Expression of endo-1, 4-beta-xylanase from Trichoderma reesei in Pichia pastoris and functional characterization of the produced enzyme.
BACKGROUND
In recent years, xylanases have attracted considerable research interest because of their potential in various industrial applications. The yeast Pichia pastoris can neither utilize nor degrade xylan, but it possesses many attributes that render it an attractive host for the expression and production of industrial enzymes.


RESULTS
The Xyn2 gene, which encodes the main Trichoderma reesei Rut C-30 endo-beta-1, 4-xylanase was cloned into the pPICZalphaA vector and expressed in Pichia pastoris. The selected P. pastoris strains produced as 4,350 nkat/ml beta-xylanase under the control of the methanol inducible alcohol oxidase 1 (AOX1) promoter. The secreted recombinant Xyn2 was estimated by SDS-PAGE to be 21 kDa. The activity of the recombinant Xyn2 was highest at 60 degrees C and it was active over a broad range of pH (3.0-8.0) with maximal activity at pH 6.0. The enzyme was quite stable at 50 degrees C and retained more than 94% of its activity after 30 mins incubation at this temperature. Using Birchwood xylan, the determined apparent Km and kcat values were 2.1 mg/ml and 219.2 S-1, respectively. The enzyme was highly specific towards xylan and analysis of xylan hydrolysis products confirmed as expected that the enzyme functions as endo-xylanase with xylotriose as the main hydrolysis products. The produced xylanase was practically free of cellulolytic activity.


CONCLUSIONS
The P. pastoris expression system allows a high level expression of xylanases. Xylanase was the main protein species in the culture supernatant, and the functional tests indicated that even the non-purified enzyme shows highly specific xylanase activity that is free of cellulolytic side acitivities. Therefore, P pastoris is a very useful expression system when the goal is highly specific and large scale production of glycosyl hydrolases.
He
Jun
J
Institute of Animal Nutrition, Sichuan Agricultural University, Ya'an, Sichuan 625014, PR China. hejun8067@163.com
Yu
Bing
B
Zhang
Keying
K
Ding
Xuemei
X
Chen
Daiwen
D
D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D013379:Substrate Specificity; D013696:Temperature; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics; D043325:Xylan Endo-1,3-beta-Xylosidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D005656:Fungal Proteins; D011994:Recombinant Proteins; D043325:Xylan Endo-1,3-beta-Xylosidase