Lukasz Peplowski

A non-native bio-amidation activity is unlocked in asparagine synthetase via biosensor-guided evolution.

Isonicotinamide has important roles and application prospects in pharmaceutical, medical, and materials sciences. The biosynthesis of isonicotinamide has great advantages over chemical methods, but the biosynthesis of isonicotinamide has been reported for three decades and has not been industrialized yet. In this study, a thermophilic NHase from Carbonactinospora thermoautotrophicus was selected and then its activity was substantially improved by a “toolbox” screening strategy. The specific activity of the βL48D mutant towards 4-cyanopyridine was 307.02 ± 3.50 U mg⁻¹, which was 13.9 times higher than that of the WT C.t NHase (22.01 ± 1.30 U mg⁻¹). The structural analysis demonstrated that introduction of Asp into the βLeu48 site resulted in a larger catalytic cavity and better substrate orientations. Finally, a recombinant strain E. coli BL21 of the βL48D mutant was cultured at high cell density in a 5 L fermenter for high-level isonicotinamide production, developing a powerful process for isonicotinamide bio-production. The yield of isonicotinamide reached 2.89 M (352 g L⁻¹), the substrate (4-cyanopyridine) was exhausted and the conversion was 100%. These results prove that C.t NHase is a promising competitor for future industrial production of biosynthetic isonicotinamide.

Studies reported evidence for opposite effects of extremely low‐frequency electromagnetic field (EMF): harmful, including the oxidative stress induction, and beneficial, such as the activation of antioxidant defense. People's exposure to EMF is often repeated or prolonged, and it is important to consider the cumulative effect of such kind of exposure on the organism. If changes evoked by repeated exposure to EMF are permanent, responsiveness to other stress factors can be modified. The aims of our study were (1) to evaluate changes in the levels of oxidative stress and antioxidant defense markers in the prefrontal cortex of adult rats after repeated exposure to 1 and 7 mT EMF and (2) to assess whether repeated EMF exposure can modify oxidative/antioxidative status in response to other stress factors. Rats were exposed to EMF 1 h/day for 7 days, one, twice, or three times. After each exposure, 8‐isoprostanes, protein carbonyl groups, and the total antioxidant capacity were assessed. Part of the animals, after EMF treatment, was exposed to another stress factor—open field. Results showed that repeated exposure changed the oxidative/antioxidative status depending on the intensity of the EMF and the number of exposures. 1 mT EMF created weak changes in the oxidative status in the brain; however, 7 mT EMF moved the balance to a clearly higher level. The changes in the oxidative status after 1 mT EMF were enough to reduce, and after 7 mT EMF to intensify oxidative processes in response to the next stress. We concluded that the organism might adapt to “weak” EMF, while “strong” EMF exceeds the adaptive capacity of the organism and sensitizes it to subsequent stress, and thus may modulate vulnerability to diseases. Our results also provide new insights into the possible therapeutic properties of the magnetic field, as 1 mT EMF appears to have a potentially protective impact on the brain.

Sucrose phosphorylase (SPase) can specifically catalyze transglycosylation reactions and can be used to enzymatically synthesize α-D-glycosides. However, the low thermostability of SPase has been a bottleneck for its industrial application. In this study, a SPase gene from Leuconostoc mesenteroides ATCC 12,291 (LmSPase) was synthesized with optimized codons and overexpressed successfully in Escherichia coli. A semi-rational design strategy that combined the FireProt (a web server designing thermostable proteins), structure–function analysis, and molecular dynamic simulations was used to improve the thermostability of LmSPase. Finally, one single-point mutation T219L and a combination mutation I31F/T219L/T263L/S360A (Mut4) with improved thermostability were obtained. The half-lives at 50 °C of T219L and Mut4 both increased approximately two-fold compared to that of wild-type LmSPase (WT). Furthermore, the two variants T219L and Mut4 were used to produce α-D-glucosylglycerol (αGG) from sucrose and glycerol by incubating with 40 U/mL crude extracts at 37 °C for 60 h and achieved the product concentration of 193.2 ± 12.9 g/L and 195.8 ± 13.1 g/L, respectively, which were approximately 1.3-fold higher than that of WT (150.4 ± 10.0 g/L). This study provides an effective strategy for improving the thermostability of an industrial enzyme. Key points: • Predicted potential hotspot residues directing the thermostability of LmSPase by semi-rational design • Screened two positive variants with higher thermostability and higher activity • Synthesized α-D-glucosylglycerol to a high level by two screened positive variants

High thermostability and catalytic activity are key properties for nitrile hydratase (NHase, EC 4.2.1.84) as a well-industrialized catalyst. In this study, rational design was applied to tailor the thermostability of NHase from Pseudonocardia thermophila JCM3095 (PtNHase) by combining FireProt server prediction and molecular dynamics (MD) simulation. Site-directed mutagenesis of non-catalytic residues provided by the rational design was subsequentially performed. The positive multiple-point mutant, namely, M10 (αI5P/αT18Y/αQ31L/αD92H/βA20P/βP38L/βF118W/βS130Y/βC189N/βC218V), was obtained and further analyzed. The Melting temperature (T_m) of the M10 mutant showed an increase by 3.2 °C and a substantial increase in residual activity of the enzyme at elevated temperatures was also observed. Moreover, the M10 mutant also showed a 2.1-fold increase in catalytic activity compared with the wild-type PtNHase. Molecular docking and MD simulations demonstrated better substrate affinity and improved thermostability for the mutant.

The transition metal (iron or cobalt) is a mandatory part that constitutes the catalytic center of nitrile hydratase (NHase). The incorporation of the cobalt ion into cobalt‐containing NHase (Co‐NHase) was reported to depend on self‐subunit swapping and the activator of the Co‐NHase acts as a self‐subunit swapping chaperone for subunit exchange. Here we discovered that the activator acting as a metallochaperone transferred the cobalt ion into subunit‐fused Co‐NHase. We successfully isolated two activators, P14K and NhlE, which were the activators of NHases from Pseudomonas putida NRRL‐18668 and the activator of low‐molecular‐mass NHase from Rhodococcus rhodochrous J1, respectively. Cobalt content determination demonstrated that NhlE and P14K were two cobalt‐containing proteins. Substitution of the amino acids involved in the C‐terminus of the activators affected the activity of the two NHases, indicating that the potential cobalt‐binding sites might be located at the flexible C‐terminal region. The cobalt‐free NHases could be activated by either of the two activators, and both the two activators activated their cognate NHase more efficiently than did the noncognate ones. This study provided insights into the maturation of subunit‐fused NHases and confirmed the metallochaperone function of the self‐subunit swapping chaperone.