación , Glucosa , Glutamina , Concentración de Iones de Hidrógeno , Ingeniería Metabólica
3.
Int J Biol Macromol ; 179: 71-79, 2021 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-33631263

RESUMEN

l-lactate dehydrogenases (LDHs) has been widely studied for their ability to reduce 2-keto acids for the production of 2-hydroxy acids, whereby 2-hydroxybutyric acids (2-HBA) is among the most important fundamental building blocks for synthesizing pharmaceuticals and biodegradable materials. However, LDHs usually show low activity towards 2-keto acids with longer side chain such as 2-oxobutyric acid (2-OBA). Here rational engineering of the Plasmodium falciparum LDH loop with residue involved in the catalytic proton transfer was initially studied. By combining homology alignment and structure-based design approach, we found that changing the charge characteristics or hydrogen bond network interactions of this loop could improve enzymatic catalytic activities and stabilities towards 2-OBA. Compared with wild type, variant N197Dldh showed 1.15 times higher activity and 2.73 times higher Kcat/Km. The half-life of variant N197Dldh at 40 °C increased to 77.9 h compared with 50.4 h of wild type. Furthermore, asymmetric synthesis of (S)-2-HBA with coenzyme regeneration revealed 95.8 g/L production titer within 12 h for variant N197Dldh, 2.05 times higher than using wild type. Our study indicated the importance of loop with residues involved in the catalytic proton transfer process, and the engineered LDH would be more suitable for (S)-2-HBA production.


Asunto(s)
Hidroxibutiratos/síntesis química , L-Lactato Deshidrogenasa/química , Plasmodium falciparum/enzimología , Proteínas Protozoarias/química , Catálisis , Hidroxibutiratos/química , L-Lactato Deshidrogenasa/genética , Plasmodium falciparum/genética , Ingeniería de Proteínas , Estructura Secundaria de Proteína , Proteínas Protozoarias/genética
4.
Sheng Wu Gong Cheng Xue Bao ; 37(12): 4231-4242, 2021 Dec 25.
Artículo en Chino | MEDLINE | ID: mdl-34984870

RESUMEN

2-Hydroxybutyric acid (2-HBA) is an important intermediate for synthesizing biodegradable materials and various medicines. Chemically synthesized racemized 2-HBA requires deracemization to obtain optically pure enantiomers for industrial application. In this study, we designed a cascade biosynthesis system in Escherichia coli BL21 by coexpressing L-threonine deaminase (TD), NAD-dependent L-lactate dehydrogenase (LDH) and formate dehydrogenase (FDH) for production of optically pure (S)-2-HBA from bulk chemical L-threonine (L-Thr). To coordinate the production rate and the consumption rate of the intermediate 2-oxobutyric acid in the multi-enzyme cascade catalytic reactions, we explored promoter engineering to regulate the expression levels of TD and FDH, and developed a recombinant strain P21285FDH-T7V7827 with a tunable system to achieve a coordinated multi-enzyme expression. The recombinant strain P21285FDH-T7V7827 was able to efficiently produce (S)-2-HBA with the highest titer of 143 g/L and a molar yield of 97% achieved within 16 hours. This titer was approximately 1.83 times than that of the highest yield reported to date, showing great potential for industrial application. Our results indicated that constructing a multi-enzyme-coordinated expression system in a single cell significantly contributed to the biosynthesis of hydroxyl acids.


Asunto(s)
Formiato Deshidrogenasas , Hidroxibutiratos , Escherichia coli/genética , Treonina Deshidratasa
5.
Sheng Wu Gong Cheng Xue Bao ; 28(1): 65-75, 2012 Jan.
Artículo en Chino | MEDLINE | ID: mdl-22667110

RESUMEN

In order to enhance gamma-aminobutyric acid production from L-glutamate efficiently, we amplified the key enzyme glutamate decarboxylase (GAD) encoding gene lpgad from the strain Lactobacillus plantarum GB 01-21 which was obtained by way of multi-mutagenesis and overexpressed it in E. coli BL21. Then we purified GAD by Ni-NTA affinity chromatography and characterized the enzyme to optimize the conditions of the whole-cell transformation. The results showed that the recombinant E. coli BL21 (pET-28a-lpgad) produced 8.53 U/mg GAD, which was increased by 3.24 fold compared with the GAD activity in L. plantarum. The optimum pH and temperature of the enzyme were pH 4.8 and 37 degrees C, respectively. At the same time, we found that Ca2+ and Mg2+ could increase the activity significantly. Based on this, we investigated gamma-aminobutyric acid transformation in 5 L fermentor under the optimum transformation conditions. Accordingly, the yield of gamma-aminobutyric acid was 204.5 g/L at 24 h when the 600 g L-glutamate was added and the mole conversion rate had reached 97.92%. The production of gamma-aminobutyric acid was improved by 42.5% compared with that under the unoptimized transformation conditions. This paved a way for the gamma-aminobutyric acid construction of the industrial applications.


Asunto(s)
Escherichia coli/genética , Escherichia coli/metabolismo , Glutamato Descarboxilasa/biosíntesis , Ácido Glutámico/metabolismo , Ácido gamma-Aminobutírico/biosíntesis , Clonación Molecular , Escherichia coli/enzimología , Glutamato Descarboxilasa/genética , Lactobacillus plantarum/enzimología , Lactobacillus plantarum/genética , Recombinación Genética
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