BackgroundThe intracellular delivery of enzymes for therapeutic use has a promising future for the treatment of several diseases such as genetic disorders and cancer. Virus-like particles offer an interesting platform for enzymatic delivery to targeted cells because of their great cargo capacity and the enhancement of the biocatalyst stability towards several factors important in the practical application of these nanoparticles.ResultsWe have designed a nano-bioreactor based on the encapsulation of a cytochrome P450 (CYP) inside the capsid derived from the bacteriophage P22. An enhanced peroxigenase, CYPBM3, was selected as a model enzyme because of its potential in enzyme prodrug therapy. A total of 109 enzymes per capsid were encapsulated with a 70 % retention of activity for cytochromes with the correct incorporation of the heme cofactor. Upon encapsulation, the stability of the enzyme towards protease degradation and acidic pH was increased. Cytochrome P450 activity was delivered into Human cervix carcinoma cells via transfecting P22-CYP nanoparticles with lipofectamine.ConclusionThis work provides a clear demonstration of the potential of biocatalytic virus-like particles as medical relevant enzymatic delivery vehicles for clinical applications.

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Phosphotriesterase, a pesticide-degrading enzyme, from Flavobacterium sp. was cloned and expressed in Escherichia coli. The catalytic zinc ions were replaced by cobalt atoms increasing the catalytic activity of phosphotriesterase on different pesticides. This metal substitution increased the catalytic activity from 1.4 times to 4 times according to the pesticide. In order to explain this catalytic increase, QM/MM calculations were performed. Accordingly, the HOMO energy of the substrate is closer to the LUMO energy of the cobalt-substituted enzyme. The chemical modification of the enzyme surface with poly(ethylene glycol) increased the thermostability and stability against metal chelating agents of both metal phosphotriesterase preparations.

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Mealworms may serve as an alternative protein source for pet foods because of their high protein content and low environmental footprint. The amino acid (AA) content and protein quality of mealworm-based ingredients may vary depending on their composition and processing, however, so testing is required. Our objective was to measure the AA composition, AA digestibility, and protein quality of mealworm-based ingredients using the precision-fed cecectomized rooster assay. The University of Illinois Institutional Animal Care and Use Committee approved all animal procedures prior to experimentation. Sixteen cecectomized roosters (4 roosters/substrate) were randomly allotted to one of four test substrates: 1) whole lesser mealworm (A. diaperinus) meal (ADw); 2) defatted lesser mealworm (A. diaperinus) meal (ADd); 3) defatted yellow mealworm (T. molitor) meal (TMd); 4) hydrolyzed T. molitor protein meal (TMh). Ingredients were provided by Ynsect, France. After 26 h of feed withdrawal, roosters were tube-fed test substrates. Following crop intubation, excreta samples were collected for 48 h. Endogenous loss corrections for AA were made by using 5 additional cecectomized roosters. All data were analyzed using SAS version 9.4. All substrates had high AA digestibilities, with all indispensable AA digestibilities being >90% with the exception of histidine (87.9%-91.1%) and valine (77.9%-79.7%). Amino acid digestibilities were not different among substrates (P>0.05). Digestible indispensable AA score (DIAAS)-like values were calculated to determine protein quality according to Association of American Feed Control Officials (AAFCO) nutrient profiles, The European Pet Food Industry Nutritional Guidelines (FEDIAF) nutritional guidelines, National Research Council (NRC) recommended allowances for adult dogs, adult cats, growing puppies, and growing kittens, and NRC minimal requirements for growing puppies and growing kittens. In general, TMh had the highest and TMd had the lowest DIAAS-like values for most indispensable AA. Methionine (TMh; TMd; ADw) and phenylalanine (ADd) were the first-limiting AA. Our results demonstrate that mealworm-based ingredients are high-quality protein sources. Further research in dogs and cats is necessary to confirm sufficient palatability and digestibility, but these data suggest that they are valuable sources of protein for pet foods.