刺参体壁胰/类胰丝氨酸蛋白酶的性质及其在自溶中的作用

Characterization of Trypsin and Trypsin-like Serine Proteases from Sea Cucumber Stichopus japonicus and Their Roles in Autolysis

以刺参体壁中丝氨酸蛋白酶为研究对象,对其部分酶学性质及其与刺参自溶的关系进行研究。采用Tris-HCl缓冲溶液提取、硫酸铵盐析、透析及超滤获得刺参体壁粗酶液。结果显示,以Boc-Phe-Ser-Arg-MCA为底物时,粗酶液的丝氨酸蛋白酶活力最高;以其为底物,测得该类蛋白酶在pH值为6.2和8.5时呈现较强酶活力峰值,两者最适温度范围均为50~55℃;Cu^2+、Fe^3+、Pb^2+、Zn^2+强烈抑制该酶的活性,Ca^2+可提高该酶活力;邻菲咯啉、乙二胺四乙酸以及半胱氨酸修饰剂均能够部分抑制该酶活力。4-(2-氨乙基)苯磺酰氟盐酸盐、苯甲基磺酰氟、Na-对甲苯磺酰-L-苯丙氨酸氯甲基酮、(3S)-7-氨基-1-氯-3-磺酰氨基-2-庚酮盐酸盐、大豆胰蛋白酶抑制剂能够显著抑制蛋白酶的活力以及自溶过程中可溶性蛋白、三氯乙酸可溶性多肽的生成。上述结果表明,刺参体壁中存在至少2种具有一定的金属离子依赖性的胰/类胰丝氨酸蛋白酶,其活性中心或底物识别位点可能有半胱氨酸的参与,该类蛋白酶很可能参与刺参自溶过程中蛋白质的降解。

In this paper, serine protease in the body wall of sea cucumber (Stichopus japonicus) was investigated on its partial properties and role in the autolysis of sea cucumber. The serine protease was obtained after a series of processes including extraction with Tris-HCl buffer,(NH4)2SO4 precipitation, dialysis, and ultrafiltration. Results showed that the maximum protease activity was observed using Boc-Phe-Ser-Arg-MCA as substrate. Based on this substrate, the peak protease activity occurred at pH 6.2 and 8.5 and 50 55 ℃. The protease activity could be signi ficantly inhibited by Cu^2+, Fe^3+, Pb^2+ and Zn^2+ but activated by Ca^2+. The activity was partially inhibited by phenanthroline, EDTA, and cysteine- modifying reagents, while it together with the production of soluble proteins and trichloroacetic acid (TCA)-soluble peptides as a result of autolysis could be significantly inhibited by 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), phenylmethanesulfonyl fluoride (PMSF), N-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK),(3S)-1-chloro- 3-tosylamido-7-amino-2-heptanone hydrochloride (TLCK), and soybean trypsin inhibitor (SBTI). The above results suggest that there are at least two metal ion-dependent trypsin/trypsin-like serine proteases in the body wall of S. japonicus, with cysteine being involved in the active center or substrate-identifying site, and these proteases may be involved in the autolysis of S. japonicus.