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Purification and Biochemical Characterization of Alkalophilic Cellulase from the Symbiotic Bacillus subtilis BC1 of the Leopard Moth, Zeuzera pyrina (L.) (Lepidoptera: Cossidae)

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Abstract

In the current study, an extracellular cellulase belonging to symbiotic Bacillus subtilis Bc1 of the leopard moth is purified and characterized. The molecular mass of enzyme was 47.8 kDa using SDS-PAGE. The purified enzyme had optimum activity in temperature and pH around 60 °C and 8, respectively. The purified cellulase was introduced as a stable enzyme in a wide variety of temperature (20–80 °C) and pH (4–10) and remained active to more than 74% at 80 °C for 1 h. Moreover, the cellulase extremely was stabled in the presence of metal ions and organic solvents and its activity was increased by acetone (20% v/v), CaCl2 and CoCl2 and inhibited by MnCl2 and NiCl2. The values of enzyme's Km and Vmax were found to be 1.243 mg/mL and 271.3 µg/mL/min, respectively. The purified cellulase hydrolyzed cellulose, avicel and carboxymethyl cellulose (CMC) and the final product of CMC hydrolysis was cellobiose using thin-layer chromatography analysis. Consequently, owing to exo/endoglucanase activity and organic solvent, temperature and pH stability of the purified cellulase belong to B. subtilis BC1, it can be properly employed for various industrial purposes.

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Abbreviations

CMC:

Carboxymethyl cellulose

CTAB:

Cetyl trimethylammonium bromide

DNS:

Dinitrosalicylic acid

NCBI:

National center for biotechnology information

PCR:

Polymerase chain reaction

SDS-PAGE:

Sodium dodecyl sulfate–polyacrylamide gel

TLC:

Thin-layer chromatography

References

  1. Ohkuma M (2003) Termite symbiotic systems: efficient bio-recycling of lignocellulose. Appl Microbiol Biotechnol 61(1):1–9

    Article  CAS  PubMed  Google Scholar 

  2. Sindhu R, Binod P, Pandey A (2016) Biological pretreatment of lignocellulosic biomass–An overview. Bioresour Technol 199:76–82

    Article  CAS  PubMed  Google Scholar 

  3. Zhou H, Guo W, Xu B, Teng Z, Tao D, Lou Y, Gao Y (2017) Screening and identification of lignin-degrading bacteria in termite gut and the construction of LiP-expressing recombinant Lactococcus lactis. Microb Pathog 112:63–69

    Article  CAS  PubMed  Google Scholar 

  4. Cortes-Tolalpa L, Salles JF, van Elsas JD (2017) Bacterial synergism in lignocellulose biomass degradation—Complementary roles of degraders as influenced by complexity of the carbon source. Front Microbiol 8:1628

    Article  PubMed  PubMed Central  Google Scholar 

  5. Knowles J, Lehtovaara P, Teeri T (1987) Cellulase families and their genes. Trends Biotechnol 5(9):255–261

    Article  CAS  Google Scholar 

  6. Singh R, Kumar M, Mittal A, Mehta PK (2016) Microbial cellulases in industrial applications. Ann appl Biosci 3(4):23–29

    Google Scholar 

  7. Trivedi N, Reddy C, Lali A (2016) Marine microbes as a potential source of cellulolytic enzymes. Advances in food and nutrition research. Elsevier, Amsterdam, pp 27–41

    Google Scholar 

  8. Engel P, Moran NA (2013) The gut microbiota of insects–diversity in structure and function. Fems Microbiol Rev 37(5):699–735

    Article  CAS  PubMed  Google Scholar 

  9. Tartar A, Wheeler MM, Zhou X, Coy MR, Boucias DG, Scharf ME (2009) Parallel metatranscriptome analyses of host and symbiont gene expression in the gut of the termite Reticulitermes flavipes. Biotechnol Biofuels 2(1):25

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Lo N, Tokuda G, Watanabe H (2010) Evolution and function of endogenous termite cellulases. Biology of termites: a modern synthesis. Springer, New York, pp 51–67

    Chapter  Google Scholar 

  11. Prasad RK, Chatterjee S, Sharma S, Mazumder PB, Vairale MG, Raju PS (2018) Insect gut bacteria and their potential application in degradation of lignocellulosic biomass: a review. Bioremediation: applications for environmental protection and management. Springer, New York, pp 277–299

    Chapter  Google Scholar 

  12. Salari E, Karimi J, Sadeghi-Nameghi H, Hosseini M (2015) Efficacy of two entomopathogenic nematodes Heterorhabditis bacteriophora and Steinernema carpocapsae for control of the leopard moth borer Zeuzera pyrina (Lepidoptera: Cossidae) larvae under laboratory conditions. Biocontrol Sci Technol 25(3):260–275

