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Characterization of a novel hatching enzyme purified from starfish Asterina pectinifera

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ABSTRACT

Hatching enzyme is a protease which can degrade the membrane of egg. In this study, a hatching enzyme was purified from starfish (Asterina pectinifera) with 6.34 fold of purification rate, 5.04 % of yield, and 73.87 U/mg of specific activity. The molecular weight of starfish hatching enzyme was 86 kDa, which was reduced to 62 kDa after removal of N-linked oligosaccharides. The optimal pH and temperature of the hatching enzyme activity were pH 7.0 and 40 °C, respectively, while those of stability were pH 8 and 20 °C. The kinetic parameters, Vmax, Km, Kcat and Kcat/Km values were 0.197 U/ml, 0.289 mg/ml, 112.57 s−1, and 389.52 ml/mg s, respectively. Zn2+ increased the enzyme activity by 167.28 %, while EDTA, TPCK, TGCK, leupeptin, PMSF, and TLCK decreased. In addition, Ca2+, Mg2+, and Cu2+ did not affect the enzyme activity. The starfish hatching enzyme activity pretreated with EDTA was recovered by Zn2+. Therefore, the starfish hatching enzyme was classified as a serine-zinc protease.

No MeSH data available.


Recovery effect of metal ions on the EDTA pretreated starfish hatching enzyme
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Fig4: Recovery effect of metal ions on the EDTA pretreated starfish hatching enzyme

Mentions: The effects of chelators, inhibitors, and metal ions on the enzyme activity are shown in Table 2. EDTA and EGTA inhibited significantly the proteolytic activity of hatching enzyme by more than 50 % (Table 2), which was similar to the results of the frog (Fan and Katagiri 2001), flounder (Shi et al. 2010), sea squirt (D’Aniello et al. 1997), A. amurensis (Li and Kim 2013), and sea urchin (Roe and Lennarz 1990). The proteolytic activity of hatching enzyme was strongly activated by 167.28 % at 5 mM of Zn2+ (Table 2). Zn2+ also recovered the denatured hatching enzyme activity more greatly than other ion metals (Fig. 4), which was similar to the hatching enzymes of the brine shrimp (Fan et al. 2010), sea squirt (D’Aniello et al. 1997), A. amurensis (Li and Kim 2013) and shrimp (Li et al. 2006). Based on the inhibitory activity of EDTA and EGTA, the starfish hatching enzyme in this study was characterized as metalloprotease, which was similar to the hatching enzymes of sea squirt (D’Aniello et al. 1997) and sea urchin (Roe and Lennarz 1990). TLCK and TPCK are known to inhibit trypsin and chymotrypsin through the alkylation of a histidine residue at active sites, whereas PMSF and leupeptin inhibit them by sulfonylating the hydroxyl group of the serine residue at the active site, respectively (Ikegami et al. 1994). The starfish hatching enzyme was sensitive to EDTA and several metal ions (Table 2). Zn2+ recovered the proteolytic activity of starfish hatching enzyme pretreated with EDTA. Therefore, it was indicated that starfish hatching enzyme might be also a kind of Zn2+-protease, which was similar to the results of hatching enzymes from frog (Fan and Katagiri 2001; Kitamura and Katagiri 1998), O. latipes (Yasumasu et al. 1989a, b), brine shrimp (Fan et al. 2010), flounder (Shi et al. 2010), shrimp (Li et al. 2006), sea squirt (D’Aniello et al. 1997), A. amurensis (Li and Kim 2013), F. heteroclitus (DiMichele et al. 1981), sea urchin (Yasumasu et al. 1989), and pike (Schoot and Denuce 1981). Based on these results, the A. pectinifera starfish hatching enzyme was classified as a serine-zinc protease.Table 2


Characterization of a novel hatching enzyme purified from starfish Asterina pectinifera
Recovery effect of metal ions on the EDTA pretreated starfish hatching enzyme
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5120168&req=5

Fig4: Recovery effect of metal ions on the EDTA pretreated starfish hatching enzyme
Mentions: The effects of chelators, inhibitors, and metal ions on the enzyme activity are shown in Table 2. EDTA and EGTA inhibited significantly the proteolytic activity of hatching enzyme by more than 50 % (Table 2), which was similar to the results of the frog (Fan and Katagiri 2001), flounder (Shi et al. 2010), sea squirt (D’Aniello et al. 1997), A. amurensis (Li and Kim 2013), and sea urchin (Roe and Lennarz 1990). The proteolytic activity of hatching enzyme was strongly activated by 167.28 % at 5 mM of Zn2+ (Table 2). Zn2+ also recovered the denatured hatching enzyme activity more greatly than other ion metals (Fig. 4), which was similar to the hatching enzymes of the brine shrimp (Fan et al. 2010), sea squirt (D’Aniello et al. 1997), A. amurensis (Li and Kim 2013) and shrimp (Li et al. 2006). Based on the inhibitory activity of EDTA and EGTA, the starfish hatching enzyme in this study was characterized as metalloprotease, which was similar to the hatching enzymes of sea squirt (D’Aniello et al. 1997) and sea urchin (Roe and Lennarz 1990). TLCK and TPCK are known to inhibit trypsin and chymotrypsin through the alkylation of a histidine residue at active sites, whereas PMSF and leupeptin inhibit them by sulfonylating the hydroxyl group of the serine residue at the active site, respectively (Ikegami et al. 1994). The starfish hatching enzyme was sensitive to EDTA and several metal ions (Table 2). Zn2+ recovered the proteolytic activity of starfish hatching enzyme pretreated with EDTA. Therefore, it was indicated that starfish hatching enzyme might be also a kind of Zn2+-protease, which was similar to the results of hatching enzymes from frog (Fan and Katagiri 2001; Kitamura and Katagiri 1998), O. latipes (Yasumasu et al. 1989a, b), brine shrimp (Fan et al. 2010), flounder (Shi et al. 2010), shrimp (Li et al. 2006), sea squirt (D’Aniello et al. 1997), A. amurensis (Li and Kim 2013), F. heteroclitus (DiMichele et al. 1981), sea urchin (Yasumasu et al. 1989), and pike (Schoot and Denuce 1981). Based on these results, the A. pectinifera starfish hatching enzyme was classified as a serine-zinc protease.Table 2

View Article: PubMed Central - PubMed

ABSTRACT

Hatching enzyme is a protease which can degrade the membrane of egg. In this study, a hatching enzyme was purified from starfish (Asterina pectinifera) with 6.34 fold of purification rate, 5.04 % of yield, and 73.87 U/mg of specific activity. The molecular weight of starfish hatching enzyme was 86 kDa, which was reduced to 62 kDa after removal of N-linked oligosaccharides. The optimal pH and temperature of the hatching enzyme activity were pH 7.0 and 40 °C, respectively, while those of stability were pH 8 and 20 °C. The kinetic parameters, Vmax, Km, Kcat and Kcat/Km values were 0.197 U/ml, 0.289 mg/ml, 112.57 s−1, and 389.52 ml/mg s, respectively. Zn2+ increased the enzyme activity by 167.28 %, while EDTA, TPCK, TGCK, leupeptin, PMSF, and TLCK decreased. In addition, Ca2+, Mg2+, and Cu2+ did not affect the enzyme activity. The starfish hatching enzyme activity pretreated with EDTA was recovered by Zn2+. Therefore, the starfish hatching enzyme was classified as a serine-zinc protease.

No MeSH data available.