Applied Microbiology and Biotechnology

, Volume 103, Issue 4, pp 1777–1787 | Cite as

The Listeria innocua chitinase LinChi78 has a unique region that is necessary for hydrolytic activity

  • Shotaro Honda
  • Masahiro Kimura
  • Satoshi Wakita
  • Yuji Oka
  • Masao Kawakita
  • Fumitaka Oyama
  • Masayoshi SakaguchiEmail author
Biotechnologically relevant enzymes and proteins


Chitinases are generally composed of multiple domains; a catalytic domain and one or more additional domains that are not absolutely required but may modify the chitinolytic activity. The LinChi78 chitinase from Listeria innocua has a catalytic domain (CatD), a fibronectin type III-like (FnIII) domain, a chitin-binding domain (ChBD), and an unknown-function region (UFR) located between the CatD and FnIII domains. The UFR is 146 amino acid residues in length and does not have a homologous domain in the Conserved Domain Database. We performed a functional analysis of these domains and the UFR using several C-terminally and internally deleted mutants of LinChi78. Hydrolysis of an artificial substrate was almost unaffected by deletion of the ChBD and/or the FnIII domain, although the ChBD-deleted enzymes were approximately 30% less active toward colloidal chitin than LinChi78. On the other hand, deletion of the UFR led to an extensive loss of chitinase activity toward an artificial substrate as well as polymeric substrates. Upon further analysis, we found that the GKQTI stretch, between the 567th (G) and 571th (I) amino acid residues, in the UFR is critical for LinChi78 activity and demonstrated that Gln569 and Ile571 play central roles in eliciting this activity. Taken together, these results indicated that LinChi78 has a unique catalytic region composed of a typical CatD and an additional region that is essential for activity. Characterization of the unique catalytic region of LinChi78 will improve our understanding of GH18 chitinases.


Chitinase Characterization Catalytic domain Insertion region Listeria species C-terminal truncation 



We are grateful to Kazuaki Okawa and Daiya Kuribara for their valuable suggestions and technical assistance.


This study was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI (Grant Number 17K07729); the Strategic Research Foundation Grant-aided Project for Private Universities of the Ministry of Education, Culture, Sport, Science, and Technology, Japan (MEXT) (Grant Number S1411005); the Science Research Promotion Fund from the Promotion and Mutual Aid Corporation for Private Schools of Japan; and the Project Research Grant from the Research Institute of Science and Technology, Kogakuin University.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals.

Supplementary material

253_2018_9573_MOESM1_ESM.pdf (1.2 mb)
ESM 1 (PDF 1207 kb)


