Abstract
XynCDBFV from Neocallimastix patriciarum is among the most effective xylanases and holds great potentials in a wide variety of industrial applications. In the present study, several active site residues were modified referring to the instrumental information of the complex structure of XynCDBFV and xylooligosaccharides. Among the 12 single active site mutants, W125F and F163W show increased activity comparing to the wild-type protein. The double mutant W125F/F163W was then generated which displayed nearly 20 % increase in the enzyme activity. Although W125F/F163W showed 5 °C reduction in the optimal temperature, it still preserves similar thermostability and is more active than the wild-type enzyme at temperatures lower than 60 °C. These properties make the double mutant a suitable candidate for commercial applications that involve lower operating temperatures. Furthermore, we investigated the effect of N-glycosylation on the thermostability of XynCDBFV when expressed in the yeast strain Pichia pastoris for industrial use. Two potential glycosylation sites (Asn-37 and Asn-88) were examined, and their roles in enzyme performance were validated. We found that the N-glycosylations of XynCDBFV are related to both catalytic activity and heat stability, with Asn-37 motif playing a dominant role. Collectively, the enzymatic properties of XynCDBFV were improved by molecular engineering, and the influences of N-glycosylations on the enzyme have been clearly elucidated herein.
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Acknowledgments
This work was supported by grants from the National Basic Research Program of China (2011CB710800 and 2011CBA00805), the National High Technology Research and Development Program of China (2012AA022200), the National Natural Science Foundation of China (31200053 and 31300615), and the Chinese Academy of Sciences (KSZD-EW-Z-015–2).
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The authors declare that they have no competing interests.
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Ya-Shan Cheng and Chun-Chi Chen contributed equally to this work.
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Cheng, YS., Chen, CC., Huang, JW. et al. Improving the catalytic performance of a GH11 xylanase by rational protein engineering. Appl Microbiol Biotechnol 99, 9503–9510 (2015). https://doi.org/10.1007/s00253-015-6712-0
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DOI: https://doi.org/10.1007/s00253-015-6712-0