Acidophilic Xylanase from Aureobasidiumpullulans'.Efficient Expression and Secretion in Pichia pastoris and Mutational Analysis

摘要

A yeast-like fungus Aureobasidium pullulans var.melanigenum strain ATCC 20524 produces an extracellular acidophilic endo-l,4-beta-xylanase with an optimum pH of 2.0 Ohta et al.,J.Biosci.Bioeng.,92,262-270(2001).The xynl cDNA encoding the precursor protein(Xynl)was expressed in the methylotrophic yeast Pichia pastoris under the control of the alcohol oxidase I gene promoter.The 34 amino acid prepro-signal peptide of the A.pullulans Xynl directed the efficient secretion of 178 mg of active xylanase per liter of the culture medium.The secretion level of the xylanase with its own signal peptide was comparable to that of the mature protein fused to the prepro leader from Saccharomyces cerevisiae a-mating factor and twofold higher than that of the mature protein fused to the pre-type signal peptide from P.pastoris acid phosphatase.The N-ter-minal amino acid sequence and the apparent M_r of 24 kDa of the secreted recombinant protein indicated the native-like processing of the A.pullulans Xynl signal sequence in P.pastoris.The three-dimensional model and mutational analysis of the xynl gene product showed that Asp-73 and Glu-157 residues located at the upper and lower edges of the active site cleft,respectively,play a significant role in its low pH optimum.