    Article  Google Scholar 

  13. Hatefi A, Makhdoumi A, Asoodeh A, Mirshamsi O (2017) Characterization of a bi-functional cellulase produced by a gut bacterial resident of Rosaceae branch borer beetle, Osphranteria coerulescens (Coleoptera: Cerambycidae). Int J Biol Macromol 103:158–164

    Article  CAS  PubMed  Google Scholar 

  14. Wood PJ, Erfle JD, Teather RM (1988) Use of complex formation between Congo Red and polysaccharides in detection and assay of polysaccharide hydrolases. Methods Enzymol 160:59–74

    Article  CAS  Google Scholar 

  15. Murray R (1994) Determinative and cytological light microscopy. Methods for general and molecular bacteriology. American Society for Microbiology, Washington, DC, pp 7–20

    Google Scholar 

  16. Wood WA, Krieg NR (1989) Methods for general and molecular bacteriology. ASM Press, Washington DC

    Google Scholar 

  17. Garrity GM (2012) Bergey's manual of systematic bacteriology volume one: the archaea and the deeply branching and phototrophic bacteria. Springer, New York

    Google Scholar 

  18. Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31(3):426–428

    Article  CAS  Google Scholar 

  19. Lane DJ, Pace B, Olsen GJ, Stahl DA, Sogin ML, Pace NR (1985) Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc Natl Acad Sci 82(20):6955–6959

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72(1–2):248–254

    Article  CAS  PubMed  Google Scholar 

  21. Zhang Z, Xie J, Zhang F, Linhardt RJ (2007) Thin layer chromatography for the analysis of glycosaminoglycan oligosaccharides. Anal Biochem 371(1):118

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Bourtzis K, Miller TA (2003) Insect symbiosis. CRC Press, Boca Raton

    Book  Google Scholar 

  23. Yun J-H, Roh SW, Whon TW, Jung M-J, Kim M-S, Park D-S, Yoon C, Nam Y-D, Kim Y-J, Choi J-H (2014) Insect gut bacterial diversity determined by environmental habitat, diet, developmental stage, and phylogeny of host. Appl Environ Microbiol 80(17):5254–5264

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Manfredi AP, Perotti NI, Martínez MA (2015) Cellulose degrading bacteria isolated from industrial samples and the gut of native insects from Northwest of Argentina. J Basic Microbiol 55(12):1384–1393

    Article  CAS  PubMed  Google Scholar 

  25. Bhuyan PM, Sandilya SP, Nath PK, Gandotra S, Subramanian S, Kardong D, Gogoi DK (2018) Optimization and characterization of extracellular cellulase produced by Bacillus pumilus MGB05 isolated from midgut of muga silkworm (Antheraea assamensis Helfer). J Asia-Pac Entomol 21(4):1171–1181

    Article  Google Scholar 

  26. Prem Anand AA, Vennison SJ, Sankar SG, Gilwax Prabhu DI, Vasan PT, Raghuraman T, Jerome Geoffrey C, Vendan SE (2010) Isolation and characterization of bacteria from the gut of Bombyx mori that degrade cellulose, xylan, pectin and starch and their impact on digestion. J Insect Sci 10(1):107

    Google Scholar 

  27. Wei KSC, Teoh TC, Koshy P, Salmah I, Zainudin A (2015) Cloning, expression and characterization of the endoglucanase gene from Bacillus subtilis UMC7 isolated from the gut of the indigenous termite Macrotermes malaccensis in Escherichia coli. Eletron J Biotechnol 18(2):103–109

    Article  CAS  Google Scholar 

  28. Ratnadewi AAI, Santoso AB, Sulistyaningsih E, Handayani W (2016) Application of cassava peel and waste as raw materials for xylooligosaccharide production using endoxylanase from Bacillus subtilis of soil termite abdomen. Procedia Chem 18:31–38. https://doi.org/10.1016/j.proche.2016.01.007

    Article  CAS  Google Scholar 

  29. Siu-Rodas Y, de los Angeles Calixto-Romo M, Guillén-Navarro K, Sánchez JE, Zamora-Briseño JA, Amaya-Delgado L (2018) Bacillus subtilis with endocellulase and exocellulase activities isolated in the thermophilic phase from composting with coffee residues. Rev Argent Microbiol 50(3):234–243

    PubMed  Google Scholar 

  30. Yin L-J, Lin H-H, Xiao Z-R (2010) Purification and characterization of a cellulase from Bacillus subtilis YJ1. J Mar Sci Techol 18(3):466–471

