1.Semi-rational evolution of ω-transaminase from Aspergillus terreus for enhancing the thermostability.
Tingting CAI ; Jiaren CAO ; Shuai QIU ; Changjiang LYU ; Fangfang FAN ; Sheng HU ; Weirui ZHAO ; Lehe MEI ; Jun HUANG
Chinese Journal of Biotechnology 2023;39(6):2126-2140
ω-transaminase (ω-TA) is a natural biocatalyst that has good application potential in the synthesis of chiral amines. However, the poor stability and low activity of ω-TA in the process of catalyzing unnatural substrates greatly hampers its application. To overcome these shortcomings, the thermostability of (R)-ω-TA (AtTA) from Aspergillus terreus was engineered by combining molecular dynamics simulation assisted computer-aided design with random and combinatorial mutation. An optimal mutant AtTA-E104D/A246V/R266Q (M3) with synchronously enhanced thermostability and activity was obtained. Compared with the wild- type (WT) enzyme, the half-life t1/2 (35 ℃) of M3 was prolonged by 4.8-time (from 17.8 min to 102.7 min), and the half deactivation temperature (T1050) was increased from 38.1 ℃ to 40.3 ℃. The catalytic efficiencies toward pyruvate and 1-(R)-phenylethylamine of M3 were 1.59- and 1.56-fold that of WT. Molecular dynamics simulation and molecular docking showed that the reinforced stability of α-helix caused by the increase of hydrogen bond and hydrophobic interaction in molecules was the main reason for the improvement of enzyme thermostability. The enhanced hydrogen bond of substrate with surrounding amino acid residues and the enlarged substrate binding pocket contributed to the increased catalytic efficiency of M3. Substrate spectrum analysis revealed that the catalytic performance of M3 on 11 aromatic ketones were higher than that of WT, which further showed the application potential of M3 in the synthesis of chiral amines.
Transaminases/chemistry*
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Molecular Docking Simulation
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Amines/chemistry*
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Pyruvic Acid/metabolism*
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Enzyme Stability
2.Protective effect of Nigella sativa oil against binge ethanol-induced oxidative stress and liver injury in rats.
Seval DEVELI ; Betül EVRAN ; Esra BETÜL KALAZ ; Necla KOÇAK-TOKER ; Gül Özdemirler ERATA
Chinese Journal of Natural Medicines (English Ed.) 2014;12(7):495-499
AIM:
Nigella sativa L. (Ranunculaceae) is considered as a therapeutic plant-based medicine for liver damage. In this study, the aim was to study the effect of Nigella sativa oil (NSO) pretreatment on ethanol-induced hepatotoxicity in rats.
METHOD:
Rats were given Nigella sativa oil at doses of 2.5 and 5.0 mL·kg(-1), orally for 3 weeks, followed by oral ethanol (EtOH) administration (5 g·kg(-1)) every 12 h three times (binge model).
RESULTS:
Binge ethanol application caused significant increases in plasma transaminase activities and hepatic triglyceride and malondialdehyde (MDA) levels. It decreased hepatic glutathione (GSH) levels, but did not change vitamins E and vitamin C levels and antioxidant enzyme activities. NSO (5.0 mL·kg(-1)) pretreatment significantly decreased plasma transaminase activities, hepatic MDA, and triglyceride levels together with amelioration in hepatic histopathological findings.
CONCLUSION
NSO pretreatment may be effective in protecting oxidative stress-induced hepatotoxicity after ethanol administration.
Animals
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Disease Models, Animal
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Ethanol
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adverse effects
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Female
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Humans
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Liver
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drug effects
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injuries
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metabolism
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Liver Diseases, Alcoholic
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drug therapy
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enzymology
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etiology
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metabolism
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Malondialdehyde
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metabolism
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Nigella sativa
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chemistry
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Oxidative Stress
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drug effects
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Plant Oils
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administration & dosage
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Protective Agents
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administration & dosage
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Rats
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Rats, Sprague-Dawley
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Superoxide Dismutase
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metabolism
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Transaminases
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blood
3.Protective effect of total flavonoid C-glycosides from Abrus mollis extract on lipopolysaccharide-induced lipotoxicity in mice.
Yun WANG ; Zhen-Zhou JIANG ; Mi CHEN ; Mei-Juan WU ; Hong-Li GUO ; Li-Xin SUN ; Hao WANG ; Shuang ZHANG ; Tao WANG ; Lu-Yong ZHANG
Chinese Journal of Natural Medicines (English Ed.) 2014;12(6):461-468
Abrus mollis is a widely used traditional Chinese medicine for treating acute and chronic hepatitis, steatosis, and fibrosis. It was found that the total flavonoid C-glycosides from Abrus mollis extract (AME) showed potent antioxidant, anti-inflammatory, and hepatoprotective activities. To further investigate the hepatoprotective effect of AME and its possible mechanisms, lipopolysaccharide (LPS)-induced liver injury models were applied in the current study. The results indicated that AME significantly attenuated LPS-induced lipid accumulation in mouse primary hepatocytes as measured by triglyceride (TG) and total cholesterol (TC) assays and Oil Red O staining. Meanwhile, AME exerted a protective effect on LPS-induced liver injury as shown by decreased liver index, serum aminotransferase levels, and hepatic lipid accumulation. Real-time PCR and immunoblot data suggested that AME reversed the LPS-mediated lipid metabolism gene expression, such as sterol regulatory element-binding protein-1 (SREBP-1), fatty acid synthase (FAS), and acetyl-CoA carboxylase 1 (ACC1). In addition, LPS-induced overexpression of activating transcription factor 4 (ATF4), X-box-binding protein-1 (XBP-1), and C/EBP homologous protein (CHOP) were dramatically reversed by AME. Furthermore, AME also decreased the expression of LPS-enhanced interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2). Here, it is demonstrated for the first time that AME ameliorated LPS-induced hepatic lipid accumulation and that this effect of AME can be attributed to its modulation of hepatic de novo fatty acid synthesis. This study also suggested that the hepatoprotective effect of AME may be related to its down-regulation of unfolded protein response (UPR) activation.
Abrus
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chemistry
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Animals
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Anti-Inflammatory Agents
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pharmacology
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therapeutic use
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Antioxidants
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pharmacology
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therapeutic use
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Chemical and Drug Induced Liver Injury
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drug therapy
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metabolism
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Cholesterol
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metabolism
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Down-Regulation
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Flavonoids
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pharmacology
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therapeutic use
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Glycosides
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pharmacology
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therapeutic use
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Hepatocytes
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drug effects
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metabolism
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Inflammation Mediators
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metabolism
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Lipid Metabolism
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drug effects
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Lipopolysaccharides
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Liver
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cytology
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drug effects
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metabolism
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Male
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Mice, Inbred Strains
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Phytotherapy
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Plant Extracts
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pharmacology
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therapeutic use
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Transaminases
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blood
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Triglycerides
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metabolism
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Unfolded Protein Response
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drug effects