温度敏感可再生型两水相体系的构建及固定化青霉素酰化酶催化合成头孢丙烯
摘要
生物分离纯化是实现产品工业应用的关键环节,两水相体系因其含水量高、表面张力小、绿色环保、操作简单等因素而被广泛应用于蛋白、多糖、氨基酸、天然活性物质和抗生素等的分离提取,然而聚合物回收困难阻碍了两水相体系的大规模工业应用。头孢丙烯作为第二代头孢菌素因其较高的安全性而被广泛应用于儿童感染用药市场,但是化学合成步骤繁琐以及单水相中酶催化反应受限于产物的抑制作用限制了头孢丙烯的生产,寻求高效的酶催化反应成为一种趋势。
本课题利用温敏单体、疏水性单体和亲水性单体为基础合成了四种具有很高回收率的聚合物即P_(VAm)、P_(NE)、 P_(NDB)和P_(NBAa°)其中,P_(NBAa)以N-异丙基丙烯酰胺、甲基丙烯酸丁酯和丙烯醇为单体无规自由聚合；P_(NDB)以N-异丙基丙烯酰胺、甲基丙烯酸二甲氨基乙酯和甲基丙烯酸丁酯为单体；PNE以N-异丙基丙烯酰胺和甲基丙烯酸乙酯为单体；P_(VAm)以N-乙烯基己内酰胺和丙烯酰胺为单体。通过IR和1HNMR确定了聚合物的结构；通过乌氏粘度计方法测定了聚合物在水溶液中的粘度,P_(NDB)和P_(NBAa)的特性粘度为45.1]mg/ml和33.5mg/ml, P_(VAm)和PNE的特性粘度为32.5ml/g和79.1ml/g；通过凝胶渗透色谱乌氏粘度计测得了聚合物的分子量,P_(NDB)和P_(NBAa)的分子量为5.63×104Da和9.72×104Da, P_(NF)和P_(VAm)的分子量为1.86×106Da和2.75×105Da；通过称重法定了聚合物的回收率,测得聚合物PNE的回收率为97.5%,PVAm的回收率为96.3%；通过测定不同浓度聚合物的浊点-浓度关系曲线确立了聚合物P_(NDB)和P_(NBAa)的最低临界点温度分别为25.5℃和31.3℃,聚合物PNE和P_(VAm)的最低临界点温度分别为28.7℃和35.6℃。
研究了可再生型两水相体系不同条件下的相图,确立了适合P_(NE)-P_(VAm)两水相体系的理论模型。通过优化液相条件,确立了聚合物PNE和P_(VAm)的最佳液相条件为220nm、乙腈为有机相、磷酸钾缓冲液为水相、体积比为50：50,流速为0.7ml/min;通过添加无机盐和改变浓度,发现硫酸根对相图的影响较大；利用两种经验模型对取得的双节线数据进行拟合,比较拟合结果均方差根的大小发现经验模型w1=exp(a+bw20.5+Cw2+dw22)比较适合该两水相体系。
研究了头孢丙烯以及其母核在P_(VAm)-P_(NE)可再生型两水相体系中的分配,确立了成相的聚合物的最佳浓度即5%P_(NE)-10%P_(VAm);通过相图数据和系线长度与两水相上下两相性质差异的关系,还有聚合物的粘度因素,选择10%PNE-10%P_(VAm)两水相进行物质的分配；通过HPLC确立了头孢丙烯以及底物的液相检测条件：乙腈作为有机相、硫酸铵缓冲液作为水相、流速为1ml/min、相体积比为10：90为最佳条件；通过添加不同种类的无机盐以及改变添加浓度,在pH为6,添加70mMLiCl时头孢丙烯和母核的分配系数达到6.51和0.086；通过改变温度和改变添加顺序,观察分配系数的变化,发现温度和改变添加顺序对头孢丙烯以及底物的分配系数影响不大。
研究了单水相中头孢丙烯的酶催化合成,确立了在单水相中酶催化合成头孢丙烯的最佳条件为：pH6.6、温度20℃、7-APRA与D-HPGME-HCl摩尔比为1：3、酶量为6u/ml、转速为150r/min。因此,从实验结果来看,在最佳条件下得率达到75%,这表明该酶催化反应还不够彻底,还有很大提高空间,但是由于在单水相中存在产物对酶的抑制作用,底物利用率不高,还有副反应发生,所以使的最终得率不高。
采用P_(vam)-P_(NE)可再生型两水相体系进行头孢丙烯的酶催化合成,确立了最佳酶催化合成条件：温度20℃、40mM7-APRA、104mM D-HPGME-HCl、 pH6.56、190r/min、酶量6u/ml、磷酸钾缓冲液。研究不同供体形式对酶催化合成的影响,结果表明溶解性能更好的D-HPGME相比于酯类酰基供体D-HPGME具有更好的酶催化效果；通过添加不同种类的无机盐和不同浓度的无机盐,考察了其对酶催化合成结果的影响,得到在70mM LiCl条件下头孢丙烯的酶催化合成得率最高达到95.7%,这也与在该条件下头孢丙烯的分配系数达到最大相吻合。
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~(-2)_4on 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 w_1=exp(a+bw~(0.5)_2+cw_2+dw~2_2) 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.
