Cloning and expression of a novel xylanase gene (Auxyn11D) from Aspergillus usamii E001 in Pichia pastoris

摘要

A full-length complementary DNA (cDNA) of Auxyn11D, a gene that encodes a novel endo-β-1,4-D-xylanase of Aspergillus usamii E001 (abbreviated to AuXyn11D), was obtained using 3'- and 5'-rapid amplification of cDNA ends (RACE) methods. The cDNA sequence is 855 bp in length, containing 5'- and 3'-untranslated regions and a 696-bp open reading frame (ORF) that encodes a 32-aa signal peptide and a 199-aa mature peptide (namely AuXyn11D). Multiple homology alignment of amino acid sequences verified that AuXyn11D belongs to glycoside hydrolase family 11. Moreover, a mature peptide-encoding cDNA fragment of Auxyn11D was cloned and expressed in Pichia pastoris GS115. One P. pastoris transformant expressing the highest recombinant AuXyn11D (reAuXyn11D) activity of 15.0 U/mL, labeled as P. pastoris GSAuXyn4-16, was chosen by shake flask test. SDS-PAGE assay demonstrated that the reAuXyn11D, a glycosylated protein with an apparent molecular mass of 32.0 kDa, was secreted into the medium. The purified reAuXyn11D displayed the highest activity at pH 4.5 and 55 °C. It was stable at a pH range of 3.5-6.5 and at a temperature of 50 °C or below. Its activity was not significantly affected by most of metal ions tested and EDTA, but increased by Ca ~(2+) and inhibited by Mn ~(2+). The Km and Vmax of the reAuXyn11D towards birchwood xylan were 6.32 mg/mL and 391.6 U/mg, respectively.