日本曲霉酸性蛋白酶的分泌表达、性质及其在猪肉嫩化中的应用

Secretory Expression and Properties of Acid Protease from Aspergillus japonicus and Its Application in Pork Tenderization

为研究日本曲霉酸性蛋白酶的分泌表达、性质及其在猪肉嫩化中的应用。将日本曲霉来源的酸性蛋白酶基因(Ajpro A1)在毕赤酵母中高效表达,经5 L发酵罐高密度发酵后测得该酸性蛋白酶酶活力为669.6 U·mL^(-1),蛋白质量浓度为6.64 mg·mL^(-1)。氨基酸多重序列比对结果表明:该酸性蛋白酶属于天冬氨酸蛋白酶A1家族,与海枣曲霉来源的酸性蛋白酶同源性最高(73%),是一个新型的酸性蛋白酶。采用强阴离子交换层析柱对该酸性蛋白酶进行纯化得到电泳级纯酶,纯化后分析测得该重组酸性蛋白酶(AjproA1)的最适催化条件为pH值3.0,反应温度为45℃,在pH值为3.0~6.0及温度为40℃以下具有良好的稳定性。该酸性蛋白酶对不同蛋白质水解的分析结果表明,该酶底物水解特异性广泛,对酪蛋白组分中的κ-酪蛋白表现出最高水解活力。利用该酸性蛋白酶对猪肉进行处理,发现该酶能够有效降低猪肉剪切力,起到嫩化效果。研究结果旨在为新型酸性蛋白酶的开发及其在肉类嫩化中的应用提供理论参考。

In order to study secretory expression and properties of acid protease from Aspergillus japonicus and its application in pork tenderization,the acid protease gene(Ajpro A1)from Aspergillus japonicus was efficiently expressed in Pichia pastoris.After high cell-density fermentation in a 5L fermenter,theenzyme activity and protein concentration of the fermentation supernatant were 669.6 U/mL^(-1)and 6.64mg/mL^(-1),respectively.According to the results of amino acid multiple sequence alignment analysis,the acid protease belongs to the aspartic protease A1 family,which had the highest homology(73%)with acid protease from Aspergillus phoenicis,and was a novel acid protease.The acid protease was purified by strong anion exchange chromatography to obtain electrophoresis-grade pure enzyme.After purification,the optimal catalytic conditions for recombinant acid protease(Ajpro A1)was p H 3.0 and reaction temperature 45℃,and it had good stability in the p H range of 3.0-6.0 and temperature below 40℃.The results of the hydrolysis analysis of this acid protease for different proteins showed that the enzyme exhibited broad substrate hydrolysis specificity and the highest hydrolysis activity towardκ-casein for casein components.Ajpro A1 was used to tenderize pork and effectively reduced the shearing force of pork to achieve a good tenderization effect.The research results aimed to provide a theoretical reference for the development of novel acid proteases and their application in meat tenderization.