Modified pPIC9K vector-mediated expression of a family 11 xylanase gene, Aoxyn11A, from Aspergillus oryzae in Pichia pastoris

A full-length cDNA sequence of Aoxyn11A, a mesophilic xylanase-encoding gene from Aspergillus oryzae, was obtained from total RNA, using 3′ and 5′ rapid amplification of cDNA ends methods. The cDNA sequence is 1,086 base pairs in length, containing 5′-untranslated and 3′-untranslated regions and an open reading frame encoding a 20 amino acid (aa) signal peptide, a 24 aa propeptide and a 188 aa mature peptide (designated AoXyn11A). Multiple alignments verified that AoXyn11A belongs to glycoside hydrolase family 11. Its three-dimensional structure was predicted by multiple templates–based homology modeling. In addition, an AoXyn11A-encoding cDNA gene was extracellularly expressed in Pichia pastoris GS115, mediated by the modified pPIC9K vector. One P. pastoris transformant, numbered as GSAorX4-3 and having the highest recombinant AoXyn11A (reAoXyn11A) activity of 98.0 U/ml, was chosen. The reAoXyn11A showed maximum activity at pH 5.5 and 50 °C. It was highly stable at a pH range of 4.0–8.0 and at 40 °C. Its activity was not significantly affected by metal ions that were tested or EDTA, but was strongly inhibited by Mn²⁺ and Ag⁺. The K _m and V _max of the reAoXyn11A were 1.85 mg/ml and 3,018 U/mg, respectively.