大豆过氧化氢酶的分离纯化及性质研究 - 食品科学.pdf

140 2015, Vol.36, No.01 食品科学 ※生物工程 大豆过氧化氢酶的分离纯化及性质研究 方 玲，孙才云，唐云明* （西南大学生命科学学院，重庆市甘薯工程研究中心，三峡库区生态环境教育部重点实验室，重庆 400715 ） 摘  要：新鲜大豆芽经匀浆、磷酸缓冲液抽提、硫酸铵分级沉淀、DEAE-Sepharose离子交换层析、Superdex-200 凝胶过滤层析，获得电泳纯的过氧化氢酶。该酶酶活回收率为30.92 % ，纯化倍数为293.09 ，酶比活力达到 26 322.43 U/mg 。该酶全分子质量为234.9 kD ，亚基分子质量为57.42 kD ；最适pH值为7.2 ，最适温度为30 ℃， 在pH 4.0 ～10.6及25 ～35 ℃范围内有较好的稳定性；在30 ℃、pH 7.2 条件下测得该酶对过氧化氢的K m 值为 31.82 mmol/L ；十二烷基硫酸钠、草酸、Ag+ 、Cu2+对该酶有强烈的抑制作用，Pb2+对该酶有双重作用，低浓度时有 激活作用而高浓度时抑制酶活性。 关键词：大豆；过氧化氢酶；分离纯化；性质 Isolation, Purification and Partial Characterization of Catalase from Soybean (Glycine max) * FANG Ling, SUN Caiyun, TANG Yunming (Key Laboratory of Eco-environments in Three Gorges Reservoir Region, Ministry of Education, Chongqing Sweet Potato Engineering Research Center, School of Life Science, Southwest University, Chongqing 400715, China) Abstract: Electrophoresis-purity catalase (CAT) was obtained from freshly sprouted soybean (Glycine max) by the sequential steps of homogenization, buffer solution extraction, ammonium sulfate precipitation, DEAE-Sepharose ion exchange chromatography and Superdex-200 gel filtration chromatography. The purified enzyme had a specific activity of 26 322.43 U/mg, with a 30.92% activity recovery and a 293.09-fold purification. The molecular mass of the CAT enzyme comprising a 57.42 kD subunit was 234.9 kD. Its optimum pH and temperature were 7.2 and 30 ℃, respectively. This enzyme was stable at pH 4.0–10.6 and at 25–35 ℃. Its K towards H O was 31.82 mmol/L. The enzyme activity was m 2 2 strongly inhibited by sodium dodecyl sulfate (SDS), oxalic, Ag+ and Cu2+, but was activated by low con