脲酶的组合固定化研究
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摘要
脲酶是催化尿素分解为氨和二氧化碳的专一性水解酶,固定化脲酶已成为工业上以脲酶为基础催化水解底物的必要条件。通过实验研究中改变不同的环境因素,如pH值、温度、缓冲反应液等已成功的实现了脲酶的固定化；酶固定化方法有很多,每种方法既有其优点又有缺点,如吸附法操作过程虽然简单有效,但酶容易脱落；共价法和交联法有效并且储存稳定,但材料昂贵且对固定化酶酶活有损害；在膜反应器法,包埋法和微囊法中的内部传质效率较差；通常作为衡量最佳固定化酶条件的标准是对所选择的酶及其固定化方法的反复试验来得出的,对选择固定化酶的材料原则上以无毒,价格低,可再生及生物降解性为主要的标准。
本课题选取的主要载体材料包括壳聚糖和亲水性聚合物,通过将材料按不同比例混合,采用不同的方法制备酶载体,包括膜块、微球、微膜等,其中固定化技术采用吸附-交联、包埋-交联以及无载体固定等几种方法；同时对以壳聚糖为主体载体进行了形态表征以及接触角的测定,从微观和宏观上检测其固定化酶的效果,以及微膜表面及内部的孔洞形成情况。通过比较不同材料和技术固定化脲酶,综合性质最佳的是在壳聚糖与聚乙二醇为1：1时形成的微膜,从SEM图片上来看CS/PEG薄膜表面及内部形成了孔径均一,分布均匀的多孔结构,增大了比表面积,提高了固定化酶的活力回收值,达到了102.75%左右,通过接触角测量,在CS/PEG=1在吸附酶前后有明显的变化,而且其固定化酶活力回收值大于其他薄膜,其固定化效果为103.72U/(cm2·min),表明酶蛋白成功吸附在薄膜上,使薄膜表面的亲水性增加。
Urease is present in abundance of jackbean' seeds which catalyzes decomposition of urea to produce ammonia and carbon dioxide. Urease immobilization has been a prerequisite for industries based on enzyme hydrolyzed products, it has been successfully implemented at lab scales for enzyme stabilization from various environment factors including pH, temperature, buffers, etc. Several methods of enzyme immobilization have been known,each of them has their own weakness and strength.Adsorption is simple,cheap and effective but frequently reversible, covalent and cross-linking are effective and durable, but expensive and easily worsening the enzyme activity,and in membrane reactor-confinement,entrapment and micro--encapsulations diffusional problems are inherent. Principlally, as a standard the optimal immobilization conditions for a chosen enzyme and its application are done empirically by a process of trial.Meanwhile, the matrixs chosen which are non-toxic, cheap, renewable and biodegradable.
In this work, chitosan and some hydrophilic polymers were mainly chosen as matrixs.and blended with them in different ratio to form different shape including gelatin.microsphere and micro membrane. The methods of immobilization contained adsorption-crosslink, entrapment-crosslink and CLEA, meanwhile SEM and Automatic Contact Angle measure device were used to observe the structure and pore distribution on the surface and cross-section, the change of hydrophilicity on the chitosan matrix surface. Immobilization methods above were compared, the best one was chitsoan/PEG=1micro membrane. Uniform and thoroughfare pores had formed on the membrane which enlarged the specific surface area and enhanced the relative activity which had reached to102.75%. The measurement of contact angle on chitosan/PEG membrane showed that the hydrophilicity of membrane surface had changed after urease adsorption and relative activity was higer than other chitosan matrix sharply, the effect of immobilization was103.72U/(cm2·min) which revealed that urease was adsorbed on chitosan membrane successfully.
本课题选取的主要载体材料包括壳聚糖和亲水性聚合物,通过将材料按不同比例混合,采用不同的方法制备酶载体,包括膜块、微球、微膜等,其中固定化技术采用吸附-交联、包埋-交联以及无载体固定等几种方法；同时对以壳聚糖为主体载体进行了形态表征以及接触角的测定,从微观和宏观上检测其固定化酶的效果,以及微膜表面及内部的孔洞形成情况。通过比较不同材料和技术固定化脲酶,综合性质最佳的是在壳聚糖与聚乙二醇为1：1时形成的微膜,从SEM图片上来看CS/PEG薄膜表面及内部形成了孔径均一,分布均匀的多孔结构,增大了比表面积,提高了固定化酶的活力回收值,达到了102.75%左右,通过接触角测量,在CS/PEG=1在吸附酶前后有明显的变化,而且其固定化酶活力回收值大于其他薄膜,其固定化效果为103.72U/(cm2·min),表明酶蛋白成功吸附在薄膜上,使薄膜表面的亲水性增加。
Urease is present in abundance of jackbean' seeds which catalyzes decomposition of urea to produce ammonia and carbon dioxide. Urease immobilization has been a prerequisite for industries based on enzyme hydrolyzed products, it has been successfully implemented at lab scales for enzyme stabilization from various environment factors including pH, temperature, buffers, etc. Several methods of enzyme immobilization have been known,each of them has their own weakness and strength.Adsorption is simple,cheap and effective but frequently reversible, covalent and cross-linking are effective and durable, but expensive and easily worsening the enzyme activity,and in membrane reactor-confinement,entrapment and micro--encapsulations diffusional problems are inherent. Principlally, as a standard the optimal immobilization conditions for a chosen enzyme and its application are done empirically by a process of trial.Meanwhile, the matrixs chosen which are non-toxic, cheap, renewable and biodegradable.
In this work, chitosan and some hydrophilic polymers were mainly chosen as matrixs.and blended with them in different ratio to form different shape including gelatin.microsphere and micro membrane. The methods of immobilization contained adsorption-crosslink, entrapment-crosslink and CLEA, meanwhile SEM and Automatic Contact Angle measure device were used to observe the structure and pore distribution on the surface and cross-section, the change of hydrophilicity on the chitosan matrix surface. Immobilization methods above were compared, the best one was chitsoan/PEG=1micro membrane. Uniform and thoroughfare pores had formed on the membrane which enlarged the specific surface area and enhanced the relative activity which had reached to102.75%. The measurement of contact angle on chitosan/PEG membrane showed that the hydrophilicity of membrane surface had changed after urease adsorption and relative activity was higer than other chitosan matrix sharply, the effect of immobilization was103.72U/(cm2·min) which revealed that urease was adsorbed on chitosan membrane successfully.
引文
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