氯过氧化物酶的固定化及其应用研究
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摘要
氯过氧化物酶(Chloroperoxidase,CPO)因其独特的活性中心结构而成为过氧化物酶家族中应用最广泛的酶,尤其引人注目的是它对底物具有手性识别功能,因此在不对称有机合成,生物转化,手性药物制备,环境毒物降解等领域具有诱人的应用前景。但游离CPO在高温、强酸强碱等极端条件下易变性失活,严重限制了它的产业化应用。本文基于CPO的固定化研究,提高了酶的热稳定性、催化活性以及对合成环境的适应能力,从而可为固定化酶在工业化生产中的实际应用奠定基础。
酶固定化技术是实现酶重复连续使用和稳定性改善的有效手段,目前对过氧化物酶家族中其它酶的固定化报道较多,而对CPO的固定化报道较少。本文选用两种新型材料——壳聚糖膜/球和磁性微球作为载体进行CPO的固定化研究。壳聚糖作为一种高分子材料不仅来源丰富,化学性质稳定,易于通过接枝而改性,而且具有生物相容性,是一种十分理想的酶固定化载体;而磁性微球所具有的超顺磁性,使其分离过程可以通过一个钕铁硼永磁材料提供的磁场反复操作而不聚集,十分有利于亲水性的CPO与反应产物的分离及回收循环利用;同时经异硫氰酸根包覆的磁性微球表面易于酶的功能基团自组装,可达到较高的酶载量。
本文通过MCD氯化活性的测定考察了CPO固定的最适条件,并研究了固定化CPO的结构和表面性质,比较了游离CPO和固定化CPO间酶学性质的差异,并将磁粒固定化CPO应用于有机合成——邻苯三酚的催化聚合反应。主要实验结果如下:
1.以壳聚糖为载体,戊二醛为交联剂,通过共价交联法制备了壳聚糖膜(球)固定化CPO。固定化反应的最佳条件:戊二醛浓度0.8%,pH 3.5。与游离CPO相比,热稳定性、对化学变性剂尿素和过氧化氢的耐受性均有明显提高。在50℃下,温育1 h后游离CPO仅剩余0.02%的催化活性,而固定化CPO可将催化活性保留40%以上;同时当尿素浓度为1.5M时,固定化CPO基本不丧失活性,而游离CPO的活性损失超过32%;而在300μM H_2O_2中放置20 h后,固定化CPO保留酶活性58%,游离CPO只能保留42%。
2.以xMag~(TM)异硫氰酸根末端磁性微球为母体,通过共价结合的方法固定CPO。固定化反应的最佳条件:pH 5.5,绑定时间10 min,酶载量可达25.59 mg/g,固定化效率为75.5%。与游离CPO相比,米氏常数Km值增大,固定化CPO与底物亲和力减小,热稳定性、对尿素、过氧化氢及有机溶剂的抗失活能力,以及重复使用次数均有所提高。在300μM的H_2O_2溶液中储存96 h后固定化CPO存留活性仍有50%,而游离CPO仅剩9%。固定化CPO重复使用16次后,其活力仍能保持最初活性的85%以上。
3.因此本文研究了磁性微球固定化氯过氧化物酶对邻苯三酚的催化聚合反应。结果表明,固定化氯过氧化物酶可高效催化邻苯三酚转化为红棓酚。
Chloroperoxidase(CPO) is recognized as the most versatile enzyme in the group of peroxidases because of its unique active site structure,it is noticeable particularly that it has the function of chiral recognition for substrates,so it has very captivating development and application in asymmetric organic synthesis,biotransformation,chiral pharmaceuticals,detoxication of environmental pollutants and etc.However,due to its inactivation in extreme conditions,including high-temperature,strong acids and base,the industrial application of native CPO has been severely limited.In this paper,immobilized CPO improved thermal stability and catalytic activity of enzyme, and increased the adaptive ability to the synthesis environment.Meanwhile,it will provide a basis for immobilized enzyme ha the practical application.
