The infected diabetic wound ulcer is a significant problem for the diabetic patients, which leads to removal of affected foot site due to its delayed/non-healing tissues. The poly-microbial infections and active matrix metalloproteinases (MMP) are the significant influencing factors to delayed healing in diabetic mice. The main purposes of present investigation are to evaluation the targeted inactivation of MMP and avoid polymicrobial infections by using a combined therapeutic effect of metal chelating dipeptide (L-carnosine) and curcumin with the biocompatible silk protein hydrogel (L-car@cur/SF) dressing in the infected diabetic wound ulcer. The in vitro biological assay methods, such as, cell viability, anti-oxidant activity, anti-inflammatory macrophage cells and inhibition collagenase exhibited that the designed hydrogel matrix to be human cell compatible and could be accelerate for significant diabetic healing potential. The activation of cur/SF matrix by the L-carnosine was persuading the inactivation of matrix metalloproteinase-9 (MMP-9) through its potent chelating effects of Zn 2+ ions from the MMP-9 active center. The L-car@cur/SF hydrogel was demonstrated for the effective MMP-9 inactivation and bacterial inhibition via in vivo mice wound site, which indorsed the diabetic wound healing efficiency in streptozotocin-tempted diabetic mice.
The objective of the present study was to understand mechanisms of inhibitory effect of l -carnosine on lipid oxidation using an oxidized myoglobin (metMb)-mediated washed cod muscle system. Experiments were conducted to compare l -carnosine with commercial antioxidants, tert-Butylhydroquinone (TBHQ) and α-tocopherol, and metal chelating reagent EDTA for inhibition of lipid oxidation, to compare l -carnosine with TBHQ for scavenging free radicals and to evaluate the effect of l -carnosine on structure of pro-oxidant metMb with spectral methods. Results showed that the inhibitory effect of l -carnosine on lipid oxidation was comparable with TBHQ and better than α-tocopherol. However, 1,1-diphenyl-2-picrylhydrazyl assay showed that TBHQ was much more effective in scavenging free radicals than l -carnosine. Comparing with 1.5% EDTA, 1.5% l -carnosine was much more effective against lipid oxidation. Incubation of l -carnosine with metMb resulted in spectral changes of metMb in wavelengths between 370 nm and 450 nm, Soret band shifted from 409 nm to 420 nm, and reduced molar ellepticity values. These results indicate that the high inhibitory effects of l -carnosine on meat lipid oxidation may result from not only its scavenging and chelating functions but also its effect on pro-oxidant metMb structure.
L-Carnosine (L-Car) is a biologically active dipeptide with antioxidant and anti-aging effects, formed by condensation of β-alanine (β-Ala) with L-histidine (L-His), which is used widely in the pharmaceutical and cosmetic industries. Here we report a dipeptide hydrolase originating from Bacillus megaterium , designated as Bm PepD, whose synthetic activity reached 6.5 times that of Sm PepD, which is the highest active dipeptidase reported so far for L-Car biosynthesis in vitro . By site-directed saturation mutagenesis, we obtained a significantly improved variant (T171N) of Bm PepD. It showed a specific activity of 22.9 U mg −1 protein , reaching 1.4 times that of wild-type Bm PepD or 9.1 times that of Sm PepD, which is the highest ever reported among this enzyme class. After optimizing the reaction conditions of variant Bm PepD T171N , we performed the synthetic reaction of L-Car simply from the two free amino acids (0.2 M L-His and 6.5 M β-Ala), achieving a titer of 31.3 mM L-Car with a specific productivity of 283 g Car · g catalyst −1 . This work provides a highly active and substrate (β-alanine)-tolerant dipeptide hydrolase for the facile synthesis of dipeptide L-Car via reversed hydrolysis.
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