鲢鱼重组Cystatin及其酶解产物对草鱼冷藏肉片优势腐败菌的抑制作用

Inhibitory effect of recombinant Cystatin and its enzymatic hydrolyzate on the dominant spoilage strains

首先通过三羟甲基甘氨酸-聚丙烯酰胺凝胶电泳(Tricine-SDS-PAGE)和滤纸片法分别鉴定木瓜蛋白酶液(Papain)对鲢鱼重组半胱氨酸蛋白酶抑制剂(Cystatin)酶解程度及酶解产物对草鱼冷藏肉片中分离的3种优势腐败菌,即草莓假单胞菌(Pseudomonas fragi)、腐败希瓦氏菌(Shewanella putrefaciens)和中间气单胞菌(Aeromonas media)的抑菌活性。结果表明,酶解6 h后的产物,即9.5 ku以下的片段对3种菌的抑菌效果均最好。再利用凝胶过滤高效液相色谱(TSK-GEL G2000SWXLHPLC)和双向电泳对6 h酶解产物进行分离纯化分析。发现仅峰T-2对3种菌均具有明显的抑制活性,峰T-2在TSK-GEL G2000SWXLHPLC上呈现单一峰,电泳得到单一蛋白点,其分子质量为8.6 ku,p I值为6.5。最后通过RPLC-MS/MS串联质谱鉴定该蛋白点,得到3个肽片段(31QSNDAFVR38、46VQQQVAAGMKY56和81NPSIEQVIQ89)且含有Cystatin特征序列QVAAG。结果提示,Cystatin蛋白(完整序列分子质量12~14 ku)内部,存在能够发挥抑菌作用的肽片段。

Firstly,the Tricine-SDS-PAGE and filter paper diffusion were used to identify the enzymatic hydrolysis of Silver carp recombinant Cystatin with Papain and antibacterial effect of Cystatin＇s hydrolyzate on the three kinds of dominant spoilage strains,Pseudomonas fragi,Shewanella putrefaciens and Aeromonas media. The results indicated that the hydrolyzate of 6 h group was less than 9. 5 ku and its antibacterial effect was the best among the three dominant spoilage strains. Therefore,the hydrolyzate of 6 h group were purified and analyzed by HPLC with column of TSK-GEL G2000 SWXL gel and two-dimensional electrophoresis. The results indicated that only the peak T-2 had significant antibacterial activity for the dominant spoilage and exhibited a single peak on HPLC and a single protein point with the molecular weight of 8. 6 ku and the p I of 6. 5 on 2-D gel. At last,RPLC-MS/MS was used to identify the protein spot and obtained three peptides fragments including 31 QSNDAFVR38, 46 VQQQVAAGMKY56 and81 NPSIEQVIQ89,which contained the characteristic sequence of Cystatin QVAAG. These results suggested that Cystatin protein with intact sequence of 12 ku-14 ku contained some peptide fragments exerting antimicrobial activity.