Abstract
Chitooligosaccharides have important application value in the fields of food and agriculture. Chitosanase can degrade chitosan to obtain chitooligosaccharides. The marine metagenome contains many genes related to the degradation of chitosan. However, it is difficult to mine valuable genes from large gene resources. This study proposes a method to screen chitosanases directly from the marine metagenome. Chitosanase gene chis1754 was identified from the metagenome and heterologously expressed in Escherichia coli. The optimal temperature and pH of CHIS1754 were 55 °C and 5.5, respectively. A mutant, CHIS1754T, with 15 single point mutations designed based on molecular evolution data was also expressed in E. coli. The results indicated that the thermal stability of CHIS1754T was significantly improved, as the Tm showed an increase of ~ 7.63 °C. Additionally, the kcat/Km of CHIS1754T was 4.8-fold higher than that of the wild type. This research provides new theories and foundations for the excavation, modification, and industrial application of chitosanases.
Key points
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A chitosanase gene, chis1754, was firstly identified from marine metagenome.
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A multi-site mutant was designed to improve enzyme stability and activity.
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The kcat/Kmof the designed mutant was 4.8-fold higher than that of the wild type.
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Acknowledgments
We thank Dr. Lida Han in Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, for helping us to conduct the LC–QTOF-MS experiments, Prof. Yuhong Zhang for the TLC, and Prof. Bin Yao for Tm determination.
Funding
This study was funded by the Fundamental Research Funds for Central Non-profit Scientific Institution (Grant no. Y2019XK01), Central Public-Interest Scientific Institution Basal Research Fund (Grant no. Y2019XK19), and Freshwater Aquaculture Innovation Team Construction Project of Hebei Modern Agricultural Industry Technology System (HBCT2018180206).
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Jian Tian and Ningfeng Wu conceived and designed the research. Yanshuo Han and Feifei Guan conducted experiments. Jilu Sun contributed new reagents or analytical tools. Jian Tian and Feifei Guan analyzed data. Feifei Guan and Jian Tian wrote the manuscript. All authors read and approved the manuscript.
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Han, Y., Guan, F., Sun, J. et al. Identification of a chitosanase from the marine metagenome and its molecular improvement based on evolution data. Appl Microbiol Biotechnol 104, 6647–6657 (2020). https://doi.org/10.1007/s00253-020-10715-8
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DOI: https://doi.org/10.1007/s00253-020-10715-8