金银花过氧化物酶的三相分离纯化及酶学性质  

Purification of Peroxidase from Lonicera japonica Thunb. by Three Phase Partitioning and Enzymatic Properties

作　　者：罗磊[1] 董金龙[1] 朱文学[1] 薛依涵 关宁宁[1] 张宽[1] 姬青华 

机构地区：[1]河南科技大学食品与生物工程学院,河南洛阳471023

出　　处：《食品科学》2017年第24期20-27,共8页Food Science

基　　金：国家自然科学基金联合基金项目(U1304330)

摘　　要：采用三相分离法提取纯化金银花中过氧化物酶,结果表明最优纯化条件为pH?5.60、硫酸铵质量浓度39.49?g/100?mL、提取液与叔丁醇体积比1∶1.38,在该条件下纯化倍数为5.849,回收率为87.64%。金银花过氧化物酶比活力为1 021.6 U/mg,色素清除率为92%;酶学性质研究表明:金银花过氧化物酶最适温度为30℃,热稳定范围为10~40℃;最适pH值为5,pH值稳定范围为4~7。在金银花过氧化物酶催化的双底物酶促反应中,当H_2O_2浓度一定时,酶对愈创木酚的Km值为8.12?mmol/L,vmax值为1.71?mmol/(min·L)。当愈创木酚浓度一定时,H_2O_2的Km值为0.822?mmol/L,vmax值为1.38?mmol/(min·L)。金银花过氧化物酶与底物的亲和力由强到弱依次是邻苯三酚、邻苯二酚、联甲氧基苯胺、愈创木酚。Ca^(2+)、Cu^(2+)、Zn^(2+)对金银花过氧化物酶有激活作用,Mg^(2+)、Mn^(2+)、柠檬酸、抗坏血酸、L-半胱氨酸、亚硫酸钠、偏重亚硫酸钠、十二烷基磺酸钠对金银花过氧化物酶有不同程度的抑制作用。Three phase partitioning was used to extract and purify peroxidase（POD） from Lonicera japonica Thunb. The optimal conditions were obtained as follows：pH 5.60,ammonium sulfate concentration 39.49 g/100 mL,and ratio of crude extract to t-butanol（V/V）,1：1.38. Under these conditions,the activity recovery was 87.64% with a purification factor of 5.849,and the specific activity of purified peroxidase was 1 021.6 U/mg. By using three phase partitioning,92% pigment was removed. Enzymatic properties revealed that the optimum temperature for this enzyme was 30 ℃ and the optimum pH was 5. The enzyme was stable in the temperature range of 10–40 ℃ and in the pH range of 4–7. In the presence of both guaiacol and H2O2,Michaelis constants（Km） and maximum reaction rates（v max） of the enzyme were 8.12 mmol/L and 1.71 mmol/（min·L） for guaiacol,and 0.822 mmol/L and and 1.38 mmol/（min·L） for H2O2 at a fixed concentration of the other substrate,respectively. The affinity of the enzyme to various substrates was in the decreasing order：pyrogal〉lolcatechol〉bimethoxy aniline〉guaiacol. The peroxidase activity was activated by Ca^2＋,Cu^2＋ and Zn^2＋ but inhibited by Mg^2＋,Mn^2＋,citric acid,ascorbic acid,L-cysteine,sodium sulphite and sodium metabisulphite.

关 键 词：金银花 过氧化物酶 三相分离法 酶学性质 

分 类 号：TS201.2[轻工技术与工程—食品科学]