Expression of a family 10 xylanase gene fromAspergillus usamiiE001 inPichia pastorisand characterization of the recombinant enzyme

• DOI: 10.1007/s10295-012-1201-2
• Publication Date: 2012-10-04T06:51:23Z
• Authors: Jun-Qing Wang; Xin Yin; Min-Chen Wu; Hui-Min Zhang; Shu-Juan Gao; Jing-Tu Wei; Cun-Duo Tang; Jian-Fang Li
• Keywords: Xylobiose; Pichia; Heterologous expression

AbstractA cDNA gene (Auxyn10A), which encodes a mesophilic family 10 xylanase from Aspergillus usamii E001 (abbreviated to AuXyn10A), was amplified and inserted into the XhoI and NotI sites of pPIC9KM vector constructed from a parent pPIC9K. The recombinant expression vector, designated pPIC9KM-Auxyn10A, was transformed into Pichia pastoris GS115. All P. pastoris transformants were spread on a MD plate, and then inoculated on geneticin G418-containing YPD plates for screening multiple copies of integration of the Auxyn10A. One transformant expressing the highest recombinant AuXyn10A (reAuXyn10A) activity of 368.6 U/ml, numbered as P. pastoris GSX10A4-14, was selected by flask expression test. SDS-PAGE assay demonstrated that the reAuXyn10A was extracellularly expressed with an apparent M.W. of 39.8 kDa. The purified reAuXyn10A displayed the maximum activity at pH 5.5 and 50 °C. It was highly stable at a broad pH range of 4.5–8.5, and at a temperature of 45 °C. Its activity was not significantly affected by EDTA and several metal ions except Mn2+, which caused a strong inhibition. The K m and V max, towards birchwood xylan at pH 5.5 and 50 °C, were 2.25 mg/ml and 6,267 U/mg, respectively. TLC analysis verified that the AuXyn10A is an endo-β-1,4-d-xylanase, which yielded a major product of xylotriose and a small amount of xylose, xylotetraose, and xylopentose from birchwood xylan, but no xylobiose.