Skip to main content
SpringerLink
Log in

Enhancing Enzyme Activity and Thermostability of Bacillus amyloliquefaciens Chitosanase BaCsn46A Through Saturation Mutagenesis at Ser196

  • Published:
Current Microbiology Aims and scope Submit manuscript

Abstract

Chitosanase plays an important role in chitooligosaccharides (COS) production. We found that the chitosanase (BaCsn46A) of Bacillus amyloliquefacien was a good candidate for chitosan hydrolysis of COS. In order to further improve the enzyme properties of BaCsn46A, the S196 located near the active center was found to be a critical site impacts on enzyme properties by sequence alignment analysis. Herein, saturation mutation was carried out to study role of 196 site on BaCsn46A catalytic function. Compared with WT, the specific enzyme activity of S196A increased by 118.79%, and the thermostability of S196A was much higher than WT. In addition, we found that the enzyme activity of S196P was 2.41% of that of WT, indicating that the type of amino acid in 196 site could significant affect the catalytic activity and thermostability of BaCsn46A. After molecular docking analysis we found that the increase in hydrogen bonds and decrease in unfavorable bonds interacting with the substrate were the main reason for the change of enzyme properties which is valuable for future studies on Bacillus species chitosanase.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Cheng C-Y, Chang C-H, Wu Y-J, Li Y-K (2006) Exploration of Glycosyl Hydrolase Family 75, a Chitosanase from Aspergillus fumigatus. J Biol Chem. https://doi.org/10.1074/jbc.M512506200

    Article  PubMed  Google Scholar 

  2. Hoell IA, Vaaje-Kolstad G, Eijsink VGH (2010) Structure and function of enzymes acting on chitin and chitosan. Biotechnol Genetic Eng Rev. https://doi.org/10.1080/02648725.2010.10648156

    Article  Google Scholar 

  3. Shimosaka M, Sato K, Nishiwaki N, Miyazawa T, Okazaki M (2005) Analysis of essential carboxylic amino acid residues for catalytic activity of fungal chitosanases by site-directed mutagenesis. J Biosci Bioeng 100:545–550. https://doi.org/10.1263/jbb.100.545

    Article  CAS  PubMed  Google Scholar 

  4. Kang BR, Song Y-S, Jung W-J (2021) Differential expression of bio-active metabolites produced by chitosan polymers-based Bacillus amyloliquefaciens fermentation. Carbohydr Polym 260:117799. https://doi.org/10.1016/j.carbpol.2021.117799

    Article  CAS  PubMed  Google Scholar 

  5. Guo N, Sun J, Wang W, Gao L, Liu J, Liu Z, Xue C, Mao X (2019) Cloning, expression and characterization of a novel chitosanase from Streptomyces albolongus ATCC 27414. Food Chem 286:696–702. https://doi.org/10.1016/j.foodchem.2019.02.056

    Article  CAS  PubMed  Google Scholar 

  6. Cahyaningtyas HAA, Suyotha W, Cheirsilp B, Yano S (2021) Statistical optimization of halophilic chitosanase and protease production by Bacillus cereus HMRSC30 isolated from Terasi simultaneous with chitin extraction from shrimp shell waste. Biocatal Agri Biotechnol 31:101918. https://doi.org/10.1016/j.bcab.2021.101918

    Article  CAS  Google Scholar 

  7. Pal P, Pal A, Nakashima K, Yadav BK (2021) Applications of chitosan in environmental remediation: a review. Chemosphere 266:128934. https://doi.org/10.1016/j.chemosphere.2020.128934

    Article  CAS  PubMed  Google Scholar 

  8. Jafari H, Bernaerts KV, Dodi G, Shavandi A (2020) Chitooligosaccharides for wound healing biomaterials engineering. Mater Sci Eng: C 117:111266. https://doi.org/10.1016/j.msec.2020.111266

