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Gene Cloning, Functional Expression, and Characterization of a Novel GH46 Chitosanase from Streptomyces avermitilis (SaCsn46A)

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Abstract

A n ovel glycoside hydrolase (GH) family 46 chitosanase (SaCsn46A) from Streptomyces avermitilis was cloned and functionally expressed in Escherichia coli Rosetta (DE3) strains. SaCsn46A consists of 271 amino acids, which includes a 34-amino acid signal peptide. The protein sequence of SaCsn46A shows maximum identity (83.5%) to chitosanase from Streptomyces sp. SirexAA-E. Then, the mature enzyme was purified to homogeneity through Ni-chelating affinity chromatography with a recovery yield of 78% and the molecular mass of purified enzyme was estimated to be 29 kDa by SDS-PAGE. The recombinant enzyme possessed a temperature optimum of 45 °C and a pH optimum of 6.2, and it was stable at pH ranging from 4.0 to 9.0 and below 30 °C. The Km and Vmax values of this enzyme were 1.32 mg/mL, 526.32 U/mg/min, respectively (chitosan as substrate). The enzyme activity can be enhanced by Mg2+ and especially Mn2+, which could enhance the activity about 3.62-fold at a 3-mM concentration. The enzyme can hydrolyze a variety of polysaccharides which are linked by β-1,4-glycosidic bonds such as chitin, xylan, and cellulose, but it could not hydrolyze polysaccharides linked by α-1,4-glycosidic bonds. The results of thin-layer chromatography and HPLC showed that the enzyme exhibited an endo-type cleavage pattern and could hydrolyze chitosan to glucosamine (GlcN) and (GlcN)2. This study demonstrated that SaCsn46A is a promising enzyme to produce glucosamine and chitooligosaccharides (COS) from chitosan.

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Data Availability

All data generated or analyzed during this study are included in this published article and its supplementary information files.

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Funding

This work was supported by the National Natural Science Foundation of China (31700075); the Natural Science Foundation of the Jiangsu Higher Education Institution of China (19KJB180001); the Key Research and Development Program of Shandong Province, China (2019JZZY020605); the Initial Research Funding of Changzhou University (ZMF17020115); the Extracurricular Innovation and Entrepreneurship Fund for College Students of Changzhou University (ZMF19020280); the Natural Science Foundation of Jiangsu Province (BK20181465).

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Authors and Affiliations

  1. Advanced Catalysis and Green Manufacturing Collaborative Innovation Center, Changzhou University, Changzhou, China

    Jing Guo, Zaiwei Man & Zhiqiang Cai

  2. Laboratory of Applied Microbiology, School of Pharmaceutical, Changzhou University, Changzhou, China

    Jing Guo, Yi Wang, Wenjun Gao, Xinrou Wang, Xin Gao, Zhiqiang Cai & Qing Qing

  3. School of Petrochemical Engineering, School of Food Science and Technology, Changzhou University, Changzhou, China

    Zaiwei Man

  4. Zaozhuang Key Laboratory of Corn Bioengineering, Zaozhuang Science and Technology Collaborative Innovation Center of Enzyme, Shandong Hengren Gongmao Co. Ltd, Zaozhuang, China

    Zaiwei Man

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  1. Jing Guo
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  2. Yi Wang
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  3. Wenjun Gao
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  4. Xinrou Wang
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  5. Xin Gao
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  7. Zhiqiang Cai
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  8. Qing Qing
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Contributions

Conceptualization: J. Guo, Z.W. Man. Methodology: J. Guo, W.J. Gao. Data analysis: J. Guo, Y. Wang. Software: J. Guo. Writing original manuscript: J. Guo. Review and revising manuscript: Y. Wang, W.J. Gao., X.R. Wang, X. Gao, Z.W. Man, Z.Q. Cai, Q. Qing. Funding acquisition: J. Guo, Z.W. Man, Z.Q. Cai, Q. Qing. All authors reviewed and approved the final manuscript.

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Correspondence to Jing Guo, Zaiwei Man, Zhiqiang Cai or Qing Qing.

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Guo, J., Wang, Y., Gao, W. et al. Gene Cloning, Functional Expression, and Characterization of a Novel GH46 Chitosanase from Streptomyces avermitilis (SaCsn46A). Appl Biochem Biotechnol 194, 813–826 (2022). https://doi.org/10.1007/s12010-021-03687-6

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