一个新型耐热普鲁兰酶的结构与功能

生物工程学报

甄杰 等/一个新型耐热普鲁兰酶的结构与功能

Chinese Journal of Biotechnology http://www.77cn.com.cn/cjbcn DOI: 10.13345/j.cjb.130389

January 25, 2014, 30(1): 119 128

©2014 Chin J Biotech, All rights reserved

工业酶改造与应用

一个新型耐热普鲁兰酶的结构与功能

甄杰1,2*，胡政2*，李树芳2，徐健勇2，宋诙2

1 天津科技大学，天津 300457

2 中国科学院天津工业生物技术研究所，天津 300308

甄杰, 胡政, 李树芳,等. 一个新型耐热普鲁兰酶的结构与功能. 生物工程学报, 2014, 30(1): 119 128.

Zhen J, Hu Z, Li SF, et al. Structure and function of a novel thermostable pullulanase. Chin J Biotech, 2014, 30(1): 119 128.

摘 要: 新型普鲁兰酶的研究对于普鲁兰酶制剂的国产化、打破国外垄断具有非常重要的意义。从我国云南

腾冲地区轮马热泉的淤泥中分离获得了一株耐热普鲁兰酶产生菌，经16S rDNA序列系统进化树分析，确定该菌为厌氧芽胞杆菌Anoxybacillus属种，并从中克隆获得了耐热普鲁兰酶的编码基因，该酶在55 ℃ 60 ℃、pH 5.6 6.4的范围内具有最大的反应活性。此外，该酶具有较好的热稳定性，在60 ℃下处理48 h，仍可保持50%以上的活力；动力学分析该酶的Vmax和Km分别为750 U/mg和1.47 mg/mL，是目前文献报道中比活力最高的耐热普鲁兰酶。同时还对该酶进行了晶体结构分析，结果显示该酶具有 -淀粉酶家族中典型的结构，在N端具有一个特殊的底物结合域，该结构域的缺失导致比活力和底物结合力都有相应降低，Vmax和Km分别为324 U/mg和1.95 mg/mL。同时，将该普鲁兰酶编码基因导入枯草芽胞杆菌中，在P43启动子的控制下，普鲁兰酶基因获得了高效表达，胞外酶活可达42 U/mL，相比初始菌种，表达活力提高40倍以上。研究表明该普鲁兰酶具有很好的应用前景。

关键词: 普鲁兰酶，酶促动力学，晶体结构，异源表达

Structure and function of a novel thermostable pullulanase

Jie Zhen1,2*, Zheng Hu2*, Shufang Li2, Jianyong Xu2, and Hui Song2

1 Tianjin University of Science & Technology, Tianjin 300457, China

2 Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China

Abstract: Research on novel pullulanase has major significance on the domestic industrialization of pullulanase and the

Received: July 26, 2013; Accepted: October 23, 2013

Supported by: The Chinese Academy of Sciences for Key Topics in Innovation Engineering (No. KSCX2-EW-G-8), Tianjin Science and Technology Program (No. 12ZCZDSY15000).

Corresponding author: Hui Song. Tel/Fax: +86-22-84861934; E-mail: song_h@http://www.77cn.com.cn *These authors contributed equally to this study.

中国科学院知识创新工程重要方向项目 (No. KSCX2-EW-G-8), 天津市科技计划项目 (No. 12ZCZDSY15000) 资助。

ISSN 1000-3061 CN 11-1998/Q Chin J Biotech January 25, 2014 Vol.30 No.1

breakdown of foreign monopoly. A thermophilic bacteria LM 18-11 producing thermostable pullulanase was isolated from Lunma hot springs of Yunnan province. It was identified as Anoxybacillus sp. by 16S rDNA phylogenetic analysis. Full-length pullulanase gene was cloned from Anoxybacillus sp. LM18-11. The optimum temperature of the pullulanase was between 55 and 60 oC with a half-life as long as 48 h at 60 oC; and its optimum pH was between 5.6 and 6.4. Vmax and Km of the pullulanase was measured as 750 U/mg and 1.47 mg/mL, which is the highest specific activity reported so far. The pullulanase crystals structure showed a typical -amylase family structure. The N-terminal has a special substrate binding domain. Activity and substrate binding were decreased when the domain was deleted, the Vmax and Km were 324 U/mg and 1.95 mg/mL, respectively. The pullulanase was highly heterologous expressed in Bacillus subtilis by P43 promoter. The extracellular enzyme activity was 42 U/mL, which increased more than 40 times compared to the initial strain. This pullulanase has good application prospects.

Keywords: pullulanase, enzyme kinetics, crystal structure, heterologous expression

淀粉 (Starch) 是植物中普遍存在的储藏多糖，是植物体内能量的储备物质。淀粉有两种结构形式，一种是直链淀粉 (Amylose)，另一种是支链淀粉 (Amylopectin)。直链淀粉是由葡萄糖通过α-1,4-糖苷键连接组成的，是不分支类型的淀粉；支链淀粉中除有α-1,4-糖苷键外，还有4%–5%的α-1,6-糖苷键。工业淀粉制糖生产中，淀粉首先经过高温液化步骤，先经过α-淀粉酶 (α-Amylase)水解，将高聚糖水解为低聚糖，这一过程的反应条件为pH 6.0、温度95 ℃ 105 ℃。液化产生的低聚糖随后在β-淀粉酶 (β-Amylase)、葡萄糖淀粉酶 (Glucoamylase) 和普鲁兰酶 (Pullulanase)、以及异淀粉酶 (Isoamylase) 等作用下，残留的直链α-1,4-糖苷键和支链α-1,6-糖苷键被进一步水解，最终形成葡萄糖浆，该过程称为糖化步骤，其最适酶促条件为pH 4.5、温度60 ℃ 62 ℃[1-3]。

普鲁兰酶 (Pullulanase，EC 3.2.1.41)，也称去分支酶，是一种专一性水解支链淀粉分支点的α-1,6-糖苷键的葡萄糖苷水解酶，可特异性地将支链淀粉水解形成长度不同直链淀粉，是糖苷水解酶13家族成员之一[4]。在淀粉加工工业中，普鲁兰酶与糖化酶等的联合应用可有效地

提高淀粉的水解效率和葡萄糖的产量，广泛地应用于高葡萄糖浆、高麦芽糖浆和干啤酒生产等应用中，是一种重要的工业酶。

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