    Article  CAS  Google Scholar 

  9. Pei MJ, Mao J, Xu WL, Zhou YS, Xiao P (2019) Photocrosslinkable chitosan hydrogels and their biomedical applications. J Polym Sci Part A-Polym Chem 57:1862–1871. https://doi.org/10.1002/pola.29305

    Article  CAS  Google Scholar 

  10. Upadhyay U, Sreedhar I, Singh SA, Patel CM, Anitha KL (2021) Recent advances in heavy metal removal by chitosan based adsorbents. Carbohydr Polym 251:117000. https://doi.org/10.1016/j.carbpol.2020.117000

    Article  CAS  PubMed  Google Scholar 

  11. Ando A, Saito A, Arai S, Usuda S, Furuno M, Kaneko N, Shida O, Nagata Y (2008) Molecular Characterization of a Novel Family-46 Chitosanase from Pseudomonas sp. A-01. Biosci Biotechnol Biochem 72:2074–2081. https://doi.org/10.1271/bbb.80175

    Article  CAS  PubMed  Google Scholar 

  12. Park Jae K, Shimono K, Ochiai N, Shigeru K, Kurita M, Ohta Y, Tanaka K, Matsuda H, Kawamukai M (1999) Purification, Characterization, and Gene Analysis of a Chitosanase (ChoA) from Matsuebacter chitosanotabidus 3001. J Bacteriol 181:6642–6649. https://doi.org/10.1128/JB.181.21.6642-6649.1999

    Article  PubMed Central  Google Scholar 

  13. Ding M, Zhang T, Sun C, Zhang H, Zhang Y (2020) A Chitosanase mutant from Streptomyces sp. N174 prefers to produce functional chitopentasaccharide. Int J Biol Macromol 151:1091–1098. https://doi.org/10.1016/j.ijbiomac.2019.10.151

    Article  CAS  PubMed  Google Scholar 

  14. Nguyen AD, Huang C-C, Liang T-W, Nguyen VB, Pan P-S, Wang S-L (2014) Production and purification of a fungal chitosanase and chitooligomers from Penicillium janthinellum D4 and discovery of the enzyme activators. Carbohydr Polym 108:331–337. https://doi.org/10.1016/j.carbpol.2014.02.053

    Article  CAS  PubMed  Google Scholar 

  15. Shehata AN, Aty A (2015) Improved production and partial characterization of chitosanase from a newly isolated Chaetomium globosum KM651986 and its application for chitosan oligosaccharides. J Chem Pharm Res 7:727–740

    CAS  Google Scholar 

  16. Yan Q, Hua C, Yang S, Li Y, Jiang Z (2012) High level expression of extracellular secretion of a β-glucosidase gene (PtBglu3) from Paecilomyces thermophila in Pichia pastoris. Protein Expr Purif 84:64–72. https://doi.org/10.1016/j.pep.2012.04.016

    Article  CAS  PubMed  Google Scholar 

  17. Banerjee S, Salunkhe SS, Apte-Deshpande AD, Mandi NS, Mandal G, Padmanabhan S (2009) Over-expression of proteins using a modified pBAD24 vector in E. coli expression system. Biotechnol Lett 31:1031–1036. https://doi.org/10.1007/s10529-009-9976-6

    Article  CAS  PubMed  Google Scholar 

  18. Cheng F, Xu J-M, Xiang C, Liu Z-Q, Zhao L-Q, Zheng Y-G (2017) Simple-MSSM: a simple and efficient method for simultaneous multi-site saturation mutagenesis. Biotechnol Lett 39:567–575. https://doi.org/10.1007/s10529-016-2278-x

    Article  CAS  PubMed  Google Scholar 

  19. Zoller MJ, Smith M (1982) Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA. Nucleic Acids Res 10:6487–6500. https://doi.org/10.1093/nar/10.20.6487

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Kegler-Ebo DM, Polack GW, Dimaio D (1996) Use of codon cassette mutagenesis for saturation mutagenesis. Methods Mol Biol 57:297

