Abstract
Thermostable alkaline pectate lyases can be potentially used for enzymatically degumming ramie in an environmentally sustainable manner and as an alternative to the currently used chemical-based ramie degumming processes. To assess its potential applications, pectate lyase from Bacillus pumilus (ATCC 7061) was cloned and expressed in Escherichia coli. Evolutionary strategies were applied to generate efficient ramie degumming enzymes. Obtained from site-saturation mutagenesis and random mutagenesis, the best performing mutant enzyme M3 exhibited a 3.4-fold higher specific activity on substrate polygalacturonic acid, compared with the wild-type enzyme. Furthermore, the half-life of inactivation at 50 °C for M3 mutant extended to over 13 h. In contrast, the wild-type enzyme was completely inactivated in less than 10 min under the same conditions. An upward shift in the optimal reaction temperature of M3 mutant, to 75 °C, was observed, which was 10 °C higher than that of the wild-type enzyme. Kinetic parameter data revealed that the catalysis efficiency of M3 mutant was higher than that of the wild-type enzyme. Ramie degumming with M3 mutant was also demonstrated to be more efficient than that with the wild-type enzyme. Collectively, our results suggest that the M3 mutant, with remarkable improvements in thermoactivity and thermostability, has potential applications for ramie degumming in the textile industry.
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Acknowledgments
This work was supported by the Key Research Program of the Chinese Academy of Sciences Grant KSZD-EW-Z-015-2, Ministry of Science and Technology of China Grant 2013CB734003, National Natural Science Foundation of China Grant 21172095, and Grant SKLMR-20120603 supported by the State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences. We would also like to thank Mr. Ye Mao for his assistance in manuscript preparations.
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Chaoning Liang and Xiwu Gui contributed equally to this work.
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Liang, C., Gui, X., Zhou, C. et al. Improving the thermoactivity and thermostability of pectate lyase from Bacillus pumilus for ramie degumming. Appl Microbiol Biotechnol 99, 2673–2682 (2015). https://doi.org/10.1007/s00253-014-6091-y
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DOI: https://doi.org/10.1007/s00253-014-6091-y