Cloning, overexpression and characterization of a thermostable pullulanase from Thermus thermophilus HB27
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- 作者：Huawei Wu ; Xinxin Yu ; Libing Chen ; Guangxu Wu
- 关键词：Thermus thermophilus HB27 ; Pullulanase ; Soluble expression ; Purification ; pHsh expression vector
- 刊名：Protein Expression and Purification
- 出版年：March, 2014
- 年：2014
- 卷：95
- 期：Complete
- 页码：22-27
- 全文大小：1512 K
文摘
A gene encoding a special type of pullulanase from Thermus thermophilus HB27 (TTHpu) was cloned. It has an open reading frame of 1428 bp encoding a mature protein with a molecular mass of 52 kDa. 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 掳C. More than 90% activity was retained after incubation at 60-70 掳C for 2 h and the half-life was 2 h at 80 掳C. The stability of the enzyme was in a pH range from 6.0 to 8.0. Manganese at 5 mM enhanced its activity up to 298%. The Km and Vmax for the enzyme activity on pullulan were 0.0031 mg mL鈭? and 23.8 渭mol min鈭?, respectively. Unlike the most of pullulan-hydrolyzing enzymes described to date, this enzyme can attack 伪-1,6- and 伪-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.