Immobilization technology of enzyme is an effective measure to achieve repeatly using of enzyme and improve stability.In the article,two new materials were selected for the carriers of immobilized CPO,including chitosan membranes/beads and magnetic microspheres.Chitosan is a very ideal immobilized carrier because of its rich resource,good biocompatibility and susceptibility to chemical modification.Meanwhile,magnetic microspheres have super paramagnetism,so the immobilized enzyme by magnetic microspheres is easer to be separate and recycle than other carriers, and it can reach a higher-load.The results of optimized conditions of immobilization of CPO, differences between native CPO and immobilized CPO had be investigated by MCD assay,and application of immobilized CPO in catalytic polymerization of pyrogaUol had be showed as follows:
1.CPO was covalently immobilized on chitosan membranes(beads) under mild condition with glutararldehyde as a crosslinking agent.The results indicated that the optimum concentration of glutaraldehyde and pH were determined to be 0.8%and 3.5,respectively.Compared with the native CPO,immobilized CPO has excellent thermal stability,and the stability of immobilized CPO against urea and hydrogen peroxide exposure was improved.The immobilized CPO can retain about 40%of its activity after 1h incubation at 50℃,whereas native CPO could maintain only about 0.02%of its activity under the same conditions.Furthermore,immobilized CPO had unchanged residual activity (about 100%),but the 32%loss of activity for native CPO in the presence of 1.5 M urea.When incubated with 300μM H_2O_2,the immobilized CPO retained 58%after 20 h whereas the residual activity of native CPO only retained 42%.
2.Thiocyanate groups containing magnetic micropheres were used in covalent immobilization of the enzyme "CPO".The results showed the optimal conditions of immobilization:the optimal pH was 5.5,and the optimal reaction time was 10 min.The maximum amount of immobilization CPO on the magnetic micropheres was 25.59 mg/g support.Compared with the native CPO,the affinity of immobilized CPO decreased toward substrate and its stability was considerably improved. Immobilized CPO is incubated in the presence of 300μM H_2O_2 in different times,the immobilized CPO still remain about 50%of its activity after 96 h,whereas native CPO only remained around 9%. This result suggested that the covalently immobilized CPO was considerably stable on the magnetic micropheres surface and the immobilized CPO retained more than 85%of its initial activity after 16 cycles of usage.
3.So the catalyzed polymerization of pyrogallol using immobilized CPO was investigated.The result indicates that the immobilized enzyme was used for transforming pyrogallol into purpurogallin in aqueous phase.
酶固定化技术是实现酶重复连续使用和稳定性改善的有效手段,目前对过氧化物酶家族中其它酶的固定化报道较多,而对CPO的固定化报道较少。本文选用两种新型材料——壳聚糖膜/球和磁性微球作为载体进行CPO的固定化研究。壳聚糖作为一种高分子材料不仅来源丰富,化学性质稳定,易于通过接枝而改性,而且具有生物相容性,是一种十分理想的酶固定化载体;而磁性微球所具有的超顺磁性,使其分离过程可以通过一个钕铁硼永磁材料提供的磁场反复操作而不聚集,十分有利于亲水性的CPO与反应产物的分离及回收循环利用;同时经异硫氰酸根包覆的磁性微球表面易于酶的功能基团自组装,可达到较高的酶载量。
本文通过MCD氯化活性的测定考察了CPO固定的最适条件,并研究了固定化CPO的结构和表面性质,比较了游离CPO和固定化CPO间酶学性质的差异,并将磁粒固定化CPO应用于有机合成——邻苯三酚的催化聚合反应。主要实验结果如下:
1.以壳聚糖为载体,戊二醛为交联剂,通过共价交联法制备了壳聚糖膜(球)固定化CPO。固定化反应的最佳条件:戊二醛浓度0.8%,pH 3.5。与游离CPO相比,热稳定性、对化学变性剂尿素和过氧化氢的耐受性均有明显提高。在50℃下,温育1 h后游离CPO仅剩余0.02%的催化活性,而固定化CPO可将催化活性保留40%以上;同时当尿素浓度为1.5M时,固定化CPO基本不丧失活性,而游离CPO的活性损失超过32%;而在300μM H_2O_2中放置20 h后,固定化CPO保留酶活性58%,游离CPO只能保留42%。
2.以xMag~(TM)异硫氰酸根末端磁性微球为母体,通过共价结合的方法固定CPO。固定化反应的最佳条件:pH 5.5,绑定时间10 min,酶载量可达25.59 mg/g,固定化效率为75.5%。与游离CPO相比,米氏常数Km值增大,固定化CPO与底物亲和力减小,热稳定性、对尿素、过氧化氢及有机溶剂的抗失活能力,以及重复使用次数均有所提高。在300μM的H_2O_2溶液中储存96 h后固定化CPO存留活性仍有50%,而游离CPO仅剩9%。固定化CPO重复使用16次后,其活力仍能保持最初活性的85%以上。
