| 摘要: |
| 内源酶是三疣梭子蟹(Portunus trituberculatus)体内重要的酶, 往往会导致梭子蟹死后肌肉组织迅速软化, 严重影响了蟹肉的贮藏品质。为了探明该酶的基本特性, 本试验从三疣梭子蟹肝胰腺中提取了粗酶, 优化了提取方法, 并对其部分酶学性质进行初步研究。结果表明: 三疣梭子蟹肝胰腺内源酶最佳浸提时间段为4～12 h, 酶比活显著高于0～4 h和12～14 h; 以酪蛋白为水解底物, 内源酶作用最适温度为65℃、最适pH 范围为7.0～8.0; 丝氨酸蛋白酶特异性抑制剂, 包括大豆胰蛋白酶抑制剂(SBTI)和苯甲基磺酰氟(PMSF), 对内源酶活力的相对抑制率分别为100%、70.46%±6.27%, 显著高于其他抑制剂的相对抑制率, 推测丝氨酸蛋白酶为主要内源酶; 在硫酸铵分级沉淀中, 分别以酪蛋白和Boc-Phe-Ser-Arg-MCA 为底物, 前者最适盐析浓度为0～70%, 后者为30%～70%, 酶比活显著高于其他盐浓度;当硫酸铵浓度为40%～60%时, 盐析蛋白质含量、粗酶酶活和丝氨酸蛋白酶活均显著高于其他盐浓度。 |
| 关键词: 三疣梭子蟹(Portunus trituberculatus) 肝胰腺 内源酶 性质 |
| DOI：10.11759//hykx20151224003 |
| 分类号: |
| 基金项目:山东省自然科学基金项目(ZR2015CM010) |
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| Characteristics of endogenous protease from hepatopancreas of swimming crab (Portunus trituberculatus) |
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YANG Li-zhu,ZHANG Li,HUANG Lin,MENG Xiang-hong
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| Abstract: |
| Endogenous autolytic enzyme is an important enzyme in Portunus trituberculatus. Normally, after the death of swimming crabs, deterioration is very rapid in the muscle, which produces a severe effect on the storage quality of crabs. To investigate few basic properties of this autolytic enzyme, crude samples were extracted from the hepatopancreas of P. trituberculatus and the parameters are optimized. Then, a preliminary study on the enzymatic properties of the endogenous enzyme was conducted. The results showed that the optimum extraction time range of endogenous protease was 4–12 h, and the specific activity was significantly higher than that at other time points, i.e., 0–4 h and 12–14 h. The optimal temperature and pH for hydrolysis of casein were 65℃ and 7.0–8.0, respectively. Compared with other protease inhibitors, endogenous protease activity was significantly inhibited by serine protease inhibitors, including soybean trypsin inhibitor (SBTI) and phenylmethylsulfonyl fluoride (PMSF), suggesting that serine protease was the dominant content of this endogenous enzyme. Ammonium sulfate grading precipitation showed that fractions of 0%–70% and 30%–70% could be used to collect the targeted proteases for casein and Boc-Phe-Ser-Arg-MCA hydrolysis, respectively. Their specific activities were significantly higher than those of the samples precipitated by other ammonium sulfate fractions. Moreover, samples collected by ammonium sulfate fractions of 40%–60% had a higher protein content and total activity of crude and serine proteinase than those of the samples precipitated by other ammonium sulfate fractions. |
| Key words: Swimming crab (Portunus trituberculatus) Hepatopancreas Endogenous protease Characteristic |
