Characterization of a novel hatching enzyme purified from starfish Asterina pectinifera
© The Author(s) 2016
Received: 3 August 2015
Accepted: 6 October 2016
Published: 22 November 2016
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, V max , K m , K cat and K cat /K m 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.
Hatching enzyme is a protease released from hatching gland cells in hatching embryos for digesting their protective extracellular coats (Lepage and Gache 1989; Fan and Katagiri 2001; Yasumasu et al. 1989a, b). The hatching enzyme can provide a typical model in the studies of certain cell differentiation, specific protein synthesis, and special gene expression regulation during a certain stage of early embryos at the morphological and molecular level (Fan et al. 2010). The hatching enzymes from many animal species, such as echinoderm (Lepage and Gache 1989), mammalian (Sawada et al. 1990), avians (Yasumasu et al. 2005), amphibians (Fan and Katagiri 2001; Kitamura and Katagiri 1998; Urch and Hedrick 1981), teleostean (Yasumasu et al. 1989a, b; Kudo et al. 2004; Shi et al. 2010), and insect (Young et al. 2000), have been studied since 1980s. Several marine hatching enzymes have been identified as a metalloprotease from a variety of marine species; brine shrimp Artermia salina (Fan et al. 2010), flounder Paralichthys olivaceus (Shi et al. 2010), shrimp Penaeus chinensis (Li et al. 2006), and sea squirt Ciona intestinalis (D’Aniello et al. 1997), whereas the sea urchin hatching enzyme is classified as a collagenase-like (EC 126.96.36.199) enzyme. The hatching enzymes were involved in many physiological processes such as cell migration, tissue repair, angiogenesis, inflammation, tumor invasion, and metastasis (Li and Kim 2013; Roe and Lennarz 1990).
Collagens compose about 70 % human skin, where the predominant ones are types I (80–90 %) and III (10–15 %) (Ala-Kokko et al. 1987). Hence, collagenases have been used for pharmacological purpose to treat various collagen mediated diseases such as keloid and scar, which are caused by over accumulation of collagen in tissue.
Starfish is an invertebrate belonging to the class of Asteroidea, Phylum Echinodermata, which produces a variety of secondary metabolites including steroids glycosides, anthraquinones, alklaoids, phospholipids, peptides, and fatty acids (Barkhouse et al. 2007; Kurihara 1999). However, starfish has been regarded as a harmful marine animal to marine ecosystem because it causes severe loss of mussel, oyster, scallop, etc. Therefore, many countries including Korea spend a lot of budget to relieve their marine ecosystem by reducing the number of starfish. In our previous studies (Li and Kim 2013, 2014a, b), a novel hatching enzyme was purified and characterized from starfish Asterias amurensis, which has habitat in the Ocean of East Russian. However, Asterin apectinifera starfish is predominant in the Ocean of Korean peninsula. Therefore, the objective of this study was to purify and characterize a hatching enzyme from starfish A. pectinifera for the development of a more value-added material.
Starfish and reagents
The adult starfish A. pectinifera was collected in July 2013 at Samcheok, Korea. About 500,000 live eggs were kept into 1 L of Kester artificial sea water (KASW salinity, 35.00 ‰; chlorinity, 19.00 ‰; pH 7.8) (Kester et al. 1967) and were dejellied by adjusting the pH 7.8 of KASW to 5.5 with 1 N of HCl. After 10 min, the supernatant was poured off and the precipitate was washed 3 or 4 times with the same volume of KASW. The sperms were collected out of the spermatophore artificially by pressing and were stored at 4 °C until inseminated. DEAE-sepharose fast flow and Sephacryl S-200 gels were purchased from Amersham Pharmacia Biotech (Uppsala, Sweden). Peptide-N-glycosidase F (PNGase F), dimethyl casein, trichloroacetic acid and tris (hydroxylmethyl) aminomethane were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals and reagents that were used were of analytical grade.
