High-level expression of improved thermo-stable alkaline xylanase variant in Pichia Pastoris through codon optimization, multiple gene insertion and high-density fermentation.

In paper industry, xylanases are used to increase the pulp properties in bleaching process as its eco-friendly nature. The xylanases activity is hindered by high temperature and alkaline conditions with high enzyme production cost in the paper industry. Here, XynHB, an alkaline stable xylanase from Bacillus pumilus HBP8 was mutated at N188A to XynHBN188A. Expressed mutant in E. coli showed 1.5-fold higher xylanase activity than XynHB at 60 °C. The mutant expressed in Pichia pastoris was glycosylated, remained stable for 30 min at 60 °C. XynHBN188A optimized based on codon usage bias for P. pastoris (xynHBN188As) showed an increase of 39.5% enzyme activity. The strain Y16 forming the largest hydrolysis halo in the xylan plate was used in shake flask experiments produced an enzyme activity of 6,403 U/ml. The Y16 strain had 9 copies of the recombinant xynHBN188As gene in the genome revealed by qPCR. The enzymatic activity increased to 48,241 U/ml in a 5 L fermentor. Supplement of 15 U/g xylanase enhanced the brightness of paper products by 2% in bleaching experiment, and thereby improved the tensile strength and burst factor by 13% and 6.5%, respectively. XynHBN188As has a great potential in paper industries.