金线鱼鱼皮抗冻蛋白酶法制备工艺优化

Optimization of preparation of antifreeze proteins of fish skin from Nemipterus virgatus by using protease

为了探讨金线鱼鱼皮抗冻蛋白的酶法高效制备工艺,该文首先探究不同蛋白酶酶解获得的酶解产物的热滞活性(thermal hysteresis activity,THA)以及不同分子区间的酶解产物的热滞活性。随后以抗冻蛋白得率为指标优化了底物与酶的比例、酶解时间、酶解温度以及p H值,并采用响应面法探究抗冻蛋白的最佳酶解工艺。最后使用差示扫描量热仪(differential scanning calorimeter,DSC)测定验证抗冻蛋白的热滞活性。结果表明:在碱性蛋白酶、中性蛋白酶、木瓜蛋白酶以及复合蛋白酶4种酶的最佳酶解条件下,碱性蛋白酶酶解所获得抗冻蛋白热滞活性最大,选择碱性蛋白酶作为制备金线鱼鱼皮抗冻蛋白的最佳蛋白酶;截留分子量为≥5~10 k Da鱼皮蛋白的抗冻效果最佳,热滞活性达1.20?0.07℃,确定≥5~10 k Da鱼皮蛋白得率作为酶解制备工艺的指标;得到金线鱼鱼皮抗冻蛋白最佳的酶解制备工艺条件为:底物与酶比例为32∶1、酶解时间为147 min、酶解温度为50℃和p H值为9,在此条件下,金线鱼鱼皮抗冻蛋白得率为49.25%±1.34%。相比于牛血清蛋白(bovine serum albumin,BSA),其降低了溶液起始结晶的温度;在最佳的保留温度条件下,其热滞活性为1.2℃。碱性蛋白酶酶解制备鱼皮抗冻蛋白是一种快速有效的方式,酶解获得的抗冻蛋白热滞活性高。该结果为综合发展和利用金线鱼鱼皮提供一定的理论依据。

The purpose of this paper was to investigate the efficient preparation technology of antifreeze protein（AFP）of fish skin from Nemipterus virgatus by protease.At first,the effects of the protein types and the molecular weight of the hydrolysate on the thermal hysteresis activity of the AFP were studied.The yield of the AFP was selected as the evaluation index for the further study.Moreover,the ratio of substrate to enzyme,enzymolysis time,enzymolysis temperature and p H value were evaluated according to the yield of AFP.Then the optimum preparation technology of AFP of fish skin from Nemipterus virgatus by protease was investigated by the response surface methodology.Finally,the thermal hysteresis activity of AFP was confirmed and determined by differential scanning calorimeter.The results showed that the thermal hysteresis activity of AFP obtained by alkaline protease was the highest among alcalase protease,neutrase protease,papain protease and compound protease under the optimal proteolysis conditions.Therefore,the alkaline protease was selected as the best protease for AFP preparation.The proteins in the molecular weight range of≥5-10 kD a displayed the highest thermal hysteresis activity up to（1.20？0.07）℃,and the yield of the proteins was selected as the evaluation index.The optimum preparation technology of AFP of fish skin from Nemipterus virgatus by protease was as follows：The ratio of substrate to enzyme was 32：1,enzymolysis time was 147 min,enzymolysis temperature was 50℃and pH value was 9.The yield of AFP was up to 49.25%±1.34%under the optimum conditions.The great linear relationship between the predicted value and actual value indicated that the predicted value of the model could reflect the actual yield of AFP accurately.The result by differential scanning calorimeter indicated the AFP of fish skin from Nemipterus virgatus reduced the temperature of the initial crystallization of the solution compared to bovine serum albumin（BSA）.Under the optimal retention temperature condition,the thermal hysteresis activity of AFP of fish skin from Nemipterus virgatus was 1.2℃.The enzymolysis method by alkaline protease was a rapid and effective method to prepare AFP of fish skin and the AFP exhibited the high thermal hysteresis activity.These results will provide a certain theoretical basis for the comprehensive development and utilization of fish skin from Nemipterus virgatus.