Abstract

The xyn II cDNA encoding the Xylanase II (Xyn II) of Aspergillus usamii E001 was cloned into the pPIC9K vector and expressed in the methylotrophic yeast Pichia pastoris under the control of the alcohol oxidase I gene promoter and secreted via the α-mating factor leader of Saccharomyces cerevisiae. In shake-flask culture induced with methanol, the supernatant from a 96 h culture of Xyn II had specific activity of 1373.37 U/mg. The three-dimensional model and mutational analysis of the xyn II gene products showed that Glu 79 and Glu 170 were the important catalytic amino acid residues in the active site and Asp37 played a significant role in its low pH optimum. When Asp37 was mutated to asparagine, the optimum pH shifted to 5.3 and the maximum specific activity decreased to about 20% of that of the wild-type enzyme. This is the first report of functional expression and mutational analysis of A .usamii xylanase in P. pastoris.

Key words: Xylanase, Aspergillus usamii, expression; Pichia pastoris, mutational analysis.