  1. Arakane Y, Muthukrishnan S (2010) Insect chitinase and chitinase-like proteins. Cell Mol Life Sci 67:201–216. CrossRefGoogle Scholar
  2. Bhattacharya D, Nagpure A, Gupta RK (2007) Bacterial chitinases: properties and potential. Crit Rev Biotechnol 27:21–28. CrossRefGoogle Scholar
  3. Chaudhuri S, Bruno JC, Alonzo F III, Xayarath B, Cianciotto NP, Freitag NE (2010) Contribution of chitinases to Listeria monocytogenes pathogenesis. Appl Environ Microbiol 76:7302–7305. CrossRefGoogle Scholar
  4. Chaudhuri S, Gantner BN, Ye RD, Cianciotto NP, Freitag NE (2013) The Listeria monocytogenes ChiA chitinase enhances virulence through suppression of host innate immunity. MBio 4:e00617–e00612. CrossRefGoogle Scholar
  5. Chen W, Qu ML, Zhou Y, Yang Q (2018) Structural analysis of group II chitinase (ChtII) catalysis completes the puzzle of chitin hydrolysis in insects. J Biol Chem 293:2652–2660. CrossRefGoogle Scholar
  6. Chuang HH, Lin HY, Lin FP (2008) Biochemical characteristics of C-terminal region of recombinant chitinase from Bacillus licheniformis: implication of necessity for enzyme properties. FEBS J 275:2240–2254. CrossRefGoogle Scholar
  7. den Bakker HC, Bundrant BN, Fortes ED, Orsi RH, Wiedmann M (2010a) A population genetics-based and phylogenetic approach to understanding the evolution of virulence in the genus Listeria. Appl Environ Microbiol 76:6085–6100. CrossRefGoogle Scholar
  8. den Bakker HC, Cummings CA, Ferreira V, Vatta P, Orsi RH, Degoricija L, Barker M, Petrauskene O, Furtado MR, Wiedmann M (2010b) Comparative genomics of the bacterial genus Listeria: genome evolution is characterized by limited gene acquisition and limited gene loss. BMC Genomics 11:688. CrossRefGoogle Scholar
  9. Donnelly LE, Barnes PJ (2004) Acidic mammalian chitinase-a potential target for asthma therapy. Trends Pharmacol Sci 25:509–511. CrossRefGoogle Scholar
  10. Duo-Chuan L (2006) Review of fungal chitinases. Mycopathologia 161:345–360. CrossRefGoogle Scholar
  11. Glaser P, Frangeul L, Buchrieser C, Rusniok C, Amend A, Baquero F, Berche P, Bloecker H, Brandt P, Chakraborty T, Charbit A, Chetouani F, Couvé E, de Daruvar A, Dehoux P, Domann E, Domínguez-Bernal G, Duchaud E, Durant L, Dussurget O, Entian KD, Fsihi H, García-del Portillo F, Garrido P, Gautier L, Goebel W, Gómez-López N, Hain T, Hauf J, Jackson D, Jones LM, Kaerst U, Kreft J, Kuhn M, Kunst F, Kurapkat G, Madueno E, Maitournam A, Vicente JM, Ng E, Nedjari H, Nordsiek G, Novella S, de Pablos B, Pérez-Diaz JC, Purcell R, Remmel B, Rose M, Schlueter T, Simoes N, Tierrez A, Vázquez-Boland JA, Voss H, Wehland J, Cossart P (2001) Comparative genomics of Listeria species. Science 294:849–852. Google Scholar
  12. Hashimoto M, Ikegami T, Seino S, Ohuchi N, Fukada H, Sugiyama J, Shirakawa M, Watanabe T (2000) Expression and characterization of the chitin-binding domain of chitinase A1 from Bacillus circulans WL-12. J Bacteriol 182:3045–3054. CrossRefGoogle Scholar
  13. Henrissat B, Davies G (1997) Structural and sequence-based classification of glycoside hydrolases. Curr Opin Struct Biol 7:637–644. CrossRefGoogle Scholar
  14. Honda S, Wakita S, Sugahara Y, Kawakita M, Oyama F, Sakaguchi M (2016) Characterization of two Listeria innocua chitinases of different sizes that were expressed in Escherichia coli. Appl Microbiol Biotechnol 100:8031–8041. CrossRefGoogle Scholar
  15. Iseli B, Armand S, Boller T, Neuhaus JM, Henrissat B (1996) Plant chitinases use two different hydrolytic mechanisms. FEBS Lett 382:186–188. CrossRefGoogle Scholar
  16. Itoh Y, Kawase T, Nikaidou N, Fukada H, Mitsutomi M, Watanabe T, Itoh Y (2002) Functional analysis of the chitin-binding domain of a family 19 chitinase from Streptomyces griseus HUT6037: substrate-binding affinity and cis-dominant increase of antifungal function. Biosci Biotechnol Biochem 66:1084–1092. CrossRefGoogle Scholar
  17. Itoh T, Hibi T, Suzuki F, Sugimoto I, Fujiwara A, Inaka K, Tanaka H, Ohta K, Fujii Y, Taketo A, Kimoto H (2016) Crystal structure of chitinase ChiW from Paenibacillus sp. str. FPU-7 reveals a novel type of bacterial cell-surface-expressed multi-modular enzyme machinery. PLoS One 11:e0167310. CrossRefGoogle Scholar
  18. Jackson P (1990) The use of polyacrylamide-gel electrophoresis for the high-resolution separation of reducing saccharides labelled with the fluorophore 8-aminonaphthalene-1,3,6-trisulphonic acid. Detection of picomolar quantities by an imaging system based on a cooled charge-coupled device. Biochem J 270:705–713CrossRefGoogle Scholar
  19. Juárez-Hernández EO, Casados-Vázquez LE, Bideshi DK, Salcedo-Hernández R, Barboza-Corona JE (2017) Role of the C-terminal and chitin insertion domains on enzymatic activity of endochitinase ChiA74 of Bacillus thuringiensis. Int J Biol Macromol 102:52–59. CrossRefGoogle Scholar
  20. Kasprzewska A (2003) Plant chitinases-regulation and function. Cell Mol Biol Lett 8:809–824Google Scholar
  21. Katouno F, Taguchi M, Sakurai K, Uchiyama T, Nikaidou N, Nonaka T, Sugiyama J, Watanabe T (2004) Importance of exposed aromatic residues in chitinase B from Serratia marcescens 2170 for crystalline chitin hydrolysis. J Biochem 136:163–168. CrossRefGoogle Scholar
