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Purification and characterization of hatching enzyme from flounder Paralichthys  olivaceus

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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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Authors and Affiliations

  1. Department of Marine Biology, Division of Life Science and Technology, Ocean University of China, Qingdao, 266003, China

    Zhen-Ping Shi, Ting-Jun Fan, Ri-Shan Cong, Xiao-Feng Wang, Wen-Jie Sun & Ling-Ling Yang

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  1. Zhen-Ping Shi
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  2. Ting-Jun Fan
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Correspondence to Ting-Jun Fan.

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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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