Preparation of crude hatching enzyme
A crude hatching enzyme was prepared according to the method of Lepage (1989). Briefly, approximately 100,000 eggs in 500 ml of KASW were inseminated by adding a few drops of 0.005 % sperm, stirred at 16 °C overnight, and then precipitated using 70 % ammonium sulfate at 4 °C overnight. After centrifuged at 7.728×g for 30 min (5810R; Eppendorf, Hamburg, Germany), the precipitate was dissolved in a 10 ml of 0.02 M Tris–HCl buffer (pH 7.4) and was then dialyzed against above buffer at 4 °C overnight. The egg membrane was prepared according to the modified method of Li (2006). About 5000 eggs were washed, stripped through 100 μm mesh, and then squeezed using a syringe needle. After washed with distilled water, the egg membrane was sonicated at 35 kHz for 10 s (MSONIC; Mirae Ultrasonic, Seoul, Korea). After centrifuged at 1.932×g for 15 min, the collected egg membrane was washed with distilled water completely and was resuspended in a 10 ml of 0.02 M Tris–HCl buffer (pH 7.4).
Purification of hatching enzyme
The crude starfish extract (5 ml, 30 mg/ml) was loaded onto DEAE-Sepharose fast flow column (2.6 × 30.0 cm), and then eluted with a linear gradient of 0–1 M NaCl in 0.02 M Tris–HCl buffer (pH 7.4). The active fractions with more than 50 % maximal activity were pooled and were then dialyzed against 0.02 M Tris–HCl buffer (pH 7.4) overnight (DEAE active fraction). The DEAE active fraction was loaded onto Sephacryl S-200 gel filtration column (2.6 × 90 cm), and then eluted with 0.1 M Tris–HCl containing 0.05 M NaCl (pH 7.4). The active fractions with more than 50 % maximal activity were pooled and were dialyzed against 0.02 M Tris–HCl buffer (pH 7.4) overnight.
The hatching enzyme was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 12 % separating and 5 % stacking gels. The molecular marker (ELPIS Bioteck Co., Taejeon, Korea) ranged from 35 to 170 kDa was used to determine the molecular weight of hatching enzyme. The electrophoresized gel was stained using 0.05 % Coomassie Blue R-250 (Bio-Rad Lavoratories, Hercules, CA, USA) and was destained in a destaining solution (40 % methanol and 10 % acetic acid).
Deglycosylation of N-glycans
PNGase F was used to deglycosylate the N-linked carbohydrate from the glycoproteins or glycopeptides according to the method of Sanchez et al. (2007). Twenty microlitre of the starfish hatching enzyme (200 μg) was added into 50 μl of denaturing buffer (0.5 % SDS and 1 % β-mercaptoethanol) and was then boiled for 10 min. After cooled down, 10 μl of reaction buffer (0.05 mM phosphate, pH 7.5), 5 μl of 15 % TritonX-100, and 5 μl of PNGase F (500 U/ml) were added and then incubated at 37 °C for 2 h. The reaction was stopped by heating at 100 °C for 10 min. Molecular weight of the de-N-glycosylated hatching enzyme was calculated based on the results of SDS-PAGE.
Protein concentration of the hatching enzyme fractions was determined using Bradford method (1976). Bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) was used as the calibration standard. The relative protein contents of chromatography fractions were estimated by measuring absorbance at 280 nm.
Choriolytic activity was determined according to the modified method of Yamagami (1972) using 10 mg/ml egg membrane as the substrate. Each 100 μl of the hatching enzyme and egg membrane (10 mg/ml) were mixed and incubated at 30 °C for 30 min. The reaction was stopped by adding the cold TCA (20 % w/v, 2.8 ml). After centrifuged at 3000×g for 30 min, the supernatant was collected. The absorbance of supernatant at 280 nm was measured using a spectrophotometer (V-300; JASCO, Seoul, Korea). One unit (U) of choriolytic activity was defined as an increase in absorbance by 0.001/min at 280 nm.
Proteolytic activity was determined using the determination method of choriolytic activity by substituting egg membrane with casein as the substrate. Each 100 μl of the hatching enzyme and casein (10 mg/ml) were mixed and incubated at 30 °C for 30 min. The reaction was stopped by adding the cold TCA (20 % w/v, 2.8 ml). After centrifuged at 3000×g for 30 min, the supernatant was collected. The absorbance of supernatant at 280 nm was measured using a spectrophotometer (V-300; JASCO). One unit (U) of proteolytic activity was defined as an increase in absorbance by 0.001/min at 280 nm.
