4℃下鲢鱼重组Cystatin稳定性分析  

Stability of recombinant silver carp(Hypophthalmichthys molitrix)Cystatin at 4℃

作　　者：马璐阳 蒋然然 陈治光[1] 陈海[1] 李冉[1] 但静[1] 钟海霞[1] 李树红[1] 

机构地区：[1]四川农业大学食品学院,雅安625014

出　　处：《应用与环境生物学报》2015年第4期640-646,共7页Chinese Journal of Applied and Environmental Biology

基　　金：国家自然科学基金青年科学基金项目(31101249);四川农业大学本科生科研兴趣培养计划项目资助~~

摘　　要：重组鲢鱼Cystatin是一种具有明显抑菌活性的半胱氨酸蛋白酶抑制因子,研究其在4℃冷藏条件下的稳定性,可为未来将Cystatin蛋白应用于食品保藏、加工奠定必要的理论和实验基础.本文首先确定鲢鱼重组Cystatin的抑制活性单位,并研究其在4℃贮藏不同时间内的活性变化趋势;同时,分别采用Tricine-SDS-PAGE和Gelatin-Substrate-Active Tricine-SDS-PAGE及凝胶过滤高效液相色谱法,监测该重组Cystatin蛋白的分子降解情况.结果显示:重组0.18 mg/m L(即25 unit/m L)的Cystatin蛋白在4℃、p H=7.0条件下贮存15 d,活性几乎无损失且未发生降解,主要为Mr 20×103蛋白形式;17-21 d内逐步降解为Mr 14×103,但21 d时Cystatin抑制活性仍保持在80%以上;33 d时活性则迅速下降50%,Mr20×103的Cystatin蛋白完全降解,即液相检测图谱中峰2消失,有活性的Mr 14×103形式即峰3降解成低活性的Mr 12.55×103的峰3′,同时降解峰4、峰5、峰6即Mr 6.5×103、Mr 4×103左右的小肽片段比例快速升高,标志Cystatin明显失活.本研究表明,鲢鱼重组Cystatin蛋白在p H=7.0的液体环境中,4℃冷藏稳定性良好,活性及其完整性可保持约30 d.Recombinant cystatin of Hypophthalmichthys molitrix is a Cystatin protease inhibitor with obvious antimicrobial activity, whose stability at 4 oC is of great importance for the application of Cystatin protein in food preservation and processing. This research first confirmed the inhibitory activity unit of restructuring Cystatin and studied the variation trend of its activity during different storage time at 4 oC with methods including Tricine-SDS-PAGE and Gelatin-Substrate-Active Tricine-SDS-PAGE as well as Gel filtration high-performance liquid chrometagraphy （HPLC）. The results showed no degradation or loss of activity in restructuring Cystatin protein when stored for 15 d under the condition of 0.18 mg/mL and pH = 7.0, with the key content as Mr 20 × 103 protein. The key content gradually degraded to Mr 14 × 103 protein during 17 d to 21 d. Cystatin inhibitory activity was over 80% on the 21 d, but decreased steeply to 50% on the 33 d, with the Cystatin Mr 20 × 103 protein completely degraded, shown as the disappearance of Peak 2 in the liquid phase detection map, and degradation of active protein from Mr 14 × 103 to Mr 12.55 × 103 with low activity in Peak 3; in the mean time,degradation peak 4, peak 5 and peak 6, that was, the small peptidev fragments around Mr 6.5 × 103 and Mr 4 × 103 protein, their proportion increased quickly, which marked that the Cystatin lost in activity. The results showed that Cystatin protein has good stability when stored at 4 oC for up to nearly 30 d.

关 键 词：鲢鱼 重组Cystatin 4℃冷藏 稳定性 降解 

分 类 号：TS254.1[轻工技术与工程—水产品加工及贮藏工程]