    CAS  PubMed  Google Scholar 

  21. Chiang LW, Kovari I, Howe MM (1993) Mutagenic oligonucleotide-directed PCR amplification (Mod-PCR): an efficient method for generating random base substitution mutations in a DNA sequence element. PCR Methods Appl 2:210

    Article  CAS  PubMed  Google Scholar 

  22. Geddie ML, Matsumura I (2004) Rapid Evolution of β-Glucuronidase Specificity by Saturation Mutagenesis of an Active Site Loop*. J Biol Chem 279:26462–26468. https://doi.org/10.1074/jbc.M401447200

    Article  CAS  PubMed  Google Scholar 

  23. Waterhouse A, Bertoni M, Bienert S, Studer G, Tauriello G, Gumienny R, Heer FT, deBeer TAP, Rempfer C, Bordoli L et al (2018) SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res 46:W296–W303. https://doi.org/10.1093/nar/gky427

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Li Y, Gou Y, Liu Z, Xie T, Wang G (2021) Structure-based rational design of chitosanase CsnMY002 for high yields of chitobiose. Colloids Sur Biointerfaces 202:111692. https://doi.org/10.1016/j.colsurfb.2021.111692

    Article  CAS  Google Scholar 

  25. O’Donoghue SI, Goodsell DS, Frangakis AS, Jossinet F, Laskowski RA, Nilges M, Saibil HR, Schafferhans A, Wade RC, Westhof E, Olson AJ (2010) Visualization of macromolecular structures. Nat Methods. https://doi.org/10.1038/nmeth.1427

    Article  PubMed  PubMed Central  Google Scholar 

  26. Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, Olson AJ (2009) AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J Comput Chem. https://doi.org/10.1002/jcc.21256

    Article  PubMed  PubMed Central  Google Scholar 

  27. Zhang X-F, Ai Y-H, Xu Y, Yu X-W (2019) High-level expression of Aspergillus niger lipase in Pichia pastoris: characterization and gastric digestion in vitro. Food Chem 274:305–313. https://doi.org/10.1016/j.foodchem.2018.09.020

    Article  CAS  PubMed  Google Scholar 

  28. Lyu Q, Wang S, Xu W, Han B, Liu W, Jones David NM, Liu W (2014) Structural insights into the substrate-binding mechanism for a novel chitosanase. Biochem J 461:335–345. https://doi.org/10.1042/BJ20140159

    Article  CAS  PubMed  Google Scholar 

  29. Boucher I, Fukamizo T, Honda Y, Willick GE, Neugebauer WA, Brzezinski R (1995) Site-directed mutagenesis of evolutionary conserved carboxylic amino acids in the chitosanase from Streptomyces sp. N174 reveals two residues essential for catalysis (∗). J Biolo Chem 270:31077–31082. https://doi.org/10.1074/jbc.270.52.31077

    Article  CAS  Google Scholar 

  30. Thadathil N, Velappan SP (2014) Recent developments in chitosanase research and its biotechnological applications: A review. Food Chem 150:392–399. https://doi.org/10.1016/j.foodchem.2013.10.083

    Article  CAS  PubMed  Google Scholar 

  31. Goo BG, Park JK (2014) Characterization of an alkalophilic extracellular chitosanase from Bacillus cereus GU-02. J Biosc Bioeng 117:684–689. https://doi.org/10.1016/j.jbiosc.2013.11.005

    Article  CAS  Google Scholar 

  32. Kulandaisamy R, Kushwaha T, Kumar V, De S, Kumar S, Upadhyay SK, Kumar M, Inampudi KK (2020) Characterization of active/binding site residues of peptidyl-tRNA hydrolase using biophysical and computational studies. Int J Biol Macromol 159:877–885. https://doi.org/10.1016/j.ijbiomac.2020.05.133

    Article  CAS  PubMed  Google Scholar 

  33. Han Y, Yu R, Gao P, Lu X, Yu W (2018) The hydrogen-bond network around Glu160 contributes to the structural stability of chitosanase CsnA from Renibacterium sp. QD1. Int J Biol Macromol 109:880–887. https://doi.org/10.1016/j.ijbiomac.2017.11.071

