Engineering an alcohol dehydrogenase for nalancing kinetics in NADPH regeneration with 1,4-butanediol as a cosubstrate

\u3cp\u3eCofactor regeneration using diols as smart cosubstrates is one of the most promising approaches, due to the thermodynamic preference and 0.5-equiv requirement. In order to establish an efficient NADPH regeneration system with 1,4-butanediol (1,4-BD), a NADP\u3csup\u3e+\u3c/sup\u3e-dependent alcohol dehydrogenase from Kluyveromyces polysporus (KpADH) was engineered to solve the kinetic imbalance. Several hotspots were identified through molecular dynamic simulation and subjected to saturation and combinatorial mutagenesis. Variant KpADH\u3csub\u3eV84I/Y127M\u3c/sub\u3e exhibited a lower K\u3csub\u3eM\u3c/sub\u3e of 15.1 mM and a higher k\u3csub\u3ecat\u3c/sub\u3e of 30.1 min\u3csup\u3e-1\u3c/sup\u3e than \u3csup\u3eWT\u3c/sup\u3eKpADH. The oxidation of 1,4-BD to 4-hydroxybutanal was found to be the rate-limiting step, for which the k\u3csub\u3ecat\u3c/sub\u3e/K\u3csub\u3eM\u3c/sub\u3e value of double mutant KpADH\u3csub\u3eV84I/Y127M\u3c/sub\u3e was 2.00 min\u3csup\u3e-1\u3c/sup\u3e·mM\u3csup\u3e-1\u3c/sup\u3e, 11.6-fold higher than that of \u3csup\u3eWT\u3c/sup\u3eKpADH. KpADH\u3csub\u3eV84I/Y127M\u3c/sub\u3e preferred diols with a longer chain length (C5-C6). The ratio of k\u3csub\u3ecat\u3c/sub\u3e/K\u3csub\u3eM\u3c/sub\u3e toward 2-hydroxytetrahydrofuran (2-HTHF), in comparison to 1,4-BD, in KpADH\u3csub\u3eV84I/Y127M\u3c/sub\u3e was dramatically reduced by almost 100-fold compared to \u3csup\u3eWT\u3c/sup\u3eKpADH, which was advantageous for NADPH regeneration. As much as 100 mM phenylpyruvic acid could be reduced into d-phenylalanine with 99.2% conversion in 6 h using merely 0.5 equiv of 1,4-BD. Both the improved catalytic efficiency toward 1,4-BD and the balanced k\u3csub\u3ecat\u3c/sub\u3e/K\u3csub\u3eM\u3c/sub\u3e between 1,4-BD and 2-HTHF contributed to the higher NADPH regeneration efficiency. This study provides guidance for engineering alcohol dehydrogenases for cosubstrate specificity toward diols and its application in NADPH regeneration for the preparation of chiral compounds of pharmaceutical relevance.\u3c/p\u3

Heme Oxygenase-1 Protects Against Steatohepatitis in Both Cultured Hepatocytes and Mice

Background & Aims: Heme oxygenase-1 (HO-1), an antioxidant defense enzyme, has been shown to protect against oxidant-induced tissue injury. We investigated the role of HO-1 in nutritional steatohepatitis in vitro and in vivo. Methods: AML-12 hepatocytes were cultured in methionine- and choline-deficient (MCD) medium. Cells were transfected with an adenovirus vector that expressed HO-1 (Ad-HO-1) or incubated with the HO-1 inducer hemin or the HO-1 inhibitor stannic mesoporphyrin for 24 hours. C57BL6 mice and db/db mice were fed MCD or control diets, with or without hemin, for up to 4 weeks. Results: AML-12 cells exposed to MCD medium developed significant steatosis, increased release of alanine aminotransferase, and showed signs of oxidative injury. Incubation with hemin induced HO-1 protein, suppressed steatosis, and reduced levels of alanine aminotransferase and lipid peroxidation. A comparable effect was observed in cells transfected with Ad-HO-1, whereas incubation of these cells with stannic mesoporphyrin completely abolished the Ad-HO-1- or hemin-mediated protection of hepatocytes. Mice injected with hemin significantly attenuated MCD-induced steatohepatitis and increased HO-1 protein and activity. This effect was associated with up-regulation of antioxidant chaperones and enzymes, down-regulation of proinflammatory cytokines, and up-regulation of the anti-inflammatory interleukin-22. Moreover, the reduction in steatosis caused by hemin was affected by up-regulation of peroxisome proliferator-activated receptor-α and by down-regulation of sterol regulatory element binding protein-1c. Conclusions: HO-1 can interrupt progression of nutritional steatohepatitis by inducing an antioxidant pathway, suppressing production of cytokines, and modifying fatty acid turnover. Induction of HO-1 might provide a new approach for treatment of steatohepatitis

Fractal characteristics and damage evaluation of corroded beams under four-point bending tests based on acoustic emission techniques

This study investigated the relationship between the acoustic emission (AE) signals parameter sequence fractal characteristics and the damage evolution information of corroded reinforced concrete (RC) beams under four-point bending. Strength deterioration behavior and AE data can be obtained by coupling the four-point bending test and the AE monitoring. The results show that AE ringing counts of corroded and uncorroded beams had prominent fractal characteristics. The fractal dimension values of corroded RC beams all showed a fluctuating rise to a peak and then a sharp drop before the failure. The damage index corresponding to the peak point decreases with the increase of corrosion degree. Fractal dimension peak point could be used as an early warning point for corroded RC beams' failure. Moreover, the AE fractal dimension analysis can effectively reflect the pattern of crack development, which have an important value for evaluating the process of corroded RC beams rupture.Green Open Access added to TU Delft Institutional Repository 'You share, we take care!' - Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public.Concrete Structure