Cloning of a gene encoding an acidophilic endo-beta-1,4-xylanase obtained from Aspergillus niger CGMCC1067 and constitutive expression in Pichia pastoris

摘要

The gene xynB encoding an acidophilic,endo-beta-1,4-xylanase,obtained from Aspergillus niger CGMCC1067 was cloned,and then successfully expressed in Pichia pastoris under the control of the GAP promoter.The full-length gene contained 745 bp,including an intron of 67 bp,and encoded a mature protein containing 188 amino acids.The purified,recombinant xylanase showed three bands at about 21,30 and 35 kDa.The maximum yield of the recombinant xylanase was 62 IU/ml,which was about 50-fold higher than that obtained when A.niger was cultured.No cellulase or beta-D-xylosidase activity was found.The optimal temperature for the recombinant xylanase enzyme was 50 deg C.The enzyme displayed about 95% of peak activity in the temperature range found in the body of animals to which the enzyme might be fed.The optimal pH for the recombinant xylanase was about pH 5.0 and the enzyme retained about 76% of its activity after being incubated at pH 2.0 for 30 min at 37 deg C.The enzyme possessed high resistance to various metal ions and chemical reagents,with the exception of copper and iron.These enzyme properties suggest that this enzyme could be used as a feed additive for animals.