Purification and characterization of hatching enzyme from brine shrimp Artemia salina

• Published in: Acta biochimica et biophysica Sinica 2010-02, Vol.42 (2), p.165-171
• Main Authors: Fan, Tingjun, Wang, Jing, Yuan, Wenpeng, Zhong, Qiwang, Shi, Ying, Cong, Rishan
• Format: Article
• Language: English
• Subjects: Animals; Artemia - enzymology; Enzyme Activation; Enzyme Stability; Metalloendopeptidases - chemistry; Metalloendopeptidases - isolation & purification; Metalloendopeptidases - metabolism; Peptide Hydrolases - chemistry; Peptide Hydrolases - metabolism; 丝氨酸蛋白酶; 大豆胰蛋白酶抑制剂; 盐水虾; 聚丙烯酰胺凝胶电泳; 酶特性; 酶纯化
• ISSN: 1672-9145; 1745-7270
• DOI: 10.1093/abbs/gmp119

By using Artemia chorion as a specific substrate, the hatching enzyme from Artemia salina （AHE） was purified by gel-filtration and ion-exchange chromatography, and characterized biochemically and enzymatically in this study. It was found that the AHE had a molecular weight of 82.2 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and often contained 73.3 kDa molecules in preparation. The AHE had obvious choriolytic activity, which was optimal at pH 7.0 and a temperature of 40℃. The Km value of the AHE for dimethyl casein was 8.20 mg/ml. The AHE activity was almost completely inhibited by soybean trypsin inhibitor and p-amidinophenyl methane sulfonyl fluoride hydrochloride, greatly inhibited by N-tosyi-L-lysyl chloromethyl ketone, phenylmethanesulfonyl fluoride, and lima bean trypsin inhibitor, slightly inhibited by pepstatin, N-tosyl-L-phenylalanyl chloromethyl ketone, leupeptin, N-ethylmaleimide, and iodoacetamide, and not inhibited by chymostatin and bestatin. All these results imply that AHE is most probably a trypsin-type serine protease. Besides of these, AHE was also sensitive to EDTA and Zn^2＋. Combined with the results that the EDTA-pre-treated HE activity could be perfectly recovered by Zn^2＋, it is indicated that AHE might be also a kind of Zn-metalioprotease.