摘要
以奥尼罗非鱼肝脏为原料,研究加热法提取超氧化物歧化酶（SOD）的工艺条件,丙酮沉淀回收酶法和Hitrap^TM Q FF阴离子柱纯化Cu,Zn-SOD,并对其酶学性质进行分析。结果表明加热法提取SOD时,在缓冲液中加入10mmol·L^-1CuCl2,调节pH为5.6,65℃进行加热处理,可以提高SOD粗酶的稳定性和得率,其得率为57%,粗酶比活为（2580±6）U·mg^-1。对酶的酶学性质研究表明纯化后SOD比活为（4895±2）U·mg^-1,SDS-PAGE电泳为单一蛋白酶带,分子量为36ku,最大紫外吸收波长为265nm。该酶在pH6.0～9.0具有较好的稳定性,在75℃以下稳定,具有较好的耐热性。氰化钾、脲素、β-巯基乙醇、H2O2、对Cu,Zn-SOD具有明显的抑制作用;EDTA浓度小于3mmol·L^-1时,对Cu,Zn-SOD活性有明显增强作用,EDTA浓度大于3mmol·L^-1时,对Cu,Zn-SOD活性有抑制作用;DTT的修饰使Cu,Zn-SOD活性显著增加。为进一步研究和应用罗非鱼肝脏SOD提供了基础参数。
In this paper, extracting conditions, purified and character analyzed superoxide dismutase （SOD） from hybrid tilapia（ Oreochromis niloticus ♀ × Oreochromi saurea ♂ ） liver with heat treatment, followed by acetone precipitation and HitrapTM Q FF anion chromatography were studied. The results showed that the fine SOD extracting conditions were： 10 mmol·L6-1 CuCl2 in the cushion fluid, pH 5.6, and heat treatment temperature 65 ℃. The yield of rough SOD is 57 % and specific activity is （ 2580 ±6） U· mg^- 1. The purified SOD obtained from tilapia liver is pale blue-green in color and specific activity is （4895 ± 2） U·mg^- 1. It is a sole proteinasebelt and the molecular weight is 36 ku as determined with SDS-PAGE electrophoresis. There is a special ultraviolet absorption spectrum at 265 nm. It is stable below 75 ℃ and at pH 6.0 - 9.0. The effects of some inhibitors on the activity of Cu, Zn-SOD showed that the potassium cyanide, urea, β-mercaptoethanol, hydrogen peroxide and EDTA （which concentration is above 3 mmol·L^-1）can obviously inhibit the enzyme, and EDTA （whose concentration is below 3 mmol· L^-1 ） and DDT can activate the enzyme. Further work should be carded out on the sequences of the enzymes and their applications.
出处
《水产学报》
CAS
CSCD
北大核心
2007年第4期518-524,共7页
Journal of Fisheries of China
基金
广东省重点项目(2005B33201002)
广东省重点项目(2003C32803)
关键词
奥尼罗非鱼
肝脏
超氧化物歧化酶
加热法
纯化
hybrid tilapia（ Oreochromis niloticus ♀× Oreochromis aurea♂ ）
liver
superoxide dismutase（SOD）
heat treatment
purification