3.因此本文研究了磁性微球固定化氯过氧化物酶对邻苯三酚的催化聚合反应。结果表明,固定化氯过氧化物酶可高效催化邻苯三酚转化为红棓酚。
Chloroperoxidase(CPO) is recognized as the most versatile enzyme in the group of peroxidases because of its unique active site structure,it is noticeable particularly that it has the function of chiral recognition for substrates,so it has very captivating development and application in asymmetric organic synthesis,biotransformation,chiral pharmaceuticals,detoxication of environmental pollutants and etc.However,due to its inactivation in extreme conditions,including high-temperature,strong acids and base,the industrial application of native CPO has been severely limited.In this paper,immobilized CPO improved thermal stability and catalytic activity of enzyme, and increased the adaptive ability to the synthesis environment.Meanwhile,it will provide a basis for immobilized enzyme ha the practical application.
Immobilization technology of enzyme is an effective measure to achieve repeatly using of enzyme and improve stability.In the article,two new materials were selected for the carriers of immobilized CPO,including chitosan membranes/beads and magnetic microspheres.Chitosan is a very ideal immobilized carrier because of its rich resource,good biocompatibility and susceptibility to chemical modification.Meanwhile,magnetic microspheres have super paramagnetism,so the immobilized enzyme by magnetic microspheres is easer to be separate and recycle than other carriers, and it can reach a higher-load.The results of optimized conditions of immobilization of CPO, differences between native CPO and immobilized CPO had be investigated by MCD assay,and application of immobilized CPO in catalytic polymerization of pyrogaUol had be showed as follows:
1.CPO was covalently immobilized on chitosan membranes(beads) under mild condition with glutararldehyde as a crosslinking agent.The results indicated that the optimum concentration of glutaraldehyde and pH were determined to be 0.8%and 3.5,respectively.Compared with the native CPO,immobilized CPO has excellent thermal stability,and the stability of immobilized CPO against urea and hydrogen peroxide exposure was improved.The immobilized CPO can retain about 40%of its activity after 1h incubation at 50℃,whereas native CPO could maintain only about 0.02%of its activity under the same conditions.Furthermore,immobilized CPO had unchanged residual activity (about 100%),but the 32%loss of activity for native CPO in the presence of 1.5 M urea.When incubated with 300μM H_2O_2,the immobilized CPO retained 58%after 20 h whereas the residual activity of native CPO only retained 42%.
2.Thiocyanate groups containing magnetic micropheres were used in covalent immobilization of the enzyme "CPO".The results showed the optimal conditions of immobilization:the optimal pH was 5.5,and the optimal reaction time was 10 min.The maximum amount of immobilization CPO on the magnetic micropheres was 25.59 mg/g support.Compared with the native CPO,the affinity of immobilized CPO decreased toward substrate and its stability was considerably improved. Immobilized CPO is incubated in the presence of 300μM H_2O_2 in different times,the immobilized CPO still remain about 50%of its activity after 96 h,whereas native CPO only remained around 9%. This result suggested that the covalently immobilized CPO was considerably stable on the magnetic micropheres surface and the immobilized CPO retained more than 85%of its initial activity after 16 cycles of usage.
3.So the catalyzed polymerization of pyrogallol using immobilized CPO was investigated.The result indicates that the immobilized enzyme was used for transforming pyrogallol into purpurogallin in aqueous phase.
引文
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