Preparation Of Recycling Aqueous Two-Phase Systems Composed Of Two Thermo-response Polymers And Its Application On Enzymatic Synthesis Of Cefprozil

Separation and purification of biological products is a key step in the large-scale application of products. Aqueous two-phase systems are efficient purification methods for bioproducts like proteins, antibiotics, amino acids and organic acids due to low interfacial tension, fast mass transfer, high water content in two phases and mild aqueous environment. However, recovery difficulty of the copolymers forming aqueous two-phase systems becomes obstacle in scale-up application. Cefprozil with high safety is an important semi-synthetic β-lactam antibiotics traditionally manufactured using chemical synthetic routes and enzymatic synthesis in single aqueous solution. The chemical route has been criticized for using toxic and not easily biodegradable solvents, multiple steps of reactions and low temperatures. The enzymatic synthesis in single aqueous solution has low yield due to product inhibition. Therefore, searching for new ways of enzymatic synthesis is a trend.In this paper, four thermo-response polymers were synthesized and were used to construct thermo-response aqueous two-phase systems. Copolymer PNBAa was copolymerized by using N-isopropylacrylamide （NIPA）, n-butyl methacrylate （BMA） and allyl alcohol （Aa） as monomers. Copolymer P_NDB was synthesized by using N-isopropylacrylamide （NIPA）,2-（dimethylamino） ethyl methacrylate （DMAEMA） and n-butyl methacrylate （BMA） as monomers. The lower critical solution temperatures （LCST） of these two polymers are25.5°C and31.3°C, respectively. Copolymer PNE was copolymerized by using N-isopropylacrylamide and Ethyl methacrylate as monomers, and PvAm was synthesized by using N-Vinylcaprolactam and Acrylamide as monomers. The lower critical solution temperatures of P_NE and P_VAm are28.7°C and35.6°C, respectively. The structures of polymers were tested by IR and H NMR and were confirmed that the synthesized polymers were desired. The average molecular weights of two polymers were measured by ubbelohde viscometer and GPC. Molecular weights of P_NE, P_VAm, P_NDB, P_NBAa are about1.89×106Da,2.75×105Da,5.63×104Da and9.72×104Da. The recoveries of polymers at different conditions were tested and the maximal recoveries of P_VAm, P_NE, P_NDB and P_NBAa are above95%.Phase diagram of P_NE-P_VAm with different conditions was investigated and suitable experimental equation was confirmed. The conditions of polymers tested by HPLC as follows: 220nm, acetonitrile, KPB,50:50（volume ratio） and0.7ml/min. Great effect of SO4^-2₄on phase diagram was found by adding different salts and different salt concentration. Two experimental equations were used to correlate with experimental data of biondal curve and the equation w₁=exp(a+bw^0.5₂+cw₂+dw²₂) was more suitable to phase diagram of P_NE-P_VAm on the basis of RMSE.Partition coefficient of cefprozil and7-APRA in ATPS was studied and the optimal concentration of forming ATPS is5%P_NE and10%P_VAm. Due to phase diagram data, tie-line length and viscosity of ATPS, aqueous two-phase system composed of10%P_NE and10%PvAm is chosen for partition of bioproduct. The best condition for cefprozil and7-APRA tested by HPLC as follows:280nm, acetonitrile, Ammonium sulfate buffer,10:90（volume ratio） and1ml/min. The optimal partition coefficient for cefprozil and7-APRA is6.51and0.086at pH6and70mM LiCl by adding different salts and changing salt concentration. Effect of temperature and sequence of adding material on partition coefficient is small by determining partition coefficient of cefprozil and7-APRA in the ATPS.Enzymatic synthesis of cefprozil in single aqueous phase was carried out. The optimal condition for enzymatic synthesis of cefprozil in single aqueous phase as follows:pH6.6,20°C,150r/min,40mM7-APRA,104mM D-HPGME-HCl and6μ/ml （enzyme load）. From the result, we can see that the yield is75%under the optimal conditions. However, the reaction is inhibited by high concentration product, resulting that substrates cannot be utilized completely. At the same time, there is side reaction.Enzymatic synthesis of cefprozil in ATPS composed of P_NE-P_VAm was carried out. The optimal condition for enzymatic synthesis of cefprozil in single aqueous phase as follows:pH6.56,20°C,190r/min,40mM7-APRA,104mM D-HPGME-HC1and6u/ml （enzyme load）. Yield with D-HPGME-HC1is higher than that with D-HPGME because solubility of D-HPGME-HC1is higher than that of D-HPGME.95.7%yield is obtained at70mM LiCl by adding different salts and changing salt concentration. The condition with95.7%yield is the same as the condition with optimal partition coefficient of cefprozil and7-APRA.