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Molecular Biotechnology

, Volume 61, Issue 9, pp 681–693 | Cite as

Biochemical and Molecular Characterizations of a Novel pH- and Temperature-Stable Pectate Lyase from Bacillus amyloliquefaciens S6 for Industrial Application

  • Seda Bekli
  • Busra AktasEmail author
  • Donus Gencer
  • Belma Aslim
Original paper

Abstract

In this paper, we report cloning of a pectate lyase gene from Bacillus amyloliquefaciens S6 (pelS6), and biochemical characterization of the recombinant pectate lyase. PelS6 was found to be identical with B. subtilis 168 pel enzyme with 100% amino acid sequence homology. Although these two are genetically very close, they are distinctly different in physiology. pelS6 gene encodes a 421-aa protein with a molecular mass of 65,75 kDa. Enzyme activity increased from 12.8 ± 0.3 to 49.6 ± 0.4 units/mg after cloning. The relative enzyme activity of the recPel S6 ranged from 80% to 100% at pH between 4 and 14. It was quite stable at different temperature values ranging from 15 to 90 °C. The recPEL S6 showed a maximal activity at pH 10 and at 60 °C. 0.5 mM of CaCl2 is the most effective metal ion on the recPEL S6 as demonstrated by its increased relative activity with 473%. recPEL S6 remained stable at − 20 °C for 18 months. In addition recPEL S6 increased juice clarity. This study introduces a novel bacterial pectate lyase enzyme with its characteristic capability of being highly thermostable, thermotolerant, and active over a wide range of pH, meaning that it can work at both acidic and alkaline environments, which are the most preferred properties in the industry.

Keywords

Pectate lyases pH-thermotolerance pH-thermostable Bacillus amyloliquefaciens 

Notes

Acknowledgements

This research was supported by the BAP (Project No. 05/2016-25).

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

12033_2019_194_MOESM1_ESM.docx (189 kb)
Electronic supplementary material 1 (DOCX 189 kb)

