甘薯叶酸性磷酸酶的分离纯化及部分性质研究  

Isolation, Purification and Characterization of Acid Phosphatase from Sweet Potato Leaves

作　　者：李星[1] 王洁[1] 王红扬[1] 唐云明[1] 

机构地区：[1]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

出　　处：《食品科学》2015年第3期152-157,共6页Food Science

基　　金：重庆市科委重点攻关项目(CSTC2011AB1027)

摘　　要：以新鲜甘薯老叶为材料,经过匀浆、硫酸铵盐析、DEAE-Sepharose阴离子交换层析和Superdex-200凝胶层析等步骤后,经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳鉴定获得了电泳纯的酸性磷酸酶(acid phosphatase,ACP),纯化后ACP比活力为251.49 U/mg,纯化了169.07 倍,回收率为3.50%。酶学性质研究结果表明:ACP为单亚基酶,全酶分子质量为42.3 kD,单亚基分子质量为41.2 kD;最适反应温度65 ℃,最适pH 5.2;在40 ℃以下和pH 4.0～6.0范围内酶活性较稳定;以对硝基苯磷酸二钠为底物时,测得其米氏常数Km值为0.258 mmol/L;Ca2+和Mg2+对ACP酶活性有明显激活作用,K+和Li+对ACP酶活性无影响,甲醇、乙二胺四乙酸和草酸对ACP酶活性的抑制作用较大;Hg2+和Ag+对ACP酶活性的抑制作用非常明显。Electrophoresis-pure acid phosphatase (ACP) was obtained from sweet potato old leaves via a series of consecutive procedures, including homogenization, extraction, ammonium sulfate fractional precipitation, DEAEsepharose chromatography and Superdex-200 gel filtration and then identified with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In this process, the purification fold was 169.07 and the recovery of ACP activity was 3.50%. The specific activity of the purified enzyme was 251.49 U/mg. The result showed that the enzyme was a single subunit protease with a relative molecular weight of 42.3 kD, and the subunit molecular weight was 41.2 kD. The optimum temperature and pH of the ACP enzyme were 65 ℃ and 5.2, respectively. This ACP was stable below 40 ℃ and in the pH range from 4.0 to 6.0. Its Michaelis-Menten Kinetics (Km) towards p-nitrophenyl phosphate was 0.258 mmol/L. The enzyme activity could be obviously inhibited by methanol, EDTA and oxalic acid and also strongly inhibited by Hg2+ and Ag+.

关 键 词：甘薯叶 酸性磷酸酶 分离纯化 性质 

分 类 号：Q946.5[生物学—植物学]