    Article  CAS  PubMed  Google Scholar 

  34. Luo S, Qin Z, Chen Q, Fan L, Jiang L, Zhao L (2020) High level production of a Bacillus amlyoliquefaciens chitosanase in Pichia pastoris suitable for chitooligosaccharides preparation. Int J Biol Macromol 149:1034–1041. https://doi.org/10.1016/j.ijbiomac.2020.02.001

    Article  CAS  PubMed  Google Scholar 

  35. Kang LX, Chen XM, Fu L, Ma LX (2012) Recombinant expression of chitosanase from Bacillus subtilis HD145 in Pichia pastoris. Carbohyd Res 352:37–43

    Article  CAS  Google Scholar 

  36. Bata Z, Molnár Z, Madaras E, Molnár B, Sánta-Bell E, Varga A, Leveles I, Qian R, Hammerschmidt F, Paizs C et al (2021) Substrate Tunnel Engineering Aided by X-ray Crystallography and Functional Dynamics Swaps the Function of MIO-Enzymes. ACS Catal 11:4538–4549. https://doi.org/10.1021/acscatal.1c00266

    Article  CAS  Google Scholar 

  37. Hirsch M, Fitzgerald BJ, Keatinge-Clay AT (2021) How cis-Acyltransferase Assembly-Line Ketosynthases Gatekeep for Processed Polyketide Intermediates. ACS Chem Biol 16:2515–2526. https://doi.org/10.1021/acschembio.1c00598

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Song W, Xu X, Gao C, Zhang Y, Wu J, Liu J, Chen X, Luo Q, Liu L (2020) Open Gate of Corynebacterium glutamicum Threonine Deaminase for Efficient Synthesis of Bulky α-Keto Acids. ACS Catal 10:9994–10004. https://doi.org/10.1021/acscatal.0c01672

    Article  CAS  Google Scholar 

Download references

Funding

This work was supported by the applied basic Research Program of Changzhou (CJ20220080), the Key Research and Development Program of Shandong Province, China (2019JZZY020605). Applied basic Research Program of Changzhou, CJ20220080, Jing Guo,Key Research and Development Program of Sichuan Province, 2019JZZY020605, Jing Guo

Author information

Authors and Affiliations

  1. Laboratory of Applied Microbiology, School of Biological and Food Engineering, Changzhou University, Changzhou Jiangsu, 213164, China

    Guo Jing, Gao Wenjun, Wang Yi, Xu Kepan, Luo Wen, Hong Tingting & Cai Zhiqiang

  2. Advanced Catalysis and Green Manufacturing Collaborative Innovation Center and Laboratory of Applied Microbiology, School of Pharmaceutical, Changzhou University, Changzhou, 213164, Jiangsu, China

    Guo Jing, Hong Tingting & Cai Zhiqiang

Authors
  1. Guo Jing
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gao Wenjun
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Wang Yi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xu Kepan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Luo Wen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hong Tingting
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Cai Zhiqiang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Conceptualization: CZQ Methodology: GJ Data analysis: GWJ and WY Software: GJ and GWJ Writing original manuscript: GJ Review and revising manuscript: CZQ, XKP, LW and HTT Funding acquisition: GJ and CZQ. All authors reviewed and approved the final manuscript.

Corresponding author

Correspondence to Cai Zhiqiang.

Ethics declarations

Conflict of interest

The authors declare no competing interests.

Ethical Approval

Not applicable.

Consent to Participate

Not applicable.

Consent for Publication

Not applicable.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (PDF 179 KB)

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Jing, G., Wenjun, G., Yi, W. et al. Enhancing Enzyme Activity and Thermostability of Bacillus amyloliquefaciens Chitosanase BaCsn46A Through Saturation Mutagenesis at Ser196. Curr Microbiol 80, 180 (2023). https://doi.org/10.1007/s00284-023-03281-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s00284-023-03281-5

Search

Navigation