High-level expression of Aspergillus ficuum acetyl xylan esterase gene in Pichia pastoris

Acetyl xylan esterase gene (AXE) from Aspergillus ficuum was cloned and its Pichia expression plasmid, pPICZαC-AXE (4.6 kb), was constructed, in which the AXE gene was under the control of the AOX1 promoter and connected downstream of mating factor α-1 signal sequence. The plasmid linearized by SacI was integrated into the 5'AOX1 region of the chromosomal DNA of P pastoris. In the flask batch culture of P. pastoris transformant on methanol medium, the cell concentration and total AXEase activity reached at 6.0 g-dry cell weight/1 and 77 unit/ml after 36 h cultivation, respectively. In the fed-batch culture employing the optimized methanol and histidine feeding strategy, the cell concentration and total AXEase activity were significantly increased to about 97 g-dry cell weight/l and 930 unit/ml. Most of AXEase activity (>90%) was found in the extracellular medium and the majority of extracellular protein (>80%) was AXEase enzyme (33.5 kDa). This result means that about 9.8 g/l of AXEase protein was produced in the extracellular medium.