Novel Bi-Modular GH19 Chitinase with Broad pH Stability from a Fibrolytic Intestinal Symbiont of Eisenia fetida, Cellulosimicrobium funkei HY-13
Abstract
:1. Introduction
2. Materials and Methods
2.1. Substrate Compounds
2.2. Cloning of the Chitinase (ChiJ) Gene
2.3. Production and Purification of Recombinant Chitinase (rChiJ)
2.4. Analysis of Proteins
2.5. Enzyme Assays
2.6. Effects of pH, Temperature, and Chemicals on Endochitinase Activity
2.7. Analysis of the Hydrolysis Products
2.8. Binding Assay
3. Results and Discussion
3.1. Genetic Characterization of the GH19 Chitinase Gene
3.2. Purification and SDS-PAGE Analysis of rChiJ
3.3. Enzymatic Properties of rChiJ
3.4. Substrate Specificity
3.5. Binding Capacity of rChiJ to Hydrophobic Materials
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
- Yan, Q.; Fong, S.S. Bacterial chitinase: Nature and perspectives for sustainable bioproduction. Bioresour. Bioproc. 2015, 2, 31. [Google Scholar] [CrossRef] [Green Version]
- Le, B.; Yang, S.H. Microbial chitinases: Properties, current state and biotechnological applications. World J. Microbiol. Biotechnol. 2019, 35, 144. [Google Scholar] [CrossRef] [PubMed]
- Duo-Chuan, L. Review of fungal chitinases. Mycopathologia 2006, 161, 345–360. [Google Scholar] [CrossRef]
- Gomaa, E.Z. Microbial chitinases: Properties, enhancement and potential applications. Protoplasma 2021, 258, 695–710. [Google Scholar] [CrossRef] [PubMed]
- Oyeleye, A.; Normi, Y.M. Chitinase: Diversity, limitations, and trends in engineering for suitable applications. Biosci. Rep. 2018, 38. [Google Scholar] [CrossRef]
- Singh, R.V.; Sambyal, K.; Negi, A.; Sonwani, S.; Mahajan, R. Chitinases production: A robust enzyme and its industrial applications. Biocatal. Biotransfor. 2021, 39, 161–189. [Google Scholar] [CrossRef]
- Brameld, K.A.; Goddard III, W.A. The role of enzyme distortion in the single displacement mechanism of family 19 chitinases. Proc. Natl. Acad. Sci. USA 1998, 95, 4276–4281. [Google Scholar] [CrossRef] [Green Version]
- Kim, D.Y.; Han, M.K.; Lee, J.S.; Oh, H.-W.; Park, D.-S.; Shin, D.-H.; Bae, K.S.; Son, K.-H.; Park, H.-Y. Isolation and characterization of a cellulose-free endo-β-1,4-xylanase produced by an invertebrate-symbiotic bacterium, Cellulosimicrobium sp. strain HY-13. Proc. Biochem. 2009, 44, 1055–1059. [Google Scholar] [CrossRef]
- Kim, D.Y.; Han, M.K.; Oh, H.-W.; Park, D.-S.; Kim, S.-J.; Lee, S.-G.; Shin, D.-H.; Son, K.-H.; Bae, K.S.; Park, H.-Y. Catalytic properties of a GH10 endo-β-1,4-xylanase from Streptomyces thermocarboxydus HY-15 isolated from the gut of Eisenia fetida. J. Mol. Catal. B Enzym. 2010, 62, 32–39. [Google Scholar] [CrossRef]
- Bai, L.; Kim, J.; Son, K.-H.; Shin, D.-H.; Ku, B.-H.; Kim, D.Y.; Park, H.-Y. Novel anti-fungal d-laminaripentaose-releasing endo-β-1,3-glucanase with a RICIN-like domain from Cellulosimicrobium funkei HY-13. Biomolecules 2021, 11, 1080. [Google Scholar] [CrossRef] [PubMed]
- Brune, A. Symbiotic digestion of lignocellulose in termite guts. Nat. Rev. Microbiol. 2014, 12, 168–180. [Google Scholar] [CrossRef]
- Warnecke, F.; Luginbühl, P.; Ivanova, N.; Ghassemian, M.; Richardson, T.H.; Stege, J.T.; Cayouette, M.; McHardy, A.C.; Djordjevic, G.; Aboushadi, N.; et al. Metagenomic and functional analysis of hindgut microbiota of a wood-feeding higher termite. Nature 2007, 450, 560–565. [Google Scholar] [CrossRef]
