食品科学 ›› 2009, Vol. 30 ›› Issue (17): 235-238.doi: 10.7506/spkx1002-6630-200917054

• 生物工程 • 上一篇    下一篇

木瓜蛋白酶具有水解壳聚糖活性的酶组分分离纯化

陈季旺1,谭 晶1,夏文水1,2   

  1. 1.武汉工业学院食品科学与工程学院 2.江南大学食品学院
  • 收稿日期:2009-02-16 修回日期:2009-06-08 出版日期:2009-09-01 发布日期:2014-04-14
  • 通讯作者: 陈季旺 E-mail:jiwangchen@yahoo.com.cn
  • 基金资助:

    国家自然科学基金项目(20576104)

Isolation and Purification of Fraction with Chitosanase Activity from A Commercial Papain Preparation

CHEN Ji-wang1,TAN Jing1,XIA Wen-shui1,2   

  1. 1. College of Food Science and Engineering, Wuhan Polytechnic University, Wuhan 430023, China;
    2. School of Food Science, Jiangnan University, Wuxi 214122, China
  • Received:2009-02-16 Revised:2009-06-08 Online:2009-09-01 Published:2014-04-14
  • Contact: CHEN Ji-wang1, E-mail:jiwangchen@yahoo.com.cn

摘要:

采用超滤、制备电泳对木瓜蛋白酶进行分离纯化。结果显示:超滤后木瓜蛋白酶的蛋白酶和壳聚糖酶比活力分别从435.10U/mg 和2.17U/mg 提高到531.39U/mg 和2.65U/mg,纯化倍数均提高了1.22 倍。制备电泳图谱上显示有五条明显的酶条带,酶活性测定显示条带2 具有较高的壳聚糖酶比活力。采用SDS-PAGE 和HPLC 对此纯化酶组分进行纯度鉴定,结果显示为单一条带和单一峰,说明木瓜蛋白酶中存在具有水解壳聚糖活力的酶组分。HPLCMS/MS 测定该纯化酶组分的氨基酸序列为 MRITISGSPG SGTTTLGRSI AEKYSYRYVS AGEVFRGLAKERNMDLAAFG KIAETDPAID LEIADARQKEI GESSDDIILE GRLAGWMVEN ADLKILLCAS PECRSTRIAAREGLTEKQAF EMTIEREACE AGRYMEYYEI DILDFSPYDL ILNSETFSAN ELFAIVDAAV SSLLKRE。

关键词: 木瓜蛋白酶, 壳聚糖酶活力, 超滤, 制备电泳, 高效液相色谱/质谱/质谱(HPLC-MS/MS)

Abstract:

The fraction with chitosanase activity in a commercial papain preparation was prepared by ultra-filtration separation followed by purification by preparation electrophoresis and its amino acid sequence was analyzed. After ultra-filtration separation with a 100000 MWCO membrane, the protease and chitosanase activities of the papain preparation both increased by 1.22 times, from 435.10 to 531.39 U/mg and from 2.17 to 2.65 U/mg, respectively. Five obvious bands were showed on the preparation electrophoresis pattern of retentates and the brand 2 with a molecular weight between 25 and 35 kD had high chitosanase activity and its homogeneity was demonstrated by SDS-PAGE and HPLC, indicating that there was the enzyme fraction with chitosanase activity in the papain preparation. The amino acid sequence of the enzyme fraction was determined by HPLC-MS/MS as follows: MRITISGSPG SGTTTLGRSI AEKYSYRYVS AGEVFRGLAK ERNMDLAAFG KIAETDPAIDLEIADARQKEI GESSDDIILE GRLAGWMVEN ADLKILLCAS PECRSTRIAA REGLTEKQAF EMTIEREACE AGRYMEYYEI DILDFSPYDL ILNSETFSAN ELFAIVDAAV SSLLKRE.

Key words: papain, chitosanase activity, ultra-filtration, preparation electrophoresis, HPLC-MS/MS

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