References

  1. 1.
    Abu-Qarn, M., Eichler, J., & Sharon, N. (2008). Not just for Eukarya anymore: protein glycosylation in Bacteria and Archaea. Current Opinion in Structural Biology, 18, 544–550.CrossRefPubMedGoogle Scholar
  2. 2.
    Alcaraz, L. D., Moreno-Hagelsieb, G., Eguiarte, L. E., et al. (2010). Understanding the evolutionary relationships and major traits of Bacillus through comparative genomics. BMC Genomics, 11, 332.CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Biz, A., Farias, F. C., Motter, F. A., et al. (2014). Pectinase activity determination: An early deceleration in the release of reducing sugars throws a spanner in the works! PLoS ONE, 9, e109529.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Bolotin, A., & Borchert, S. (1997). The complete genome sequence of the Gram-positive bacterium Bacillus subtilis. Nature, 390, 249–256.  https://doi.org/10.1021/ic00220a054.CrossRefPubMedGoogle Scholar
  5. 5.
    Bonnin, E., Ralet, M.-C., Thibault, J.-F., & Schols, H. A. (2009). Enzymes for the valorisation of fruit-and vegetable-based co-products. Handbook of waste management and co-product recovery in food processing (pp. 257–285). Cambridge: Woodhead Publishing.CrossRefGoogle Scholar
  6. 6.
    Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254.  https://doi.org/10.1016/0003-2697(76)90527-3.CrossRefPubMedGoogle Scholar
  7. 7.
    Cheng, Z., Chen, D., Lu, B., et al. (2016). A novel acid-stable endo-polygalacturonase from Penicillium oxalicum CZ1028: Purification, characterization, and application in the beverage industry. Journal of Microbiology and Biotechnology, 26, 989–998.CrossRefPubMedGoogle Scholar
  8. 8.
    Crawford, M. S., & Kolattukudy, P. E. (1987). Pectate lyase from Fusarium solani f. sp. pisi: Purification, characterization, in vitro translation of the mRNA, and involvement in pathogenicity. Archives of Biochemistry and Biophysics, 258, 196–205.CrossRefPubMedGoogle Scholar
  9. 9.
    De Lima Damásio, A. R., Maller, A., Márcio Da Silva, T., et al. (2011). Biotechnological potential of alternative carbon sources for production of pectinases by rhizopus microsporus var. rhizopodiformis. Archives of Biology and Technology, 54154, 141–148.CrossRefGoogle Scholar
  10. 10.
    Dubey, A. K., Yadav, S., Kumar, M., et al. (2016). Molecular biology of microbial pectate lyase: A review. British Biotechnology Journal, 13, 1–26.CrossRefGoogle Scholar
  11. 11.
    Fuchs, A. (1965). The trans-eliminative breakdown of Na-polygalacturonate by Pseudomonas fluorescens. Antonie van Leeuwenhoek, 31, 323–340.CrossRefPubMedGoogle Scholar
  12. 12.
    Fujiwara, S. (2002). Extremophiles: Developments of their special functions and potential resources. ShinsukeFujiwara, 94, 518–525.Google Scholar
  13. 13.
    Garg, G., Singh, A., Kaur, A., et al. (2016). Microbial pectinases an ecofriendly tool of nature for industries. 3 Biotech, 6, 47.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Garron, M. L., & Cygler, M. (2010). Structural and mechanistic classification of uronic acid-containing polysaccharide lyases. Glycobiology, 20, 1547–1573.  https://doi.org/10.1093/glycob/cwq122.CrossRefPubMedGoogle Scholar
  15. 15.
    Gómez-Plaza, E., Gil-Muñ Oz, R., López-Roca, J. M., & Martínez, A. (2000). Color and phenolic compounds of a young red wine. Influence of wine-making techniques, storage temperature, and length of storage time. Journal of Agriculture and Food Chemistry, 48, 736–741.CrossRefGoogle Scholar
  16. 16.
    Gummadi, S. N., & Panda, T. (2003). Purification and biochemical properties of microbial pectinases—A review. Process Biochemistry, 38, 987–996.CrossRefGoogle Scholar
  17. 17.
    Haki, G. D., & Rakshit, S. K. (2003). Developments in industrially important thermostable enzymes: A review. Bioresource Technology, 89, 17–34.CrossRefPubMedGoogle Scholar
  18. 18.
    Hoondal, G., Tiwari, R., Tewari, R., et al. (2002). Microbial alkaline pectinases and their industrial applications: A review. Applied Microbiology and Biotechnology, 59, 409–418.CrossRefPubMedGoogle Scholar
  19. 19.
    Hugouvieux-Cotte-Pattat, N., Condemine, G., Nasser, W., & Reverchon, S. (1996). Regulation of pectinolysis in Erwinia chrysanthemi. Annual Review of Microbiology, 50, 213–257.CrossRefPubMedGoogle Scholar
  20. 20.
    Hugouvieux-Cotte-Pattat, N., Condemine, G., & Shevchik, V. E. (2014). Bacterial pectate lyases, structural and functional diversity. Environmental Microbiology Reports, 6, 427–440.CrossRefPubMedGoogle Scholar
  21. 21.
    Jacob, N. (2009). Biotechnology for agro-industrial residues utilisation: Utilisation of agro-residues. In P. S. Nigam & A. Pandey (Eds.), Biotechnology for agro-industrial residues utilisation: Utilisation of agro-residues (pp. 1–466). Dordrecht: Springer.Google Scholar
  22. 22.
    Jurick, W. M., Vico, I., McEvoy, J. L., et al. (2009). Isolation, purification, and characterization of a polygalacturonase produced in Penicillium solitum-decayed ‘golden delicious’ apple fruit wayne. Phytopathology, 99, 636–641.CrossRefPubMedGoogle Scholar
  23. 23.
    Kashyap, D. R., Vohra, P. K., Chopra, S., & Tewari, R. (2001). Applications of pectinases in the commercial sector: A review. Bioresource Technology, 77, 215–227.CrossRefPubMedGoogle Scholar
  24. 24.
    Kita, N., Boyd, C. M., Garrett, M. R., et al. (1996). Differential effect of site-directed mutations in pelC on pectate lyase activity, plant tissue maceration, and elicitor activity. Journal of Biological Chemistry, 271, 26529–26535.CrossRefPubMedGoogle Scholar
  25. 25.