- Kim, D.Y.; Shin, D.-H.; Jung, S.; Lee, J.S.; Cho, H.Y.; Bae, K.S.; Sung, C.-K.; Rhee, Y.H.; Son, K.-H.; Park, H.-Y. Biocatalytic properties and substrate-binding ability of a modular GH10 β-1,4-xylanase from an insect-symbiotic bacterium, Streptomyces mexicanus HY-14. J. Microbiol. 2014, 52, 863–870. [Google Scholar] [CrossRef] [PubMed]
- Kim, D.Y.; Shin, D.-H.; Jung, S.; Kim, H.; Lee, J.S.; Cho, H.Y.; Bae, K.S.; Sung, C.-K.; Rhee, Y.H.; Son, K.-H.; et al. Novel alkali-tolerant GH10 endo-β-1,4-xylanase with broad substrate specificity from Microbacterium trichothecenolyticum HY-17, a gut bacterium of the mole cricket Gryllotalpa orientalis. J. Microbiol. Biotechnol. 2014, 24, 943–953. [Google Scholar] [CrossRef] [PubMed]
- Park, D.S.; Oh, H.-W.; Jeong, W.-J.; Kim, H.; Park, H.-Y.; Bae, K.S. A culture-based study of the bacterial communities within the guts of nine longicorn beetle species and their exo-enzyme producing properties for degrading xylan and pectin. J. Microbiol. 2007, 45, 394–401. [Google Scholar]
- Scully, E.D.; Geib, S.M.; Hoover, K.; Tien, M.; Tringe, S.G.; Barry, K.W.; Glavina del Rio, T.; Chovatia, M.; Herr, J.R.; Carlson, J.E. Metagenomic profiling reveals lignocellulose degrading system in a microbial community associated with a wood-feeding beetle. PloS One 2013, 8, e73827. [Google Scholar] [CrossRef] [Green Version]
- Talamantes, D.; Biabini, N.; Dang, H.; Abdoun, K.; Berlenmont, R. Natural diversity of cellulases, xylanases, and chitinases in bacteria. Biotechnol. Biofuels 2016, 9, 133. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Jang, S.; Kikuchi, Y. Impact of the insect gut microbiota on ecology, evolution, and industry. Curr. Opin. Insect Sci. 2020, 41, 30–39. [Google Scholar] [CrossRef] [PubMed]
- Kim, D.Y.; Han, M.K.; Park, D.-S.; Lee, J.S.; Oh, H.-W.; Shin, D.-H.; Jeong, T.-S.; Kim, S.U.; Bae, K.S.; Son, K.-H.; et al. Novel GH10 xylanase, with a fibronectin type 3 domain, from Cellulosimicrobium sp. strain HY-13, a bacterium in the gut of Eisenia fetida. Appl. Environ. Microbiol. 2009, 75, 7275–7279. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Kim, D.Y.; Ham, S.-J.; Lee, H.J.; Kim, Y.-J.; Shin, D.-H.; Rhee, Y.H.; Son, K.-H.; Park, H.-Y. A highly active endo-β-1,4-mannanase produced by Cellulosimicrobium sp. strain HY-13, a hemicellulolytic bacterium in the gut of Eisenia fetida. Enzyme Microb. Technol. 2011, 48, 365–370. [Google Scholar] [CrossRef]
- Kim, D.Y.; Ham, S.-J.; Lee, H.J.; Cho, H.-Y.; Kim, J.-H.; Kim, Y.-J.; Shin, D.-H.; Rhee, Y.H.; Son, K.-H.; Park, H.-Y. Cloning and characterization of a modular GH5 β-1,4-mannanase with high specific activity from the fibrolytic bacterium Cellulosimicrobium sp. strain HY-13. Bioresour. Technol. 2011, 102, 9185–9192. [Google Scholar] [CrossRef]
- Kim, D.Y.; Ham, S.-J.; Kim, H.J.; Kim, J.; Lee, M.-H.; Cho, H.-Y.; Shin, D.-H.; Rhee, Y.H.; Son, K.-H.; Park, H.-Y. Novel modular endo-β-1,4-xylanase with transglycosylation activity from Cellulosimicrobium sp. strain HY-13 that is homologous to inverting GH family 6 enzymes. Bioresour. Technol. 2012, 107, 25–32. [Google Scholar] [CrossRef]
- Kim, D.Y.; Lee, M.J.; Cho, H.-Y.; Lee, J.S.; Lee, M.-H.; Chung, C.W.; Shin, D.-H.; Rhee, Y.H.; Son, K.-H.; Park, H.-Y. Genetic and functional characterization of an extracellular modular GH6 endo-β-1,4-glucanase from an earthworm symbiont, Cellulosimicrobium funkei HY-13. Antonie van Leeuwenhoek 2016, 109, 1–12. [Google Scholar] [CrossRef]
