Abstract
The gene xynB from Aspergillus sulphureus encoding the endo-β-1,4-xylanase was de novo synthesized by splicing overlap extension polymerase chain reaction according to Pichia pastoris protein’s codon bias. The synthetic DNA and wild-type DNA were placed under the control of a glyceraldehyde-3-phosphate dehydrogenase gene promoter (GAP) in the constitutive expression vector plasmid pGAPzαA and electrotransformed into the P. pastoris X-33 strain, respectively. The transformants screened by Zeocin were able to constitutively secrete the xylanase in YPD liquid medium. The maximum yield of the recombinant xylanase produced by the synthetic DNA was 105 U ml−1, which was about 5-fold higher than that by wild-type DNA under the flask culture at 28 °C for 3 days. The enzyme showed optimal activity at 50 °C and pH 5.0. The residual activity remained above 90% after the recombinant xylanase was pretreated in Na2HPO4–citric acid buffer (pH 2.4) for 2 h. The xylanase activity was significantly improved by Zn2+. These biochemical characteristics suggest that the recombinant xylanase has a prospective application in feed industry as an additive.
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Acknowledgements
This work was supported by the Program for New Century Excellent Talents in University (NCET-07-0807), the National High Technology Research and Development Program (2007AA100601), and the Project of State Key Laboratory of Animal Nutrition (2004DA125184 (team) 0806).
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Li, Y., Zhang, B., Chen, X. et al. Improvement of Aspergillus sulphureus Endo-β-1,4-Xylanase Expression in Pichia pastoris by Codon Optimization and Analysis of the Enzymic Characterization. Appl Biochem Biotechnol 160, 1321–1331 (2010). https://doi.org/10.1007/s12010-009-8621-0
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DOI: https://doi.org/10.1007/s12010-009-8621-0