N-Terminal Domain Truncation and Domain Insertion-Based Engineering of a Novel Thermostable Type I Pullulanase from Geobacillus thermocatenulatus
N-Terminal Domain Truncation and Domain Insertion-Based Engineering of a Novel Thermostable Type I Pullulanase from Geobacillus thermocatenulatus
复制标题
N-基于热链地芽孢杆菌的新型耐热 I 型普鲁兰酶的末端结构域截断和结构域插入工程
DOI：
10.1021/acs.jafc.8b03331
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发表时间：
2018-10-17
影响因子：
6.1
通讯作者：
Wei, Dongzhi
中科院分区：
文献类型：
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作者：
Li, Lingmeng;Dong, Fengying;Wei, Dongzhi
从热链土芽孢杆菌DSMZ 730中克隆了一个新的耐热I型普鲁兰酶基因pul（GT）。该基因的开放阅读框长2154 bp，编码718个氨基酸。G.发现热小链菌普鲁兰酶（Pul（GT））在pH 6.5和70 ℃下具有最佳活性。在pH5.5 -7.0范围内，该酶活性稳定。Pul（GT）与产酸普鲁兰芽孢杆菌（2 WAN）的普鲁兰酶相比，缺少三个结构域（CBM 41结构域、X25结构域和X45结构域）。构建了不同的N端结构域截短（730 T）或剪接（730 T-U1和730 T-U2）突变体。截短N-末端85个氨基酸降低了K-m值，并且不改变其最适pH，这在某些应用中是有利的生物化学性质。与2 WAN相比，Pul（GT）可直接用于玉米淀粉糖化，无需调节pH，降低了成本，提高了效率。
A novel thermostable type I pullulanase gene (pul(GT)) from Geobacillus thermocatenulatus DSMZ730 was cloned. It has an open reading frame of 2154 bp encoding 718 amino acids. G. thermocatenulatus pullulanase (Pul(GT)) was found to be optimally active at pH 6.5 and 70 degrees C. It exhibited stable activity in the pH range of 5.5-7.0. Pul(GT) lacked three domains (CBM41 domain, X25 domain, and X45 domain) compared with the pullulanase from Bacillus acidopullulyticus (2WAN). Different N-terminally domain truncated (730T) or spliced (730T-U1 and 730T-U2) mutants were constructed. Truncating the N-terminal 85 amino acids decreased the K-m value and did not change its optimum pH, an advantageous biochemical property in some applications. Compared with 2WAN, Pul(GT) can be used directly for maize starch saccharification without adjusting the pH, which reduces cost and improves efficiency.