Subtilisin

Shettar, Shreya S., et al. Saudi Journal of Biological Sciences, 2024, 31(7), 104009.

The choice of alginate-immobilized recombinant subtilisin in this work enhances the performance and durability of encapsulated subtilisin in the alkaline pH range (9-11) and thermostability at 80°C. Silver and zinc oxide nanoparticles conjugated with recombinant subtilisin were further synthesized, which imparted significant antimicrobial efficacy against a variety of organisms and convincing antioxidant properties.
Sodium alginate entrapment method for subtilisin
The consistency of the beads was examined through the immobilization of sodium alginate. The solution made from 3 g of sodium alginate in 100 mL of distilled water created a 3% solution. 1mL of processed recombinant subtilisin was added to 10 mL of the prepared sodium alginate solution, and the mixture was inverted two to four times to achieve a uniform blend. Subsequently, the combination of subtilisin and carrier was gradually released from approximately 15 cm in height using a 10 mL syringe into a cold solidifying buffer of 0.2 M calcium chloride while being stirred on a magnetic stirrer. New beads were then cured in the fridge at 4 °C for one hour. The solidified beads were rinsed twice or three times with sterile distilled water and held in distilled water at 4°C until use. All experiments were performed aseptically in a laminar flow hood.
Moreover, the recombinant subtilisin was reacted with solutions of silver nitrate and zinc acetate dihydrate to form corresponding silver and zinc oxide nanoparticles, respectively.

Prabhawathi, Veluchamy, et al. Materials Science and Engineering: C, 2019, 94, 656-665.

Subtilisin is a non-toxic serine protease used in skin care ointments, cosmetics, food processing, and pharmaceuticals. In this work, subtilisin was combined with gold core-silver shell nanoparticles (AuAgNPs) and coated or immobilized on the surface of polycaprolactam to prepare antimicrobial nanocomposites. When subtilisin was bound to the surface of AuAgNPs, it improved the biocompatibility while retaining its antimicrobial film properties. In addition, the immobilization of the nanoparticles on the polymer surface (rather than coating them) greatly prolonged the activity of the nanocomposites.
Preparation of subtilisin-conjugated AuAgSNPs and nanocomposites
· The gold dispersion was incrementally mixed with a silver nitrate stock solution in increasing volumes. Following this, the reducing solution was added swiftly. To optimize the formation of AuAgNP, varying amounts of AgNO3 (ranging from 0.50 to 8.00 cm3) and ascorbic acid solution (from 0.30 to 5.00 cm3) were utilized.
· To make AuAgNP with the addion of subtilisin, 10 M of subtilisin was added to 15 nM of the nanoparticles. After 20 minutes of incubation at 4 °C with gentle shaking, 5 L of 10% SDS solution was added into the mixture and gently stirred over night. It was then spun at 5000 rpm for 10 minutes at 4°C to remove any unbound subtilisin, and was redissolved in 25 mM phosphate buffer at pH 7.
· Two types of polycaprolactam (PCL) nanocomposites with AuAgSNP were prepared: one involved coating the nanoparticles (AuAgSNP), while the other involved their immobilization (designated as AuAgSNPI) on the polymer.