Biosynthesis, Characteristics And Applications Of β-glucosidase-silk Fibroin Nanoparticles Conjugates

Silk fibroin derived from Bombyx mori is a biomacromolecular protein with outstanding biocompatibility. When dissolved in highly concentrated CaCl2 solution, and with the protein-salt mixture subjected to lengthy desalting treatments in flowing water, the resulting liquid silk was water-soluble polypeptides with different molecular masses of range 10-200 kDa. When the liquid silk was introduced rapidly into acetone, silk protein nanoparticles with a range of 40-120 nm in diameter could be obtained. The crystallization of silk fibroin nanoparticles is insoluble in water, but after ultrasonic treatment can be a good dispersed in aqueous solution. In this paper, these silk fibroin nanoparticles as carrier, respectively, cross-linking and embedding are two ways to prepare two kinds ofβ-glucosidase-silk fibroin nanoparticles (βG-SFNs). In vitro properties of theβ-glucosidase-silk fibroin nanoparticles (βG-SFNs) bioconjugates were determined by p-nitrophenyl-β-D-glucoside (p-NPG) as a substrate. The optimal conditions for the biosynthesis ofβG-SFNs bioconjugates were investigated.The experimental results showed that the optimal modifying condition ofβG-SFNs bioconjugates synthesized with glutaraldehyde as cross-linking reagent was set up as follows: glutaraldehyde concentration was 0.25%, duration was 5 h, temperature was 37 oC, the U: mg ratio ofβ-glucosidase and SFNs was 75:100; and that ofβG-SFNs prepared by embedding was set up as follow: the U: mg ratio ofβ-glucosidase and silk fibroin was 1:1. The recoveries ofβG-SFNs bioconjugates were 46.0% and 59.2% respectively. Kinetic analyses showed that performances of the two kinds ofβG-SFNs were the same as for the freeβ-glucosidase. The optimal pH was 5.0 and the optimal temperature was 60°C. The apparent Michaelis Constant of freeβ-glucosidase (Km) was 7.26×10?3 mol·L-1, and the apparent Michaelis Constants ofβG-SFNs bioconjugates were 1.41×10-3 mol·L-1 and 1.01×10?2 mol·L^–1, respectively. The apparent Km ofβG-SFNs (cross-linking) was near five-fold less than that of the free enzyme, this fully shows that after the free enzyme conjugated with silk fibroin nanoparticles, the enzyme affinity with substrate increased; The Michaelis Constant ofβG-SFNs (embedding) was higher than for the free enzyme, suggesting that affinity between the enzyme and its substrate p-NPG considerably decreased whenβ-glucosidase was embedded in silk fibroin. The binding of free enzyme with silk fibroin nanoparticles, either embedded or linked manner, and their thermal stability are improved; but also have good operational stability, can be used repeatedly. These results fully demonstrated that silk protein nanoparticles were good carriers as bioconjugation for modification of enzymes.Naringinase is an enzyme complex consisting ofα-L-rhamnosidase and ?avonoid-β-glucosidase. With cross-linking of reagent glutaraldehyde, these two kinds of enzymes could be coimmobilized at the surface of silk fibroin nanoparticles and naringinase-silk fibroin nanoparticles (N-SFNs) were obtained. In this work, the enzyme activity of N-SFN conjugates was determined by naringin as a substrate and we used high performance liquid chromatography (HPLC) to analyze the biological activities of the N-SFNs. The results show that N-SFNs still could well hydrolysis naringin when naringinase was coupled covalently with silk nanoparticles, indicating that the activity did not affected. The kinetic parameters of N-SFNs were the same as for the free naringinase and the optimal temperatures both were 55°C. Naringin by N-SFNs digested, you can use high-speed centrifugal sedimentation method by combining enzyme and product (naringenin, rhamnose and glucose) to separate. When the precipitated fibroin nanoparticles conjugating were treated by ultrasound suspended, the enzymatic reaction could be proceeded. In our study, after eight cycles the N-SFNs retained > 70% residual activity. Therefore, silk fibroin nanoparticles were good carriers as the bioconjugation or modification of enzyme, not only for a single enzyme but also for two enzymes and have the potential values for research and development in the role of debittering of citrus fruit, grapes and other fruits.