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山东大学学报(理学版) ›› 2015, Vol. 50 ›› Issue (01): 50-55.doi: 10.6040/j.issn.1671-9352.0.2014.182

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Paenibacillus sp. K1 α-半乳糖苷酶酶学性质

辛永平1, 孔文涛2, 陆文伟1, 孔健1   

  1. 1. 山东大学微生物技术国家重点实验室, 山东 济南 250100;
    2. 伊利诺斯大学香槟分校生物工程系, 美国 乌尔班纳 伊利诺伊 61801
  • 收稿日期:2014-04-23 修回日期:2014-11-10 出版日期:2015-01-20 发布日期:2015-01-24
  • 通讯作者: 孔健(1964-),女,教授,博士生导师,研究方向为乳酸菌应用基础.E-mail:kongjian@sdu.edu.cn E-mail:kongjian@sdu.edu.cn
  • 作者简介:辛永平(1991-),男,硕士,研究方向为乳酸菌应用基础.E-mail:18769781107@163.com
  • 基金资助:
    国家科技支撑计划课题资助项目(2012BAD39B01-6);国家863课题资助项目(2011AA100902)

Characterization of the alpha-galactosidase of Paenibacillus sp. K1

XIN Yong-ping1, KONG Wen-tao2, LU Wen-wei1, KONG Jian1   

  1. 1. State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, Shandong, China;
    2. Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana 61801, IL, USA
  • Received:2014-04-23 Revised:2014-11-10 Online:2015-01-20 Published:2015-01-24

摘要: 利用CODEHOP PCR和Anchor-ligated PCR方法从类芽孢杆菌Paenibacillus sp. K1中克隆得到一个α-半乳糖苷酶基因agaP1, 大小为2 190 bp,同源性分析显示,该基因与其他α-半乳糖苷酶基因的序列相似低,是一个新的α-半乳糖苷酶基因。将agaP1在大肠杆菌Origami B (DE3)中表达并纯化获得AgaP1,酶学性质分析显示:以pNPG为底物时,AgaP1最适反应温度为40 ℃,最适pH 6.5~10,Km值为0.75 mmol/L,最大反应速率Vmax为1.96 μmol·min-1·mg-1。同时Fe2+、Mg2+、Ca2+、K+和甘油能使α-半乳糖苷酶酶活提高1~3倍,而Cu2+、Zn2+、Fe3+和还原型谷胱甘肽则抑制该酶的活性。SDS-PAGE检测AgaP1蛋白大小约为80 ku,与理论预测值基本一致;Native-PAGE分析表明正常条件下AgaP1蛋白以二聚体或六聚体形式存在。以上结果显示,Paenibacillus sp. K1产生的α-半乳糖苷酶为一个新的低温α-半乳糖苷酶。

关键词: 类芽孢杆菌, 酶学性质, -半乳糖苷酶, &alpha

Abstract: A novel alpha-galactosidase encoded by agaP1 gene was amplilfied in the genome of Paenibacillus sp. K1 by CODEHOP PCR and Anchor-ligated PCR techniques. It was 2 190 bp in size and composed of 729 amino acid residues with 82.7 ku of predicted molecular weight,and pI 5.16. The AgaP1 was expressed in E.coli Origami B (DE3) and purified with His-tag chromatography column. The enzymic properties of alpha-galactosidase AgaP1 was analysized and showed optimum temperature of 40 ℃, optimal pH of 6.5-10, Km 0.75 mmol/L and Vmax 1.96 μmol·min-1·mg-1. The AgaP1 activity could be improved one to three folds by Fe2+, Mg2+, Ca2+, K+ and glycerol, whereas Cu2+, Zn2+, Fe3+ and glutathione could completely inhibit the AgaP1 activity. SDS-PAGE revealed a single band of 80 ku, consistant with the predicted size, while two bands of 150 ku and 440 ku was observed on the native-PAGE gel, corresponding to two and six oligomers of AgaP1. All of these results suggested that the AgaP1 produced by Paenibacillus sp. K1 was a novel alpha-galactosidase with the highest enzymic activity at low temperature.

Key words: enzymic properties, alpha-galactosidase, Paenibacillus

中图分类号: 

  • Q78
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