Paenibacillus sp. K1 α-半乳糖苷酶酶学性质

doi:10.6040/j.issn.1671-9352.0.2014.182

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

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. 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. 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/Abstract

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,K_m值为0.75 mmol/L,最大反应速率V_max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, K_m 0.75 mmol/L and V_max 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

引用本文

辛永平, 孔文涛, 陆文伟, 孔健. Paenibacillus sp. K1 α-半乳糖苷酶酶学性质[J]. 山东大学学报（理学版）, 2015, 50(01): 50-55.

XIN Yong-ping, KONG Wen-tao, LU Wen-wei, KONG Jian. Characterization of the alpha-galactosidase of Paenibacillus sp. K1[J]. JOURNAL OF SHANDONG UNIVERSITY(NATURAL SCIENCE), 2015, 50(01): 50-55.

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[1]	武翠玲,刘丹,杨兴昊,吴日帮,黄嘉丰,张姜,伦梓丰,何海伦. Salinivibrio sp.YH4胞外蛋白酶EYHⅠ的分离纯化、酶学性质及基因克隆[J]. 山东大学学报（理学版）, 2016, 51(5): 36-42.
[2]	王尧, 张玖琳, 任艳丽. 幂零p.p.-环和幂零Baer环的Ore扩张[J]. 山东大学学报（理学版）, 2015, 50(04): 76-81.
[3]	魏杰, 董珺. 强α-可逆环的推广[J]. 山东大学学报（理学版）, 2015, 50(03): 88-94.
[4]	陆文伟,孔文涛,孙芝兰,孔健*,季明杰 . Paenibacillus sp. K1 β-半乳糖苷酶基因的克隆及在大肠杆菌中的表达[J]. J4, 2008, 43(7): 69-73 .

Paenibacillus sp. K1 α-半乳糖苷酶酶学性质

Characterization of the alpha-galactosidase of Paenibacillus sp. K1

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