NK-lysin is an effector protein of the innate immune system and an important component of host protection. We isolated a SNP in the NK-lysin coding sequence among different chicken breeds. This A to G substitution at the position 271 nucleotide in the ORF results in an Asn (N) to Asp (D) amino acid alteration. We synthesized two 30-aa peptides (N29N and N29D) to compare the biological activity of the helix 2-loop-helix 3 region of NK-lysin resulting from the polymorphic gene. Both peptides were found to be cytotoxic in bacteria and tumor cell cultures at micromolar concentrations. The N29N peptide, however, exhibited greater antibacterial and anticancer activity than the N29D peptide. Circular dichroism spectroscopy of the two peptides in negatively charged single unilamellar vesicles showed spectra typical of α-helical peptides. The helical profile of N29D was reduced substantially compared with that of N29N. However, no structural change was observed in neutral vesicles. ζ-Potential measurements of liposomes incubated with increasing peptide concentrations allowed surface charge neutralization with a negatively charged lipid, but not with a zwitterionic lipid. This result suggests that a difference in electrostatic interaction between lipid membranes and the helical peptides results from the polymorphic gene and is subsequently an important factor in cell lytic activity of variant NK-lysin peptides.antimicrobial peptides | genetic variation | innate immunity
In vitro biodegradation of Bombyx mori silk fibroin (SF) was studied using food-grade proteolytic enzymes to replace acid hydrolysis. Based on the residual protein quantity and yield of amino acids (AAs) after enzymatic hydrolysis, we evaluated the proteolytic enzyme process of SF. FoodPro and Alcalase that are classified as alkaline proteases are selected as two of the best candidate enzymes for hydrolysis of SF. e activity of these enzymes exhibits a broad range of pH (6.5 to 9.0) and temperature (50°C to 65°C). e single enzyme treatment of SF using FoodPro exhibited a hydrolytic efficiency of 20-25%, and >2 g/L AAs were released after reaction for 3 h. A 2-stage enzymatic treatment using a combination of FoodPro and Flavourzyme in a sequence for a reaction time of 6 h was developed to enhance the efficiency of the proteolytic process. e yield of AAs and residual protein quantity in the enzymatic hydrolysates obtained from FoodPro-treated regenerated SF in the 1st step was 2,040 ± 23.7 mg/L and 70.6%, respectively. e yield of AAs was > two-fold (4,519 ± 42.1 mg/L), whereas the residual protein quantity decreased to 55.1% after the Flavourzyme treatment (2nd step) compared to those of the single FoodPro treatment. In the mixed treatment by simultaneously using FoodPro and Flavourzyme, approximately 45% of SF was degraded and 4.5 g/L of AAs were released within 3 h of reaction time. e regenerated SF and its enzymatic hydrolysates were characterized by performing UV-visible spectra, gel electrophoresis, and size-exclusion chromatography analyses. In the 2-stage treatment using FoodPro initially and subsequently Flavourzyme, the aggregates and high molecular weight proteins of SF were dissociated and degraded into the low molecular weight proteins/peptides (10-15 kDa and 27 kDa). SF hydrolysates as functional food might be enzymatically produced using the commercial food-grade proteolytic enzymes.
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