Purification,Characterization and in vitro Anthelmintic Activity of a Neutral Metalloprotease from Laccocephalum mylittae

CIESC Journal ›› 2010, Vol. 18 ›› Issue (1): 122-128.

Purification,Characterization and in vitro Anthelmintic Activity of a Neutral Metalloprotease from Laccocephalum mylittae

周立华, 许勤勤, 张一琼, 周振兴, 管文军, 李永泉

College of Life Sciences, Zhejiang University, Hangzhou 310058, China

收稿日期:2009-03-16 修回日期:2009-07-11 出版日期:2010-02-28 发布日期:2010-12-30
通讯作者: GUAN Wenjun, E-mail: guanwj@zju.edu.cn
作者简介:
基金资助:
Supported by the National High Technology Research and Development Program of China(2007AA021506);the Natural Science Foundation of Zhejiang Province(R207609)

Purification,Characterization and in vitro Anthelmintic Activity of a Neutral Metalloprotease from Laccocephalum mylittae

ZHOU Lihua, XU Qinqin, ZHANG Yiqiong, ZHOU Zhenxing, GUAN Wenjun, LI Yongquan

College of Life Sciences, Zhejiang University, Hangzhou 310058, China

Received:2009-03-16 Revised:2009-07-11 Online:2010-02-28 Published:2010-12-30
Supported by:
Supported by the National High Technology Research and Development Program of China(2007AA021506);the Natural Science Foundation of Zhejiang Province(R207609)

摘要/Abstract

摘要： A neutral metalloprotease was purified from the cultured mycelia of Laccocephalum mylittae,an effective medicinal fungus widely used in anthelmintic therapy.The protease was purified to homogeneity with 31.85-fold purification and a final yield of 21.76%.The subunit molecular weight of the protease is about 40000 estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE).The optimum reaction pH and temperature are 7.5 and 50℃,respectively.The protease activity is largely enhanced by Ca²⁺,but highly inhibited by tetrasodium ethylenediaminetetraacetate(EDTA),a metal-chelator,suggesting that the enzyme is a metalloprotease.The Michaelis-Menten constan Km and Vmax value for casein substrate are 6.09mg·ml^-1and 21.32μg·min^-1·ml^-1,respectively.In vitro anthelmintic tests of the protease exhibit distinct lethal effects on the third stage larvae(L3)of Ascaris suum.Scanning electron microscopy and SDS-PAGE analysis indicates that the proteolysis of larvae proteins caused by this protease may relate to the anthelmintic activity of L.mylittae.

关键词: metalloprotease, Laccocephalum mylittae, purification, characterization, anthelmintic activity

Abstract: A neutral metalloprotease was purified from the cultured mycelia of Laccocephalum mylittae,an effective medicinal fungus widely used in anthelmintic therapy.The protease was purified to homogeneity with 31.85-fold purification and a final yield of 21.76%.The subunit molecular weight of the protease is about 40000 estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE).The optimum reaction pH and temperature are 7.5 and 50℃,respectively.The protease activity is largely enhanced by Ca²⁺,but highly inhibited by tetrasodium ethylenediaminetetraacetate(EDTA),a metal-chelator,suggesting that the enzyme is a metalloprotease.The Michaelis-Menten constan Km and Vmax value for casein substrate are 6.09mg·ml^-1and 21.32μg·min^-1·ml^-1,respectively.In vitro anthelmintic tests of the protease exhibit distinct lethal effects on the third stage larvae(L3)of Ascaris suum.Scanning electron microscopy and SDS-PAGE analysis indicates that the proteolysis of larvae proteins caused by this protease may relate to the anthelmintic activity of L.mylittae.

Key words: metalloprotease, Laccocephalum mylittae, purification, characterization, anthelmintic activity

周立华, 许勤勤, 张一琼, 周振兴, 管文军, 李永泉. Purification,Characterization and in vitro Anthelmintic Activity of a Neutral Metalloprotease from Laccocephalum mylittae[J]. CIESC Journal, 2010, 18(1): 122-128.

ZHOU Lihua, XU Qinqin, ZHANG Yiqiong, ZHOU Zhenxing, GUAN Wenjun, LI Yongquan. Purification,Characterization and in vitro Anthelmintic Activity of a Neutral Metalloprotease from Laccocephalum mylittae[J]. , 2010, 18(1): 122-128.

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Purification,Characterization and in vitro Anthelmintic Activity of a Neutral Metalloprotease from Laccocephalum mylittae

Purification,Characterization and in vitro Anthelmintic Activity of a Neutral Metalloprotease from Laccocephalum mylittae

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