本课题利用温敏单体、疏水性单体和亲水性单体为基础合成了四种具有很高回收率的聚合物即P_(VAm)、P_(NE)、 P_(NDB)和P_(NBAa°)其中,P_(NBAa)以N-异丙基丙烯酰胺、甲基丙烯酸丁酯和丙烯醇为单体无规自由聚合；P_(NDB)以N-异丙基丙烯酰胺、甲基丙烯酸二甲氨基乙酯和甲基丙烯酸丁酯为单体；PNE以N-异丙基丙烯酰胺和甲基丙烯酸乙酯为单体；P_(VAm)以N-乙烯基己内酰胺和丙烯酰胺为单体。通过IR和1HNMR确定了聚合物的结构；通过乌氏粘度计方法测定了聚合物在水溶液中的粘度,P_(NDB)和P_(NBAa)的特性粘度为45.1]mg/ml和33.5mg/ml, P_(VAm)和PNE的特性粘度为32.5ml/g和79.1ml/g；通过凝胶渗透色谱乌氏粘度计测得了聚合物的分子量,P_(NDB)和P_(NBAa)的分子量为5.63×104Da和9.72×104Da, P_(NF)和P_(VAm)的分子量为1.86×106Da和2.75×105Da；通过称重法定了聚合物的回收率,测得聚合物PNE的回收率为97.5%,PVAm的回收率为96.3%；通过测定不同浓度聚合物的浊点-浓度关系曲线确立了聚合物P_(NDB)和P_(NBAa)的最低临界点温度分别为25.5℃和31.3℃,聚合物PNE和P_(VAm)的最低临界点温度分别为28.7℃和35.6℃。
研究了可再生型两水相体系不同条件下的相图,确立了适合P_(NE)-P_(VAm)两水相体系的理论模型。通过优化液相条件,确立了聚合物PNE和P_(VAm)的最佳液相条件为220nm、乙腈为有机相、磷酸钾缓冲液为水相、体积比为50：50,流速为0.7ml/min;通过添加无机盐和改变浓度,发现硫酸根对相图的影响较大；利用两种经验模型对取得的双节线数据进行拟合,比较拟合结果均方差根的大小发现经验模型w1=exp(a+bw20.5+Cw2+dw22)比较适合该两水相体系。
研究了头孢丙烯以及其母核在P_(VAm)-P_(NE)可再生型两水相体系中的分配,确立了成相的聚合物的最佳浓度即5%P_(NE)-10%P_(VAm);通过相图数据和系线长度与两水相上下两相性质差异的关系,还有聚合物的粘度因素,选择10%PNE-10%P_(VAm)两水相进行物质的分配；通过HPLC确立了头孢丙烯以及底物的液相检测条件：乙腈作为有机相、硫酸铵缓冲液作为水相、流速为1ml/min、相体积比为10：90为最佳条件；通过添加不同种类的无机盐以及改变添加浓度,在pH为6,添加70mMLiCl时头孢丙烯和母核的分配系数达到6.51和0.086；通过改变温度和改变添加顺序,观察分配系数的变化,发现温度和改变添加顺序对头孢丙烯以及底物的分配系数影响不大。
研究了单水相中头孢丙烯的酶催化合成,确立了在单水相中酶催化合成头孢丙烯的最佳条件为：pH6.6、温度20℃、7-APRA与D-HPGME-HCl摩尔比为1：3、酶量为6u/ml、转速为150r/min。因此,从实验结果来看,在最佳条件下得率达到75%,这表明该酶催化反应还不够彻底,还有很大提高空间,但是由于在单水相中存在产物对酶的抑制作用,底物利用率不高,还有副反应发生,所以使的最终得率不高。
采用P_(vam)-P_(NE)可再生型两水相体系进行头孢丙烯的酶催化合成,确立了最佳酶催化合成条件：温度20℃、40mM7-APRA、104mM D-HPGME-HCl、 pH6.56、190r/min、酶量6u/ml、磷酸钾缓冲液。研究不同供体形式对酶催化合成的影响,结果表明溶解性能更好的D-HPGME相比于酯类酰基供体D-HPGME具有更好的酶催化效果；通过添加不同种类的无机盐和不同浓度的无机盐,考察了其对酶催化合成结果的影响,得到在70mM LiCl条件下头孢丙烯的酶催化合成得率最高达到95.7%,这也与在该条件下头孢丙烯的分配系数达到最大相吻合。
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~(-2)_4on 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 w_1=exp(a+bw~(0.5)_2+cw_2+dw~2_2) 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.
引文
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