RESUMEN
Nanocapsuling organophosphorus hydrolase (OPH) is a promising strategy, which can improve OPH stability and put it into practical application. Nanocapsule can protect OPH from deactivation, but on the other hand it may block the contact of enzymes and substrates to some extent. Thus, it is worth exploring the influences of nanocapsule density on the enzyme activity and stability. In this study, OPH surface was modified by N-acryloxysuccinimide (NAS), and then the in situ radical polymerization technique was applied to construct a polymer shell around the surface to form the OPH nanocapsule (nOPH). Transmission electron microscope (TEM) and Fourier Transform Infrared (FTIR) was used to characterize the structure of nOPH. The nOPH activity is not influenced by the presence of nanocapsule when the feed ratio of NAS to OPH is below 75. On the contrary, it decreases with the increasing of NAS feed ratio when it is above 75. Furthermore, nOPH activities at high temperatures and in 20% DMSO solutions both first increase and then decrease with the increasing of NAS feed ratio. The results showed that the appropriate density of the nanocapsule could retain the enzyme activity to the maximum extent during the preparation of nOPH nanocapsule, and significantly improve its thermal stability and organic solvent stability. Hence, the results are of great significance to further realize the OPH practical application.
RESUMEN
A bacterial strain (PAP04) isolated from cattle farm soil was shown to produce an extracellular, solvent-stable protease. Sequence analysis using 16S rRNA showed that this strain was highly homologous (99%) to Brevibacillus laterosporus. Growth conditions that optimize protease production in this strain were determined as maltose (carbon source), skim milk (nitrogen source), pH 7.0, 40°C temperature, and 48 h incubation. Overall, conditions were optimized to yield a 5.91-fold higher production of protease compared to standard conditions. Furthermore, the stability of the enzyme in organic solvents was assessed by incubation for 2 weeks in solutions containing 50% concentration of various organic solvents. The enzyme retained activity in all tested solvents except ethanol; however, the protease activity was stimulated in benzene (74%) followed by acetone (63%) and chloroform (54.8%). In addition, the plate assay and zymography results also confirmed the stability of the PAP04 protease in various organic solvents. The organic solvent stability of this protease at high (50%) concentrations of solvents makes it an alternative catalyst for peptide synthesis in non-aqueous media.