摘要
以研究低温胁迫小麦苗冰结构蛋白(ISP)纯化为目的,以小麦苗为对象,经低温胁迫,并采用粗分离、饱和硫酸铵溶液沉淀、DEAE-Cellulose 52层析、sephadex-G75凝胶层析4个步骤对其中ISP进行纯化,分别采用差示扫描量热法(DSC)、热休克处理法和聚丙烯酰氨凝胶电泳(SDS-PAGE)测定各纯化步骤所得ISP的热滞活性(THA)、冰晶重结晶抑制能力(ICP)和分子质量(MW),并计算小麦苗存活率和各纯化步骤的得率。实验结果表明,梯度低温胁迫的小麦苗存活率与恒温低温胁迫相比提高了240%,小麦苗提取物(AWSE)中蛋白质含量与未低温胁迫小麦苗提取物(NAWSE)相比提高了7.8%;以THA表征的纯化倍数(T_1)和以ICP表征的纯化倍数(T_2)分别为451.15和137.60,得率为2.85%,THA为0.180℃(5 mg/mL),ICP为70.03%,分子质量范围为20.1~31.0 ku;与THA相比,ISP在较低浓度下即可达到ICP的峰值。小麦苗可作为制备冰结构蛋白的天然植物资源。
To investigate purification of ice structuring protein （ISP）from cold-acclimated wheat seedlings, wheat seedlings underwent cold acclimation and ISP from cold-acclimated wheat seedlings was purified by 4 sequential steps including coarse separation, saturated ammonium sulphate precipitation, DEAE- Cellulose 52 chromatography and sephadex- G75 gel filtration chromatography. Differential scanning calorimetry （ DSC ） , heat shock treatments and polyacrylamide gel electro- phoresis （ SDS- PAGE） were employed respectively for determination of thermal hysteresis activity（THA）, ice recrystallization inhibition power （ICP） and molecular weight（MW） of ISPs during process of purification.Survival rate of cold-acclimated wheat seedlings and purification times of ISP during each purification step were calculated as well.Results showed an increase of 240% in survival rate of wheat seedlings using gradient cold- acclimation compared with constant cold- acclimation. Protein content of cold- acclimated wheat seedlings extract（AWSE）was increased by 7.8% in comparison to non-cold-acclimated wheat seedlings extract（NAWSE）.Eventual purification times based on THA and ICP were 451.15 and 137.60 respectively. Yield, THA at 5 mg/mL,ICP and MW range of ISP were 2.85% ,0.180 ℃ ,70.03% and 20.1-31.0 ku respectively.Maximal ICP of ISP was achieved at lower concentration compared with THA.Wheat seedlings can be a potential plant resource in production of ISP.
作者
焦宇知
徐桃
梁家冰
朱云
何志勇
张晓晓
刘永博
孙芝杨
翟玮玮
JIAO Yu- zhi XU Tao LIANG Jia- bing ZHU Yun HE Zhi- yong ZHANG Xiao- xiao LIU Yong- bo SUN Zhi- yang ZHAI Wei- wei(Jiangsu Food ＆ Pharmaceutical Science College, Huai＇ an 223005, China Purdue University, West Lafayette, IN, USA ,47907 Civil Hospital of Juxian, Juxian 276511, China Jiangnan University, Wuxi 214122, China Chaoneng Freda Bio Co.Ltd. ,Jinan 250031 ,China)
出处
《食品工业科技》
CAS
CSCD
北大核心
2017年第3期102-107,共6页
Science and Technology of Food Industry
基金
淮安市产学研协同创新计划(HAC2015006)
江苏省“青蓝工程”中青年学术带头人计划(苏教师(2014)23号)
淮安市食品技术研究院(HAP201301)
江苏省首批高校优秀中青年教师境外研修计划(苏教师[2011]34号)
企业委托横向项目(苏食院合字(2015)432号)
