Role of Q177A and K173A/Q177A substitutions in thermostability and activity of the ELBn12 lipase.

Thermostable lipases have many applications in detergent industries and in organic synthesis. There are many ways to improve thermal stability of enzymes, for example, higher hydrophobicity, greater structural packing, higher content of the charged residues, and lower thermolabile ones. In this study, thermolabile Gln (sensitive to higher temperatures) was substituted with Ala in native ELBn12 and mutated K173A lipases to examine its effect on thermal stability and activity of the lipases. Single (Q177A) and double mutants (K173A/Q177A) were expressed in Escherichia coli pLysS. The Q177A variant increased both activity and thermostability of the lipase, whereas K173A/Q177A had a negative effect on the lipase activity and only had better thermal stability than the native at 50 °C. pH stability of the double mutant between 9.0 and 11 was also lower than the other variants. Stability analysis in the presence of chemicals showed that Q177A mutant had better activity with 50% (v/v) organic solvents. On the other hand, K173A lipase showed increased activity with 1% (w/v) nonionic surfactant, and finally K173A/Q177A had better stability with 10 mM metal ions compared to the native lipase.

α-Ketoglutarate stimulates cell growth through the improvement of glucose and glutamine metabolism in C2C12 cell culture.

Introduction: Cellular adaptation to physical training and energy metabolism play an important role during physical exercise. This study sought to investigate the effects of α-KG on cell growth and energy metabolism in C2C12 cell culture. Methods: C2C12 cells were cultured in media pretreated without (control) or with α-KG at different concentrations, and cells and media were harvested every 24 h for 8 days. From cell counts, specific cell growth rate (SGR) and doubling time were calculated. The content of glucose, glutamine, lactate, and ammonia in media was determined, and the specific consumption rate (SCR) or production rate (SPR) was calculated. Additionally, cell colony-forming efficiency (CFE) was determined. Results: The control cells showed a CFE at 50%, a typical cell growth curve in the first 5 days with a mean SGR at 0.86/day, and a mean cell count doubling time at 19.4 h. In the group with α-KG at 100 mM, the cells underwent rapid cell death, and thus no further analysis was made. The treatment with α-KG at lower concentrations (0.1 mM and 1.0 mM) led to a higher CFE at 68 and 55%, respectively, whereas those in groups with higher α-KG concentration decreased (10 and 6% for 20 mM and 30 mM α-KG, respectively). The mean SGR was 0.95/day, 0.94/day, 0.77/day, 0.71/day, and 0.65/day for groups treated with α-KG at 0.1, 1.0, 10.0, 20.0, and 30.0 mM, respectively, and the corresponding cell count doubling time was 17.6, 17.8, 20.9, 24.6, and 24.7 h, respectively. In comparison with that of the control group, the mean glucose SCR decreased in all the groups treated with α-KG, while the mean glutamine SCR remained unchanged; the mean lactate SPR increased in the groups treated with α-KG ≥ 20.0 mM. Finally, the mean SPR of ammonia was lower in all α-KG groups than that in the control. Discussion and conclusion: The treatment with α-KG at lower concentrations increased cell growth whereas at higher concentrations decreased cell growth, and α-KG reduced glucose consumption and ammonia production. Therefore, α-KG stimulates cell growth in a dose-dependent manner, which is likely through the improvement of glucose and glutamine metabolism in a C2C12 culture setting.

Hypoxia Induces Saturated Fatty Acids Accumulation and Reduces Unsaturated Fatty Acids Independently of Reverse Tricarboxylic Acid Cycle in L6 Myotubes.

Obstructive sleep apnea syndrome, characterized by repetitive episodes of tissue hypoxia, is associated with several metabolic impairments. Role of fatty acids and lipids attracts attention in its pathogenesis for their metabolic effects. Parallelly, hypoxia-induced activation of reverse tricarboxylic acid cycle (rTCA) with reductive glutamine metabolism provides precursor molecules for de novo lipogenesis. Gas-permeable cultureware was used to culture L6-myotubes in chronic hypoxia (12%, 4% and 1% O2) with 13C labelled glutamine and inhibitors of glutamine uptake or rTCA-mediated lipogenesis. We investigated changes in lipidomic profile, 13C appearance in rTCA-related metabolites, gene and protein expression of rTCA-related proteins and glutamine transporters, glucose uptake and lactate production. Lipid content increased by 308% at 1% O2, predominantly composed of saturated fatty acids, while triacylglyceroles containing unsaturated fatty acids and membrane lipids (phosphatidylcholines, phosphatidylethanolamines, phosphatidylinositol) decreased by 20-70%. rTCA labelling of malate, citrate and 2-hydroxyglutarate increased by 4.7-fold, 2.2-fold and 1.9-fold in 1% O2, respectively. ATP-dependent citrate lyase inhibition in 1% O2 decreased lipid amount by 23% and increased intensity of triacylglyceroles containing unsaturated fatty acids by 56-80%. Lactate production increased with hypoxia. Glucose uptake dropped by 75% with progression of hypoxia from 4% to 1% O2. Protein expression remained unchanged. Altogether, hypoxia modified cell metabolism leading to lipid composition alteration and rTCA activation.

Production of phosphinothricin-tolerant rice (Oryza sativa L.) through the application of phosphinothricin as growth regulator.

A novel procedure has been developed to produce rice (Oryza sativa L.) tolerant to the herbicide phosphinothricin (PPT) by means of in vitro selection. First, sublethal and lethal concentrations of PPT on 7-day-old seedlings were determined and morphogenetic events in response to the PPT treatment evaluated. Differentiation of 6-30 microshoots on 5-40% of the treated plant material was observed on a hormone-free culture medium supplemented with a sublethal concentration of PPT. We proved that PPT is morphogenetically active, similar to the action of many other herbicides, showing cytokinin-like effects in rice tissue culture. Fertile plants were grown from those microshoots having PPT tolerance under greenhouse conditions. To the best of our knowledge, this is the first report on the production of rice plants tolerant to this herbicide without genetic transformation. Since PPT is a competitive inhibitor of glutamine synthetase (GS), total GS activity in PPT-tolerant and PPT-sensitive plants was examined comprehensively in order to decide whether this enzyme has any role in PPT tolerance. An elevated GS activity was detected in PPT-tolerant plant material which could result in an elevated PPT tolerance at unchanged concentrations of the herbicide.