- Miller, G.L. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 1959, 31, 426–428. [Google Scholar] [CrossRef]
- Watanabe, T.; Kanai, R.; Kawase, T.; Tanabe, T.; Mitsutomi, M.; Sakuda, S.; Miyashita, K. Family 19 chitinases of Streptomyces species: Characterization and distribution. Microbiology 1999, 145, 3353–3363. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Kawase, T.; Yokokawa, S.; Saito, A.; Fujii, T.; Nikaidou, N.; Miyashita, K.; Watanabe, T. Comparison of enzymatic and antifungal properties between family 18 and 19 chitinases from S. coelicolor A3(2). Biosci. Biotechnol. Biochem. 2006, 70, 988–998. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Huang, L.; Garbulewska, E.; Sato, K.; Kata, Y.; Nogawa, M.; Taguchi, G.; Shimosaka, M. Isolation of genes coding for chitin-degrading enzymes in the novel chitinolytic bacterium, Chitiniphilus shinanonensis, and characterization of a gene coding for a family 19 chitinase. J. Biosci. Bioeng. 2012, 113, 293–299. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- García-Fraga, B.; da Silva, A.F.; López-Seijas, J.; Sieiro, C. A novel family 19 chitinase from the marine-derived Pseudoalteromonas tunicata CCUG 44952T: Heterologous expression, characterization and antifungal activity. Biochem. Eng. J. 2015, 93, 84–93. [Google Scholar] [CrossRef]
- Lv, C.; Gu, T.; Ma, R.; Yao, W.; Huang, Y.; Gu, J.; Zhao, G. Biochemical characterization of a GH19 chitinase from Streptomyces alfalfae and its applications in crystalline chitin conversion and biocontrol. Int. J. Biol. Macromol. 2021, 167, 193–201. [Google Scholar] [CrossRef]
- Honda, Y.; Taniguchi, H.; Kitaoka, M. A reducing-end-acting chitinase from Vibrio proteolyticus belonging to glycoside hydrolase family 19. Appl. Microbiol. Biotechnol. 2008, 78, 627–634. [Google Scholar] [CrossRef]
- Wang, Y.-T.; Wu, P.-L. Gene cloning, characterization, and molecular simulations of a novel recombinant chitinase from Chitinibacter tainanensis CT01 appropriate for chitin enzymatic hydrolysis. Polymers 2020, 12, 1648. [Google Scholar] [CrossRef] [PubMed]
- Yano, S.; Honda, A.; Rattanakit, N.; Noda, Y.; Wakayama, M.; Plikomol, A.; Tachiki, T. Cloning and expression of chitinase A gene from Streptomyces cyaneus SP-27: The enzyme participates in protoplast formation of Schizophyllum commune. Biosci. Biotechnol. Biochem. 2008, 72, 1853–1859. [Google Scholar] [CrossRef] [Green Version]
- Zhang, W.; Liu, Y.; Ma, J.; Yan, Q.; Jiang, Z.; Yang, S. Biochemical characterization of a bifunctional chitinase/lysozyme from Streptomyces sampsonii suitable for N-acetylchitobiose production. Biotechnol. Lett. 2020, 42, 1489–1499. [Google Scholar] [CrossRef]
- Ueda, M.; Kojima, M.; Yoshikawa, T.; Mitsuda, N.; Araki, K.; Kawaguchi, T.; Miyatake, K.; Arai, M.; Fukamizo, T. A novel type of family 19 chitinase from Aeromonas sp. No. 10S-24: Cloning, sequence, expression, and the enzymatic properties. Eur. J. Biochem. 2003, 270, 2513–2520. [Google Scholar] [CrossRef] [Green Version]
- Han, B.; Zhou, K.; Li, Z.; Sun, B.; Ni, Q.; Meng, X.; Pan, G.; Li, C.; Long, M.; Zhou, C.; et al. Characterization of the first fungal glycosyl hydrolase family 19 chitinase (NbchiA) from Nosema bombycis (Nb). J. Eukaryot. Microbiol. 2016, 63, 37–45. [Google Scholar] [CrossRef] [PubMed]
- Hoster, F.; Schmitz, J.; Daniel, R. Enrichment of chitinolytic microorganisms: Isolation and characterization of a chitinase exhibiting antifungal activity against phytopathogenic fungi from a novel Streptomyces strain. Appl. Microbiol. Biotechnol. 2005, 66, 434–442. [Google Scholar] [CrossRef]