    Google Scholar 

  31. Nimchua T, Thongaram T, Uengwetwanit T, Pongpattanakitshote S, Eurwilaichitr L (2012) Metagenomic analysis of novel lignocellulose-degrading enzymes from higher termite guts inhabiting microbes. J Microbiol Biotechnol 22(4):462–469

    Article  CAS  PubMed  Google Scholar 

  32. Fagbohunka B, Okonji R, Adenike AZ (2016) Purification and characterization of cellulase from termite Ametermes eveuncifer (Silverstri) soldiers. Int J Biol 9(1):1

    Article  CAS  Google Scholar 

  33. Zi-Zhong T, Zhen-Fang W, Hui C, Xin L, Xue-yi H, Qi W (2013) Characterization of novel EGs reconstructed from Bacillus subtilis endoglucanase. Appl Biochem Biotechol 169(6):1764–1773

    Article  CAS  Google Scholar 

  34. Annamalai N, Rajeswari MV, Elayaraja S, Balasubramanian T (2013) Thermostable, haloalkaline cellulase from Bacillus halodurans CAS 1 by conversion of lignocellulosic wastes. Carbohyd Polym 94(1):409–415

    Article  CAS  Google Scholar 

  35. Zaks A, Klibanov AM (1988) Enzymatic catalysis in nonaqueous solvents. J Biol Chem 263(7):3194–3201

    CAS  PubMed  Google Scholar 

  36. Zafar M, Ahmed S, Khan MIM, Jamil A (2014) Recombinant expression and characterization of a novel endoglucanase from Bacillus subtilis in Escherichia coli. Mol Biol Rep 41(5):3295–3302

    Article  CAS  PubMed  Google Scholar 

  37. Gaur R, Tiwari S (2015) Isolation, production, purification and characterization of an organic-solvent-thermostable alkalophilic cellulase from Bacillus vallismortis RG-07. BMC Biotechnol 15(1):19

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. Javaheri-Kermani M, Asoodeh A (2019) A novel beta-1, 4 glucanase produced by symbiotic Bacillus sp. CF96 isolated from termite (Anacanthotermes). Int J Biol Macromol 131:752–759

    Article  CAS  PubMed  Google Scholar 

  39. Li Y-H, Ding M, Wang J, Xu G-j, Zhao F (2006) A novel thermoacidophilic endoglucanase, Ba-EGA, from a new cellulose-degrading bacterium, Bacillus sp. AC-1. Appl Microbiol Biotechnol 70(4):430–436

    Article  CAS  PubMed  Google Scholar 

  40. Han SJ, Yoo YJ, Kang HS (1995) Characterization of a bifunctional cellulase and its structural gene The cel gene of Bacillus sp. D04 has exo-and endoglucanase activity. J Biol Chem 270(43):26012–26019

    Article  CAS  PubMed  Google Scholar 

  41. Bao L, Huang Q, Chang L, Zhou J, Lu H (2011) Screening and characterization of a cellulase with endocellulase and exocellulase activity from yak rumen metagenome. J Mol Catal B 73(1–4):104–110

    Article  CAS  Google Scholar 

  42. Zhang F, Zhang X-M, Yin Y-R, Li W-J (2015) Cloning, expression and characterization of a novel GH5 exo/endoglucanase of Thermobifida halotolerans YIM 90462T by genome mining. J Biosci Bioeng 120(6):644–649

    Article  CAS  PubMed  Google Scholar 

  43. Wu B, Zheng S, Pedroso MM, Guddat LW, Chang S, He B, Schenk G (2018) Processivity and enzymatic mechanism of a multifunctional family 5 endoglucanase from Bacillus subtilis BS-5 with potential applications in the saccharification of cellulosic substrates. Biotechnol Biofuels 11(1):20

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Plant Protection, Faculty of Agriculture, Lorestan University, Khorramabad, Iran

    Fahimeh Dehghanikhah & Jahanshir Shakarami

  2. Department of Chemistry, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran

    Ahmad Asoodeh

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  1. Fahimeh Dehghanikhah
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  2. Jahanshir Shakarami
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  3. Ahmad Asoodeh
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Correspondence to Jahanshir Shakarami.

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Dehghanikhah, F., Shakarami, J. & Asoodeh, A. Purification and Biochemical Characterization of Alkalophilic Cellulase from the Symbiotic Bacillus subtilis BC1 of the Leopard Moth, Zeuzera pyrina (L.) (Lepidoptera: Cossidae). Curr Microbiol 77, 1254–1261 (2020). https://doi.org/10.1007/s00284-020-01938-z

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