Junfen Wan

Preparation of a novel light-sensitive copolymer and its application in recycling aqueous two-phase systems.

Aqueous two-phase systems (ATPSs) are potential bioseparation techniques in industry. However, a key problem is that aqueous two-phase systems could not be effectively recycled to result in high cost and environment pollution. Recently, how to prepare recycling copolymers forming aqueous two-phase systems is focused on in the area. In this study, a light-sensitive copolymer (P(NNC)) was synthesized by using N-isopropylacrylamide (NIPA), N-vinyl-2-pyrrolidone (NVP), chlorophyllin sodium copper salt (CHL) as monomers. The copolymer P(NNC) can form ATPSs with another novel pH-sensitive copolymer (P(ADB)) which was synthesized by co-worker in our laboratory. Over 98% of the P(NNC) copolymer could be recovered by using laser radiation at 488 nm. The copolymer P(ADB) could be recovered by adjusting the isoelectric point (pI) to 4.1, with a recovery of 97%. Bovine serum albumin (BSA) and Tyr were partitioned in the P(NNC)-P(ADB) aqueous two-phase systems to examine the systems. It was found that partition coefficient of BSA and L-Tyr could reach 4.1 and 0.12 in the systems, respectively.

Synthesis of thermo-responsive polymers recycling aqueous two-phase systems and phase formation mechanism with partition of ε-polylysine.

Aqueous two-phase systems (ATPS) have the potential application in bioseparation and biocatalysis engineering. In this paper, a recyclable ATPS was developed by two thermo-responsive copolymers, PVBAm and PN. Copolymer PVBAm was copolymerized using N-vinylcaprolactam, Butyl methacrylate and Acrylamide as monomers, and PN was synthesized by N-isopropylacrylamide. The lower critical solution temperature (LCST) of PVBAm and PN were 45.0°C and 33.5°C, respectively. The recoveries of both polymers could achieve over 95.0%. The phase behavior and formation mechanism of PVBAm/PN ATPS was studied. Low-field nuclear magnetic resonance (LF-NMR) was applied in the phase-forming mechanism study in ATPS. In addition, combining the analysis results of surface tension, transmission electron microscopy and dynamic light scattering, the phase-forming of the PVBAm/PN ATPS was proved. The application was performed by partition of ε-polylysine in the 2% PVBAm/2% PN (w/w) ATPS. The results demonstrated that ε-polylysine was extracted into the PN-rich phase, the maximal partition coefficient (1/K) and extraction recovery of pure ε-polylysine were 6.87 and 96.36%, respectively, and 7.41 partition coefficient and 97.85% extraction recovery for ε-polylysine fermentation broth were obtained in the presence of 50mM (NH4)2SO4 at room temperature. And this method can effectively remove the most impurities from fermentation broth when (NH4)2SO4 exists in the ATPS. It is believed that the thermo-responsive recycling ATPS has a good application prospect in the field of bio-separation.

Preparation of a Light‐Sensitive and Reversible Dissolution Copolymer and Its Application in Lysozyme Purification

A novel light‐sensitive and cation‐exchange copolymer (PNBCC) has been synthesized by random copolymerization of chlorophyllin sodium copper salt, crylic acid, n‐butyl acrylate, and N‐isopropylacrylamide. The PNBCC copolymer showed reversible dissolution and could be precipitated by 488 nm laser irradiation with the least light density of 1.70 × 105 W/m2. By optimizing the ratio of monomers, pH, and ion concentration, over 95% copolymer was recovered by laser irradiation. The copolymer was used to purify lysozyme as light‐sensitive cation exchanger, and its adsorption matched a Langmuir adsorption isotherm with maximum adsorption capacity of 98 900 U/g and dissociated constant of 852 U/mL. By applying the copolymer to the separation of lysozyme from egg white, the specific activity of lysozyme was improved from 399 to 6346 U/mg and the recovery of lysozyme achieved 81.3%.

Molecular interaction mechanisms in reverse micellar extraction of microbial transglutaminase

Reverse micellar extraction is an efficient and economical alternative for protein purification. 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 work might provide valuable reference to the MTGase purification and downstream processing.