- Roberge, M.; Shareck, F.; Morosoli, R.; Kluepfel, D.; Dupont, C. Characterization of active-site aromatic residues in xylanase A from Streptomyces lividans. Protein Eng. 1999, 12, 251–257. [Google Scholar] [CrossRef] [Green Version]
- Zolotnitsky, G.; Cogan, U.; Adir, N.; Solomon, V.; Shoham, G.; Shoham, Y. Mapping glycoside hydrolase substrate subsites by isothermal titration calorimetry. Proc. Natl. Acad. Sci. USA 2004, 101, 11275–11280. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Hao, Z.; Wu, H.; Yang, M.; Chen, J.; Xi, L.; Zhao, W.; Yu, J.; Liu, J.; Liao, X.; Huang, Q. Cloning, expression and 3D structure prediction of chitinase from Chitinolyticbacter meiyuanensis SYBC-H1. Int. J. Mol. Sci. 2016, 17, 825. [Google Scholar] [CrossRef]
- Wu, X.; Wang, J.; Shi, Y.; Chen, S.; Yan, Q.; Jiang, Z.; Jing, H. N-Acetyl-chitobiose ameliorates metabolism dysfunction through Erk/p38 MAPK and histone H3 phosphorylation in type 2 diabetes mice. J. Funct. Foods 2017, 28, 96–105. [Google Scholar] [CrossRef]
Strain | Enzyme | Mr (kDa) | Optimum pH | Optimum Temp. (°C) | Specific Activity (U/mg) | Reference |
---|---|---|---|---|---|---|
Cellulosimicrobium funkei HY-13 | rChiJ | 30.0 | 5.5 | 55 | 338.8 a, 16.0 b | This study |
Streptomyces griseus HUT6037 | chitinase C | 28.5 | 4.5–6.0 | 55 | 255.0 a, 24.5 b | [25] |
Aeromonas sp. No. 10S-24 | chitinase | 70.0 | 5.0 | 40 | 0.9 a | [34] |
Nosema bombycis (Nb) | NbchiA | NI | 7.0 | 40 | 58.6 a, 0.7 b | [35] |
Streptomyces griseus MG3 | ChiIS | 29.0 | 5.0–7.0 | 45 | 1.8 b | [36] |
Streptomyces coelicolor A3(2) | Chi19F | <30.0 | 6.0–7.0 | 50 | NI c | [26] |
Chitiniphilus shinanonensis SAY3T | rChiN | 41.4 | 5.6 | 50 | 8.6 b | [27] |
Pseudoalteromonas tunicata | PtChi19p | 53.5 | 7.5 | 43 | <0.1 b | [28] |
Streptomyces cyaneus SP-27 | chitinase A | 29.0 | 7.0 | 60 | 25.0 a | [32] |
Streptomyces sampsonii XY2-7 | SsChi28 | 30.0 | 6.0 | 55 | 222.3 a, 20.1 b | [33] |
Streptomyces alfalfae ACCC 40021 | SaChiB | 29.0 | 8.0 | 45 | 286.6 a, 28.4 b | [29] |
Substrate | Specific activity (U/mg) a of rChiJ | Relative activity (%) of rChiJ |
---|---|---|
Ethylene glycol chitin | 338.8 ± 1.2 | 100.0 |
Colloidal shrimp shell chitin | 16.0 ± 0.3 | 4.7 |
Colloidal crab shell chitin | 8.1 ± 0.2 | 2.4 |
Carboxymethylcellulose | ND b | - |
Soluble starch | ND | - |
Beechwood xylan | ND | - |
Locust bean gum | ND | - |
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Bai, L.; Kim, J.; Son, K.-H.; Chung, C.-W.; Shin, D.-H.; Ku, B.-H.; Kim, D.Y.; Park, H.-Y. Novel Bi-Modular GH19 Chitinase with Broad pH Stability from a Fibrolytic Intestinal Symbiont of Eisenia fetida, Cellulosimicrobium funkei HY-13. Biomolecules 2021, 11, 1735. https://doi.org/10.3390/biom11111735
Bai L, Kim J, Son K-H, Chung C-W, Shin D-H, Ku B-H, Kim DY, Park H-Y. Novel Bi-Modular GH19 Chitinase with Broad pH Stability from a Fibrolytic Intestinal Symbiont of Eisenia fetida, Cellulosimicrobium funkei HY-13. Biomolecules. 2021; 11(11):1735. https://doi.org/10.3390/biom11111735
Chicago/Turabian StyleBai, Lu, Jonghoon Kim, Kwang-Hee Son, Chung-Wook Chung, Dong-Ha Shin, Bon-Hwan Ku, Do Young Kim, and Ho-Yong Park. 2021. "Novel Bi-Modular GH19 Chitinase with Broad pH Stability from a Fibrolytic Intestinal Symbiont of Eisenia fetida, Cellulosimicrobium funkei HY-13" Biomolecules 11, no. 11: 1735. https://doi.org/10.3390/biom11111735