Abstract
Using chorion of Paralichthys as a specific substrate, hatching enzyme (HE) from Paralichthys olivaceus (PHE) was purified by gel-filtration and ion-exchange chromatography, and characterized in terms of its molecular weight and enzymatic properties in this study. It was found that the molecular size of PHE is about 34.8 kDa in SDS-PAGE. The PHE had obvious choriolytic activity, which was optimal at pH 7.0 and temperature of 35 °C, respectively. The Km value of the PHE for casein was 4.28 mg ml−. The PHE was very sensitive to trypsin-specific inhibitors, especially serine protease-specific inhibitors, such as LBTI, SBTI, bestatin and p-APMSF, leupeptin, ovomucoid, PMSF, pepstatin A and TLCK, indicates that it is a trypsin-type serine protease. The PHE was also extremely sensitive to Cu2+ and Ca2+, combined with the results that it was inhibited by EDTA in a dose-dependent manner, indicates this PHE is also a kind of metalloprotease.
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Abbreviations
- IAM:
-
iodoacetamide
- LBTI:
-
limabean trypsin inhibitor
- NEM:
-
N-ethylmaleimide
- p-APMSF :
-
p-Amidinophenyl methane sulfonyl fluoride hydrochloride
- PMSF:
-
phenylmethane sulforyl fluoride
- SBTI:
-
soybean trypsin inhibitor
- TLCK:
-
N-tosyl-l-lysyl choromethyl ketone
- TPCK:
-
N-tosyl-l-phenylalanyl chloromethyl ketone
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Shi, ZP., Fan, TJ., Cong, RS. et al. Purification and characterization of hatching enzyme from flounder Paralichthys olivaceus . Fish Physiol Biochem 32, 35–42 (2006). https://doi.org/10.1007/s10695-005-5250-6
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DOI: https://doi.org/10.1007/s10695-005-5250-6