多酚氧化酶的固定化及其酶学性质的研究  

Immobilization of Polyphenol Oxidase and Its Enzymatic Properties

作　　者：王涵[1] 李瑾[1,2] 程华丽[1] 马启敏[1,2] 

机构地区：[1]中国海洋大学环境科学与工程学院,山东青岛266100 [2]中国海洋大学海洋环境与生态教育部重点实验室,山东青岛266100

出　　处：《中国海洋大学学报（自然科学版）》2015年第8期90-96,共7页Periodical of Ocean University of China

基　　金：国家自然科学基金项目(41106067);山东省自然科学基金项目(ZR2010BQ013);青岛市基础研究计划项目(13-1-4-252-jch);中国海洋大学中央高校基本科研业务费专项资金项目(201013032)资助

摘　　要：以壳聚糖/蒙脱土插层复合物为载体,采用吸附法和戊二醛交联法固定多酚氧化酶(PPO),考察了固定化酶的最佳制备条件及其酶学性质。结果表明,吸附法固定化多酚氧化酶(A-PPO)的最佳制备条件为pH=5.0,酶与载体质量比20mg/g,固定化时间6h,固定化酶的载酶量为12.12mg/g,单位载体酶活为12.76×103 U/g,酶活回收率为81.7%。交联法固定化酶(C-PPO)的最佳制备条件为pH=4.0,酶与载体质量比75 mg/g,固定化时间6h,固定化酶的载酶量为18.33mg/g,单位载体酶活为2.78×103 U/g,酶活回收率为12.9%。A-PPO和C-PPO的最适酶促反应条件为pH=8.0,温度分别为40和30℃。与游离多酚氧化酶相比,固定化酶的操作稳定性、贮存稳定性均有所提高。固定化酶重复使用5次后,可保留37.6%以上的相对酶活。PPO,A-PPO和C-PPO的米氏常数Km值分别为0.48,0.56和3.06mmol/L,较游离酶的Km值均有所增加;最大反应速率Vmax分别为2.70,2.08和0.18U/g,较游离酶的Vmax值均有所减小。Polyphenol oxidase（PPO）was immobilized on chitosan/montmorillonite intercalation composites by adsorption and glutaraldehyde cross-linking.The immobilization conditions and enzymatic properties were investigated in detail.Optimal preparation conditions of the immobilization of polyphenol oxidase by adsorption（A-PPO）were pH=5.0,enzyme/support weight ratio 20mg/g,immobilization time 6h.And the protein loading yield of A-PPO was 12.12mg/g,enzyme activity of the unit support 12.76×103U/g,and activity recovery 81.7%.Optimal preparation conditions of enzyme immobilizated by glutaraldehyde cross-linking（C-PPO）were found to be pH=4.0,enzyme/support weight ratio 75mg/g,immobilization time 6h.The protein loading yield of C-PPO was 18.33mg/g,enzyme activity of the unit support2.78×103 U/g,and activity recovery 12.91%.A-PPO and C-PPO showed the optimum activity at pH=8.0,and 40℃and 30℃,respectively.Compared with free PPO,immobilization enhanced enzyme stability against changes in pH and temperature.Immobilized PPO retained more than 37.6% of its initial activity after five cycles.A-PPO and C-PPO had a higher Kmvalues（Michaelis constant）than free enzyme,and the values were 0.56,3.06 and 0.48mmol/L,respectively.The maximum reaction rate Vmaxof A-PPO and C-PPO were decreased compared with free enzyme,and determined as 2.08,0.18 and 2.70U/g,respectively.

关 键 词：多酚氧化酶 壳聚糖 固定化 插层复合物 酶学性质 

分 类 号：O625.31[理学—有机化学]