鲢鱼卵中低分子CPIs的纯化鉴定及改善鱼糜凝胶强度的效果研究

Purification and Characterization of Low-Molecular-Weight Cysteine Proteinase Inhibitors(CPIs) from Silver Carp Eggs and Their Effects on Improving Gel Strength of Surimi

通过比较鲢鱼卵半胱氨酸蛋白酶抑制因子(cysteine proteinase inhibitors,CPIs)粗提过程中的比活力(Azocasein法),确定其最佳粗提条件为:以含0.1 mmol/L苯甲基磺酰氟(phenylmethanesulfonyl fluoride,PMSF)的20 mmol/L磷酸盐缓冲液(pH 6.0)作为匀浆缓冲液,并于30℃对匀浆样进行pH 3.0酸处理10 min,然后碱回调至pH 8.0,CPIs纯度提高了15.54倍。进一步经SP Sepharose Fast Flow阳离子层析验证表明,与pH 4.0相比,pH 3.0酸处理能高效除去活性峰Ⅱ中的酸碱及热不稳定杂蛋白,使CPIs比活力提高了48.22倍。采用Sephacryl S-200分子筛层析,获得部分纯化的低分子CPIs组分Ⅱ-b和Ⅱ-c,比活力和纯化倍数分别为1 098.59 U/mg、312.10倍和1 769.23 U/mg、502.62倍。电泳分析表明,明胶底物-SDS-反相酶谱法无法鉴定Ⅱ-b和Ⅱ-c的抑制活性;而与木瓜蛋白酶在适宜条件下反应后进行的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecylsulfate polyacrylamide gel electrophoresis,SDS-PAGE)则初步鉴定Ⅱ-b和Ⅱ-c中均含有至少7 kD和10 kD两种低分子CPIs。最后经质构分析表明,Ⅱ-b和Ⅱ-c以5 U/g添加入鲢鱼鱼糜时,均能够极显著提高鲢鱼鱼糜凝胶强度(48.77%和55.55%),抑制软化。

In this study, the optimum isolation condition of crude cysteine proteinase inhibitors(CPIs) from silver carp eggs that provided increased specific activity as measured by Azocasein assay was determined as homogenization using 20 mmol/L phosphate buffer containing 0.1 mmol/L phenylmethanesulfonyl fluoride(PMSF) at pH 6.0, and pH readjustment to 8.0 after acid(pH 3.0) treatment of the homogenate at 30 ℃ for 10 min. Under the optimized conditions, thepurity of CPIs was enhanced 16.54 folds. SP Sepharose fast fl ow chromatography analysis demonstrated effective removal of acid, alkali and protein impurities with poor thermal stability using acid treatment at pH 3.0 than at pH 4.0, giving rise to a purifi cation factor of 48.22. After further chromatography on a Sephacryl S-200 column, partially purifi ed low-molecularweight fractions named as Ⅱ-b and Ⅱ-c were obtained with a specifi c activity of 1 098.59 and 1 769.23 U/mg, respectively, and their purifi cation folds were 312.10 and 502.62 times, respectively. The electrophoresis analysis showed that Ⅱ-b and Ⅱ-c could not be identifi ed by gelatin substrate-SDS-reverse zymography. However, after reaction with papain under the appropriate condition, both fractions were found to contain at least two low-molecule-weight CPIs(7 and 10 kD) as indicated by SDS-PAGE analysis. When added to silver carp surimi at a level of 5 U/g, each fraction signifi cantly improved the gel strength signifi cantly by 48.77% and 55.55%, respectively, and inhibited softening.