Abstract
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.
Highlights
The chemicals used for paper bleaching process resulted in the release of large amounts of chlorinated organic compounds that are known to have toxic, mutagenic and carcinogenic effects, causing serious environmental pollution
Developing a cost-efficient bleaching process with xylanase represents a positive benefit to paper industry, in which the low-cost alkaline and thermo-stable xylanase is a key issue
Xylanase was effective in alkaline condition with 90% activity and its activity in the supernatant of the recombinant P. pastoris expressing xynHB was 644 U/ml[30]
Summary
DNA shuffling and error prone PCR were applied to engineer recombinant xylanase with improved thermo-stability[18,19,20] These findings prompted the engineering of xylanase for large scale industrial application in paper industry, but further work is needed to develop a highly thermo-stable xylanase having better enzyme activity in alkaline condition. Several studies have reported the role of different host strains produce high level of exogenous xylanase, like Bacillus, Trichoderma reesei, Pichia pastoris, Saccharomyces cerevisiae and Escherichia coli[23,24,25,26,27]. A site mutagenesis was performed to improve thermo-stability of recombinant XynHB as well as better protein expression level in P. pastoris by gene codon usage optimization and multiple gene insertion
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