Silk fibroin derived from Bombyx mori is a biomacromolecular protein with outstanding biocompatibility. When it was dissolved in highly concentrated CaCl2 solution and then the mixture of the protein and salt was subjected to desalting treatments for long time in flowing water, the resulting liquid silk was water-soluble polypeptides with different molecular masses, ranging from 10 to 200 kDa. When the liquid silk were introduced rapidly into acetone, silk protein nanoparticles (SFNs) with a range of 40~120 nm in diameter could be obtained. The crystalline silk nanoparticles could be conjugated with β-glucosidase with cross-linking of reagent glutaraldehyde. In this work, the activity of β-glucosidase-silk fibroin nanoparticles (βG-SFNs) bioconjugates was determined by p-nitrophenyl-β-D-glucoside (p-NPG) as a substrate and the optimal conditions for the biosynthesis of βG-SFNs bioconjugates were investigated. βG-SFN constructs obtained by 5h of covalent cross-linking time at the experimental conditions of 0.25% cross-linking reagent, 37 °C and the proportion of β-glucosidase and SFNs (75 : 100, U/mg) showed 46% of recoveries. Results showed that kinetic parameters of βG-SFNs were the same as for the free β-glucosidase. The optimal pH was 5.0 and the optimal temperature was 60°C. When β-glucosidase was coupled covalently with silk nanoparticles, the thermal stability of βG-SFNs was slightly enhanced as compared with free β-glucosidase. The apparent Km of βG-SFNs (1.41×10-3 mol•L-1) was near five-fold less than that of the free enzyme (7.26×10−3 mol•L-1), this fully shows that after the free enzyme conjugated with silk fibroin nanoparticles, the enzyme affinity with substrate increased. These results fully demonstrated the silk protein nanoparticles were good carriers as bioconjugation or modification of enzymes. Moreover, they had potential values for research and development in food processing and flavor processing agents.
A comparative study of horseradish peroxidase conjugates prepared with a one-step and a two-step method.