Cloning, overexpression and characterization of a thermostable pullulanase from Thermus thermophilus HB27.
Protein Expr Purif
; 95: 22-7, 2014 Mar.
Article
em En
| MEDLINE
| ID: mdl-24316447
ABSTRACT
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.
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Proteínas de Bactérias
/
Proteínas Recombinantes
/
Thermus thermophilus
/
Glicosídeo Hidrolases
Idioma:
En
Ano de publicação:
2014
Tipo de documento:
Article