Effects of pH and temperature on the proteolytic activity and stability of hatching enzyme
The effect of pH profile on the proteolytic activity of hatching enzyme was determined at different ranges of pH 4.0–10.0: sodium acetic acetate buffer (pH 4.0–6.0), phosphate buffer (pH 7.0–8.0), and glycine-NaOH buffer (pH 9.0–10.0) (Li and Kim 2013). The effect of temperature on the enzyme activity was determined at different temperatures of 20–50 °C. Casein was used as the substrate. The effect of pH on the hatching enzyme stability was determined by pre-incubating enzyme over a range of pH 4.0–10.0 for 30 min. Subsequently, the enzyme mixture was adjusted to pH 7.4 using a 0.1 N NaOH or HCl. The effect of temperature on the enzyme stability was determined by pre-incubating the enzyme at 20–50 °C for 30 min. The remaining proteolytic activity for hatching enzyme activity and stability was measured under the same condition as the determination of proteolytic activity described above. The relative activity was defined as the percentage of activity determined with respect to the maximum hatching enzyme activity.
Determination of kinetic parameters
The kinetic parameters (K m and V max ) of the purified enzyme were determined by measuring proteolytic activity at different concentrations of casein under the same condition as described above. K m and V max were calculated from the Lineweaver–Burk plot. The K cat and K cat /K m values were calculated based on the K m and V max values.
Effect of inhibitors and metal ions on the hatching enzyme activity
The effects of various inhibitors on the proteolytic activity of hatching enzyme were determined. Each 100 μl of the hatching enzyme and casein (10 mg/ml) were mixed with inhibitors (5 mM for EDTA and EGTA, and 0.1 mM for leupeptin, TLCK, TPCK and PMSF) and incubated at 30 °C for 30 min. The reaction was stopped by adding the cold TCA (20 % w/v, 2.8 ml). After centrifuged at 3000×g for 30 min, the supernatant was collected. The absorbance of supernatant at 280 nm was measured using a spectrophotometer (V-300; JASCO). In addition, the purified hatching enzyme was pre-incubated at 30 °C for 30 min in the absence and the presence of bivalent cations such as Mg2+, Ca2+, Cu2+, and Zn2+. Then, the remaining proteolytic activity was measured under the same condition as described above. The relative proteolytic activity of hatching enzyme pre-incubated with no inhibitors or metal ions was used as the control.
Recovery effect of metal ions on the EDTA-pretreated hatching enzyme
The hatching enzyme was pretreated with 10 mM of EDTA at 4 °C for 30 min. Afterwards, metal ions (Mg2+, Ca2+, Cu2+, and Zn2+) at 5 mM were added and the enzyme mixture was incubated at 4 °C for 3 h. The proteolytic activity was measured under the same condition as described above. The relative proteolytic activity of hatching enzyme pre-incubated with no metal ions was used as the control.
Experimental results were tested in triplicates and presented as mean values ± standard error (SD).
Results and discussion
Purification of the starfish hatching enzyme
Purification of hatching enzyme from starfish Asterinapectinifera
Total protein (mg)
Total choriolyticactivity (U)
Specificchoriolytic activity (U/mg)
Effect of pH and temperature on the hatching enzyme activity and stability
Effects of chelators, inhibitors, and metal ions on the enzyme activity
Effect of metal ions and inhibitors on the proteolyticactivity of hatching enzyme
Inhibitors or metal ions
Relative activity (%)
38.15 ± 9.86
42.31 ± 8.41
75.38 ± 7.01
71.22 ± 4.65
167.28 ± 12.69
86.47 ± 2.50
56.29 ± 2.57
56.20 ± 4.15
56.72 ± 2.34
40.47 ± 8.40
A novel hatching enzyme with 86 kDa of molecular weight was purified from starfish (A. pectinifera). De-N-glycosylation of the enzyme leads to a loss of 24 kDa as observed by the migration behavior in SDS-PAGE. The purification rate and yield of starfish hatching enzyme were 6.34 fold and 5.04 %, respectively. The optimal pH and temperature of hatching enzyme activity were 7.0 and 40 °C, respectively, while those of stability were pH 7.0 and 20 °C. The starfish hatching enzyme was classified was a serine-zinc protease. Therefore, the A. pectinifera hatching enzyme might be utilized as a cosmeceutical because its optimum pH and temperature stability were similar to those of human skin.
JHC, a first author, carried out the purification and characterization of starfish hatching enzyme, participated in the sequence alignment, and drafted the manuscript. SMK, a corresponding author, participated in its design and coordination, and helped to draft the manuscript. Both authors read and approved the final manuscript.
This research was partially supported by the Korea Sea Grant Program (GangWon Sea Grant) funded by the Ministry of Oceans and Fisheries in Korea.
The authors declare that they have no competing interests.
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