Thermostable carbohydrate binding module increases the thermostability and substrate-binding capacity of Trichoderma reesei xylanase 2.
To improve the thermostability of Trichoderma reesei xylanase 2 (Xyn2), the thermostabilizing domain (A2) from Thermotoga maritima XynA were engineered into the N-terminal region of the Xyn2 protein. The xyn2 and hybrid genes were successfully expressed in Pichia pastoris using the strong methanol inducible alcohol oxidase 1 (AOX1) promoter and the secretion signal sequence from S. cerevisiae (alpha-factor). The transformants expressed the hybrid gene produced clearly increased both the thermostability and substrate-binding capacity compared to the corresponding strains expressed the native Xyn2 gene. The activity of the hybrid enzyme was highest at 65 degrees C that was 10 degrees C higher than the native Xyn2. The hybrid enzyme was stable at 60 degrees C and retained more than 85% of its activity after 30-min incubation at this temperature. The hybrid enzyme was highly specific toward xylan and analysis of the products from birchwood xylan degradation confirmed that the enzyme was an endo-xylanase with xylobiose and xylotriose as the main degradation products. These attributes should make it an attractive applicant for various applications. Our results also suggested that the N-terminal domain A2 is responsible for both the thermostability and substrate-binding capacity of T. maritima XynA.
Jun
He
H
Institute of Animal Nutrition, Sichuan Agricultural University, Yaan, Sichuan 625014, PR China.
Bing
Yu
Y
Keying
Zhang
Z
Xuemei
Ding
D
Daiwen
Chen
C
D002482:Cellulose / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability / Q000187:drug effects; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D006868:Hydrolysis / Q000187:drug effects; D010843:Pichia / Q000187:drug effects / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity / Q000187:drug effects; D013696:Temperature; D020124:Thermotoga maritima / Q000201:enzymology; D014242:Trichoderma / Q000187:drug effects / Q000201:enzymology*; D014990:Xylans / Q000494:pharmacology; D014995:Xylosidases / Q000378:metabolism*
D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D014990:Xylans; C055213:saccharide-binding proteins; D002482:Cellulose; D014995:Xylosidases