A Novel Hatching Enzyme from Starfish Asterias amurensis : Purification, Characterization, and Cleavage Specificity
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A Novel Hatching Enzyme from Starfish Asterias amurensis: Purification, Characterization, and Cleavage Specificity Zhi Jiang Li & Sang Moo Kim
Received: 28 August 2012 / Accepted: 28 December 2012 / Published online: 11 January 2013 # Springer Science+Business Media New York 2013
Abstract Hatching enzyme (HE) is of importance to degrade egg membrane to let the larvae be free. HE was purified and characterized from starfish blastula. The specific activity and the purification ratio of the purified HE with 110.9 kDa of molecular weight were 449.62 U/mg and 7.42-fold, respectively. Its optimal pH and temperature for activity were pH8.0 and 30 °C, respectively. This enzyme was relatively stable in the range of pH4.0–6.0 and 30–40 °C. This enzyme was inhibited by ethylene diamine tetraacetic acid (EDTA) and ethylene glycol tetraacetic acid, and also done moderately by Leupeptin, tosyl-lysine chloromethyl ketone, tosylphenylalanine chloromethyl ketone, and phenyl-methanesulfonyl fluoride. Zn2+ ion activated HE activity strongly and recovered the EDTA-pretreated activity more than did Ca2+, Mg2+, and Cu2+. Based on the results above, the starfish HE was classified as a zinc metallo- and trypsinlike serine protease. The values of Km, Vmax, and Kcat of the starfish HE on dimethyl casein were 0.31 mg/ml, 0.17 U/ml, and 122.70 s−1, respectively, whereas 1.09 mg/ml, 0.12 U/ml, and 771.98 s−1 on type I collagen. Therefore, the starfish HE could be a potential cosmeceutical because of its strong cleavage specificity on type I collagen. Keywords Hatching enzyme . Starfish . Asterias amurensis . Purification . Characterization . Cleavage specificity
Introduction Hatching enzyme (HE) is a special enzyme secreted from the inner hatching gland cells at the blastula stage, which can digest the protective extracellular coat of egg and let the larva be free in vertebrate or invertebrate animals [1–4]. Since 1960s, there have been many Z. J. Li Department of Food and Engineering, College of Food Science, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang 163-319, People’s Republic of China Z. J. Li : S. M. Kim (*) Department of Marine Food Science and Technology, Gangneung-Wonju National University, 7 Jukheon-gil, Gangneung 210-702, Republic of Korea e-mail: [email protected]
Appl Biochem Biotechnol (2013) 169:1386–1396
1387
reports about the purification and characterization of HE from many species, such as insect [5], echinoderm [6], amphibian [7, 8], teleostean [9, 10], and mammalian [11]. HE is of great importance for marine species because almost all of them are fertilized in the seawater. So far, several marine HEs have been characterized as metalloprotease from various marine species including sea urchin Strongylocentrotus purpuratus and Strongylocentrotus intermedius [12, 13], shrimp Penaeus chinensis [14], sea squirt Ciona intestinalis [15], and flounder Paralichthys olivaceus [16]. On the other hand, the sea urchin HE is classified as collagenase-like (EC 3.4.24.12), which is involved in many physiological or p
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