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

摘要

Acetyl xylan esterase gene (AXE) from Aspergillus ficuum was cloned and its Pichia expression plasmid, pPICZ alpha C-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 alpha-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 AXEaseactivity reached at 6.0 g-dry cell weight/l 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 significantlyincreased to about 97 g-dry cell weight/l and 930 unit/ml. Most of AXEase activity (>90 percent) was found in the extracellular medium and the majority of extracellular protein (>80 percent) was AXEase enzyme (33.5 kDa). This result means that about 9.8g/l of AXEase protein was produced in the extracellular medium.