  22. Leisner JJ, Larsen MH, Jørgensen RL, Brøndsted L, Thomsen LE, Ingmer H (2008) Chitin hydrolysis by Listeria spp., including L. monocytogenes. Appl Environ Microbiol 74:3823–3830. CrossRefGoogle Scholar
  23. Li H, Greene LH (2010) Sequence and structural analysis of the chitinase insertion domain reveals two conserved motifs involved in chitin-binding. PLoS One 5:e8654. CrossRefGoogle Scholar
  24. Lin FP, Chuang HH, Liu YH, Hsieh CY, Lin PW, Lin HY (2009) Effects of C-terminal amino acids truncation on enzyme properties of Aeromonas caviae D1 chitinase. Arch Microbiol 191:265–273. CrossRefGoogle Scholar
  25. Lin FP, Wu CY, Chen HN, Lin HJ (2015) Effects of C-terminal domain truncation on enzyme properties of Serratia marcescens chitinase C. Appl Biochem Biotechnol 175:3617–3627. CrossRefGoogle Scholar
  26. Liu T, Chen L, Zhou Y, Jiang X, Duan Y, Yang Q (2017) Structure, catalysis, and inhibition of OfChi-h, the lepidoptera-exclusive insect chitinase. J Biol Chem 292:2080–2088. CrossRefGoogle Scholar
  27. Matsumoto T, Nonaka T, Hashimoto M, Watanabe T, Mitsui Y (1999) Three-dimensional structure of the catalytic domain of chitinase A1 from Bacillus circulans WL-12 at a very high resolution. Proc Japan Acad 75:269–274CrossRefGoogle Scholar
  28. Niesen FH, Berglund H, Vedadi M (2007) The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat Protoc 2:2212–2221. CrossRefGoogle Scholar
  29. Sakaguchi M, Osaku K, Maejima S, Ohno N, Sugahara Y, Oyama F, Kawakita M (2014) Highly conserved salt bridge stabilizes a proteinase K subfamily enzyme, Aqualysin I, from Thermus aquaticus YT-1. AMB Express 4:59. CrossRefGoogle Scholar
  30. Sakaguchi M, Shimodaira S, Ishida S, Amemiya M, Honda S, Sugahara Y, Oyama F, Kawakita M (2015) Identification of GH15 family thermophilic archaeal trehalases that function within a narrow acidic pH range. Appl Environ Microbiol 81:4920–4931. CrossRefGoogle Scholar
  31. Sambrook J, Russell DW (2012) Molecular cloning: a laboratory manual, 4th edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  32. Sha L, Shao E, Guan X, Huang Z (2016) Purification and partial characterization of intact and truncated chitinase from Bacillus thuringiensis HZP7 expressed in Escherichia coli. Biotechnol Lett 38:279–284. CrossRefGoogle Scholar
  33. Shimahara K, Takiguchi Y (1988) Preparation of crustacean chitin. Methods Enzymol 161:417–423CrossRefGoogle Scholar
  34. Shirai A, Matsuyama A, Yashiroda Y, Hashimoto A, Kawamura Y, Arai R, Komatsu Y, Horinouchi S, Yoshida M (2008) Global analysis of gel mobility of proteins and its use in target identification. J Biol Chem 283:10745–10,752. CrossRefGoogle Scholar
  35. Suzuki K, Taiyoji M, Sugawara N, Nikaidou N, Henrissat B, Watanabe T (1999) The third chitinase gene (chiC) of Serratia marcescens 2170 and the relationship of its product to other bacterial chitinases. Biochem J 343:587–596. CrossRefGoogle Scholar
  36. Tews I, Terwisscha van Scheltinga AC, Perrakis A, Wilson KS, Dijkstra BW (1997) Substrate-assisted catalysis unifies two families of chitinolytic enzymes. J Am Chem Soc 119:7954–7959CrossRefGoogle Scholar
  37. Todd AE, Orengo CA, Thornton JM (2001) Evolution of function in protein superfamilies, from a structural perspective. J Mol Biol 307:1113–1143. CrossRefGoogle Scholar
  38. Uchiyama T, Katouno F, Nikaidou N, Nonaka T, Sugiyama J, Watanabe T (2001) Roles of the exposed aromatic residues in crystalline chitin hydrolysis by chitinase A from Serratia marcescens 2170. J Biol Chem 276:41343–41,349. CrossRefGoogle Scholar
  39. Vaaje-Kolstad G, Horn SJ, Sørlie M, Eijsink VG (2013) The chitinolytic machinery of Serratia marcescens – a model system for enzymatic degradation of recalcitrant polysaccharides. FEBS J 280:3028–3049. CrossRefGoogle Scholar
  40. van Aalten DM, Synstad B, Brurberg MB, Hough E, Riise BW, Eijsink VG, Wierenga RK (2000) Structure of a two-domain chitotriosidase from Serratia marcescens at 1.9-Å resolution. Proc Natl Acad Sci U S A 97:5842–5847. CrossRefGoogle Scholar
  41. Wakita S, Kimura M, Kato N, Kashimura A, Kobayashi S, Kanayama N, Ohno M, Honda S, Sakaguchi M, Sugahara Y, Bauer PO, Oyama F (2017) Improved fluorescent labeling of chitin oligomers: chitinolytic properties of acidic mammalian chitinase under somatic tissue pH conditions. Carbohydr Polym 164:145–153. CrossRefGoogle Scholar
  42. Watanabe T, Ito Y, Yamada T, Hashimoto M, Sekine S, Tanaka H (1994) The roles of the C-terminal domain and type III domains of chitinase A1 from Bacillus circulans WL-12 in chitin degradation. J Bacteriol 176:4465–4472CrossRefGoogle Scholar
  43. Zees AC, Pyrpassopoulos S, Vorgias CE (2009) Insights into the role of the (α+β) insertion in the TIM-barrel catalytic domain, regarding the stability and the enzymatic activity of chitinase A from Serratia marcescens. Biochim Biophys Acta 1794:23–31. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Chemistry and Life ScienceKogakuin UniversityTokyoJapan

Personalised recommendations