纸纤维固定木瓜蛋白酶的研究及应用  

Study on the paper cellulose immobilized papain and its exploration

作　　者：易喻[1] 李慧娟[1] 陈建澍[1] 梅建凤[1] 朱克寅 应国清[1] 

机构地区：[1]浙江工业大学药学院,浙江杭州310032 [2]杭州博林生物技术有限公司,浙江杭州300018

出　　处：《浙江工业大学学报》2013年第2期147-151,共5页Journal of Zhejiang University of Technology

基　　金：浙江省科技厅资助项目(2011C23070)

摘　　要：利用纸纤维为载体,制备高活性的固定化木瓜蛋白酶并应用于抗HCG抗体的水解.利用戊二醛交联法,制备固定化木瓜蛋白酶,并通过一系列优化,得到最佳固定化条件,包括pH﹑给酶量﹑温度﹑戊二醛体积分数,将制备的固定化木瓜蛋白酶应用于抗HCG抗体水解并制备F(ab′)2片段.研究表明:反应温度为40℃时,固定化酶在pH 5.0,0.01mol/L的醋酸缓冲液条件下,给酶量为46mg/g载体,戊二醛体积分数为0.01%时,固定化酶的酶活达到最高19 250U/g,相对于优化前1 940U/g提高了近10倍,成功应用于抗HCG抗体水解制备F(ab′)2片段:经SDS-PAGE电泳检测,F(ab′)2片段达到电泳纯.ELISA反应检测表明:片段仍保留较高的活性,效价为1∶12 000.To use paper cellulose as a carrier, immobilization of papain on paper cellulose was studied. Thus, papain was immobilized onto glutaraldehyde-activated paper cellulose under acid conditions. The enzymatic pi＇operties, such as proteolytic activity, heat-stability, glutaraldehyde volume fraction, enzyme amount and pH-activity profile, of the immobilized papain preparation were examined. The immobilization conditions were optimized by orthogonal experiments. The prepared products were applied to digest anti-HCG to obtain F（ab＇）2 fragments. The results indicated that the immobilized enzyme has an high activity at 40 ℃ when the pH of the buffer solution is 5.0 as well as with the glutaraldehyde volume fraction is 0.01 %. Besides the enzyme concentration is 46 mg/g. The activity of the products can reach up to 19 250 U/g which are ten times higher than those came from the processes not optimized. The preparation and characterization Of the F（abl）2 fragments of anti-HCG are presented in detail. The monoclonal antibody was digested with immobilized papain to produce antibody fragment. The F（ab＇）2 fragments were then purified by using DEAE-FF size exclusion chromatography. The F（ab＇）2 fragment（ll0 KDa） maintained potent suppressive activity and the titer can reaches 1 ：12 000.

关 键 词：抗HCG抗体 固定化酶 木瓜蛋白酶 F(ab’)2片段 ELISA 纸纤维 

分 类 号：R981.3[医药卫生—药品]