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Heterologous expression of β-xylosidase gene from Paecilomyces thermophila in Pichia pastoris

Juturu, Veeresh, Wu, Jin Chuan
World journal of microbiology & biotechnology 2013 v.29 no.2 pp. 249-255
Paecilomyces, Pichia pastoris, alcohols, alpha-N-arabinofuranosidase, amino acid composition, amino acid sequences, genes, glycosylation, hydrolysis, molecular weight, pH, polyacrylamide gel electrophoresis, post-translational modification, thermophilic fungi
beta-xylosidase from thermophilic fungi Paecilomyces thermophila was functionally expressed in Pichia pastoris with a his tag in the C-terminal under the alcohol oxidase 1 (AOX1) promoter and secreted into the medium at 0.22 mg l(-1). Its molecular mass was estimated to be 52.3 kDa based on the SDS-PAGE analysis, which is 1.3 times higher than the predicted 39.31 kDa from its amino acid compositions, although no potential N- or O- glycosylation sites were predicted from its amino acid sequence. This is presumed to be caused by some unpredictable posttranslational modifications based on mass spectrum analysis of the recombinant protein. The enzyme was most active at 60 A degrees C and pH 7. It showed not only a beta-xylosidase activity with a K-m of 8 mM and a V-max of 54 mu mol min(-1) mg(-1) for hydrolysis of p-nitrophenyl beta-d-xylopyranoside but also an arabinofuranosidase activity (6.2 U mg(-1)) on p-nitrophenyl arabinofuranoside.