Wu Huawei, Yu Xinxin, Chen Libing, Wu Guangxu
College of Life Sciences, Yangtze University, Jingzhou 434025, Hubei, China.
College of Life Sciences, Yangtze University, Jingzhou 434025, Hubei, China.
Protein Expr Purif. 2014 Mar;95:22-7. doi: 10.1016/j.pep.2013.11.010. Epub 2013 Dec 4.
A gene encoding a special type of pullulanase from Thermus thermophilus HB27 (TTHpu) was cloned. It has an open reading frame of 1428bp encoding a mature protein with a molecular mass of 52kDa. The gene was expressed in Escherichia coli using pHsh and pET28a vectors. The pHsh expression system produced a 3.6-fold higher recombinant pullulanase than pET28a. The recombinant TTHpu was purified to homogeneity by heat treatment and Ni-NTA affinity chromatography. The purified TTHpu exhibited highest activity at pH 6.5 and 70°C. More than 90% activity was retained after incubation at 60-70°C for 2h and the half-life was 2h at 80°C. The stability of the enzyme was in a pH range from 6.0 to 8.0. Manganese at 5mM enhanced its activity up to 298%. The Km and Vmax for the enzyme activity on pullulan were 0.0031mgmL(-1) and 23.8μmolmin(-1), respectively. Unlike the most of pullulan-hydrolyzing enzymes described to date, this enzyme can attack α-1,6- and α-1,4-glycosidic linkages in pullulan, and produce a mixture of maltotriose, maltose and glucose. The enzyme could be further employed for industrial saccharification of starch.
克隆了来自嗜热栖热菌HB27的一种特殊支链淀粉酶（TTHpu）的编码基因。它有一个1428bp的开放阅读框，编码一种分子量为52kDa的成熟蛋白。该基因利用pHsh和pET28a载体在大肠杆菌中表达。pHsh表达系统产生的重组支链淀粉酶比pET28a高3.6倍。重组TTHpu通过热处理和Ni-NTA亲和层析纯化至同质。纯化后的TTHpu在pH 6.5和70°C时表现出最高活性。在60 - 70°C孵育2小时后，保留了超过90%的活性，在80°C时半衰期为2小时。该酶在pH 6.0至8.0范围内稳定。5mM的锰可使其活性提高至298%。该酶对支链淀粉活性的Km和Vmax分别为0.0031mgmL(-1)和23.8μmolmin(-1)。与迄今为止描述的大多数支链淀粉水解酶不同，这种酶可以作用于支链淀粉中的α-1,6-和α-1,4-糖苷键，并产生麦芽三糖、麦芽糖和葡萄糖的混合物。该酶可进一步用于淀粉的工业糖化。
