The Effect Of Chaotropes On Reverse Micellar Backward Extraction And The Molecular Interaction In Reverse Micellar Extraction

Reverse micellar extraction is an efficient and economical alternative for protein purification.Chaotropes(urea and guanidine hydrochloride)could significantly enhance enzyme recovery in reverse micellar back extraction.In this study,we added chaotropes in the process of lipase reverse micelle extraction,and obtained favorable back extraction recovery.In back extraction,at 0.6 M urea in stripping solution,94.60%total extraction recovery was obtained.Meanwhile at 0.3 M guanidine hydrochloride,nearly 65%lipases were released into stripping solution.DLS and Karl Fischer method results showed that presence of urea in stripping solution could weaken the electrostatic interaction between lipase and CTAB molecules,promoted lipase get out of reverse micelles.And GuHCl could make enzymes and free water molecules enwrapped in reverse micelles easily release to the stripping solution.In this study,microbial transglutaminase(MTGase)from crude materials was purified using reverse micellar extraction,and the molecular interaction mechanism in reverse micellar extraction of MTGase was explored.By using a molecular simulation study,the interaction mechanism of forward extraction was investigated.The molecular simulation results reveal the interaction of MTGase-water-surfactant is the major driving force for the forward extraction.Further,the effect of ionic strength on molecular interactions in backward extraction was investigated using 1H low-field nuclear magnetic resonance(LF-NMR)and circular dichroism(CD)spectra.In backward extraction,the interactions between water and the other two molecules(MTGase and surfactant molecules)are enhanced while the interactions between target molecules(MTGase)and the other two molecules(water and surfactant molecules)are weakened as the ionic strength increases.Moreover,the effect of size exclusion on backward extraction was also investigated.The results demonstrate size exclusion has limit effect at high ionic strength,and the weakened interaction of MTGase-water-surfactant is the main reason causing the release of the target molecules in backward extraction.This study was carried out to immobilize the partially purified laccase from trametes versicolor on pH-sensitive polyacrylate amphiphilic copolymers and subsequently using the immobilized laccase in the reverse micellar solvent.Under optimized conditions,the activity recovery of the immobilized laccase was 71.90%and its recovery was 96.8%by adjusting the pH of the aqueous solution to 3.3.The Km of the immobilized laccase is 9.32mmol/L and the relative activity of the immobilized laccase in the reverse micelles is 50%.This work might provide valuable reference to the reverse micellar extraction and downstream processing.