一个新型耐热普鲁兰酶的结构与功能

Structure and function of a novel thermostable pullulanase

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LM 18-11，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a-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。

Abstract:

Research on novel pullulanase has major significance on the domestic industrialization of pullulanase and the 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 a-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.