    Kleerebezem, M., Hols, P., Bernard, E., et al. (2010). The extracellular biology of the lactobacilli. FEMS Microbiology Reviews, 34, 199–230.CrossRefPubMedGoogle Scholar
  26. 26.
    Kobayashi, T., Hatada, Y., Higaki, N., et al. (1999). Enzymatic properties and deduced amino acid sequence of a high-alkaline pectate lyase from an alkaliphilic Bacillus isolate. Biochimica et Biophysica Acta, 1427, 145–154.CrossRefPubMedGoogle Scholar
  27. 27.
    Kobayashi, T., Koike, K., Yoshimatsu, T., et al. (1999). Purification and Properties of a low-molecular-weight, high-alkaline pectate lyase from an alkaliphilic strain of Bacillus. Bioscience Biotechnology and Biochemistry, 63, 65–72.CrossRefGoogle Scholar
  28. 28.
    Kohli, P., & Gupta, R. (2015). Alkaline pectinases: A review. Biocatalysis and Agricultural Biotechnology, 4, 279–285.CrossRefGoogle Scholar
  29. 29.
    Kozianowski, G., Canganella, F., Rainey, F. A., et al. (1997). Purification and characterization of thermostable pectate-lyases from a newly isolated thermophilic bacterium, Thermoanaerobacter italicus sp. nov. Extremophiles, 1, 171–182.CrossRefPubMedGoogle Scholar
  30. 30.
    Lombard, V., Bernard, T., Rancurel, C., et al. (2010). A hierarchical classification of polysaccharide lyases for glycogenomics. Biochemical Journal, 432, 437–444.CrossRefPubMedGoogle Scholar
  31. 31.
    Ma, G., Zhu, W., & Liu, Y. (2016). QM/MM studies on the calcium-assisted β-elimination mechanism of pectate lyase from bacillus subtilis. Proteins: Structure, Function, and Bioinformatics, 84, 1606–1615.  https://doi.org/10.1002/prot.25103.CrossRefGoogle Scholar
  32. 32.
    Macmillan, J. D., & Vaughn, R. R. (1962). Purification and properties of a polygalacturonic acid-trans-eliminase produced. Biochemistry, 3, 564–572.CrossRefGoogle Scholar
  33. 33.
    Mukhopadhyay, A., Dasgupta, A. K., Chattopadhyay, D., & Chakrabarti, K. (2012). Improvement of thermostability and activity of pectate lyase in the presence of hydroxyapatite nanoparticles. Bioresource Technology, 116, 348–354.CrossRefPubMedGoogle Scholar
  34. 34.
    Nasser, W., Awade, A. C., Reverchon, S., & Robert-Baudouy, J. (1993). Pectate lyase from Bacillus subtilis: Molecular characterization of the gene, and properties of the cloned enzyme. FEBS Letters, 335, 319–326.CrossRefPubMedGoogle Scholar
  35. 35.
    Nasser, W., Chalet, F., & Robert-Baudouy, J. (1990). Purification and characterization of extracellular pectate lyase from Bacillus subtilis. Biochimie, 72, 689–695.CrossRefPubMedGoogle Scholar
  36. 36.
    Pedrolli, D. B., Monteiro, A. C., Gomes, E., & Carmona, E. C. (2009). Pectin and pectinases: Production, characterization and industrial application of microbial pectinolytic enzymes. Open Biotechnology Journal, 3, 9–18.CrossRefGoogle Scholar
  37. 37.
    Phrommao, E., Yongsawatdigul, J., Rodtong, S., & Yamabhai, M. (2011). A novel subtilase with NaCl-activated and oxidant-stable activity from Virgibacillus sp. SK37. BMC Biotechnology, 11, 1–15.CrossRefGoogle Scholar
  38. 38.
    Pickersgill, R., Jenkins, J., Harris, G., et al. (1994). The structure of Bacillus subtilis pectate lyase in complex with calcium. Nature Structural & Molecular Biology, 1, 717–723.CrossRefGoogle Scholar
  39. 39.
    Poondla, V., Bandikari, R., Subramanyam, R., & Reddy Obulam, V. S. (2015). Low temperature active pectinases production by Saccharomyces cerevisiae isolate and their characterization. Biocatalysis and Agricultural Biotechnology, 4, 70–76.CrossRefGoogle Scholar
  40. 40.
    Pušić, T., Tarbuk, A., & Dekanić, T. (2015). Bio-innovation in cotton fabric scouring- acid and neutral pectinases. Fibres & Textiles in Eastern Europe, 23, 98–103.Google Scholar
  41. 41.
    Sakai, T., Sakamoto, T., Hallaert, J., & Vandamme, E. J. (1993). Pectin, pectinase, and protopectinase: Production, properties, and applications. Advances in Applied Microbiology, 39, 213–294.CrossRefPubMedGoogle Scholar
  42. 42.
    Sambrook, J., Russell, D. W. (2006). Isolation of high-molecular-weight DNA from mammalian cells using formamide. In: Press CSHL (ed) Cold Spring Harb Protoc, 3rd edn. Cold Spring Harbor, NYGoogle Scholar
  43. 43.
    Sato, M., & Kaji, A. (1975). Purification and properties of pectate lyase produced by Streptomyces fradiae IFO 3439. Agricultural and Biological Chemistry, 39, 819–824.Google Scholar
  44. 44.
    Sharma, H. P., & Patel, H. (2017). Critical reviews in food science and nutrition enzymatic added extraction and clarification of fruit juices—A review enzymatic added extraction and clarification of fruit juices—A review. Critical Reviews in Food Science and Nutrition, 57, 1215–1227.CrossRefPubMedGoogle Scholar
  45. 45.
    Sharma, R., Lee, D.-W., Xu, Z., et al. (2016). Comparative genomic analysis of Bacillus amyloliquefaciens and Bacillus subtilis reveals evolutional traits for adaptation to plant-associated habitats. Frontiers in Microbiology, 7, 2039.Google Scholar
  46. 46.
    Sharon, N. (2007). Celebrating the golden anniversary of the discovery of bacillosamine, the diamino sugar of a Bacillus. Glycobiology, 17, 1150–1155.CrossRefPubMedGoogle Scholar
  47. 47.
    Singh Jayani, R., Saxena, S., & Gupta, R. (2005). Microbial pectinolytic enzymes: A review. Process Biochemistry, 40, 2931–2944.CrossRefGoogle Scholar
  48. 48.
    Singh, S. A., Plattner, H., & Diekmann, H. (1999). Exopolygalacturonate lyase from a thermophilic Bacillus sp. Enyzme and Microbial Technology, 25, 420–425.CrossRefGoogle Scholar
  49. 49.
    Soriano, M., Blanco, A., Dıaz, P., & Pastor, F. I. J. (2000). An unusual pectate lyase from a Bacillus sp. with high activity on pectin: Cloning and characterization. Microbiology, 146, 89–95.CrossRefPubMedGoogle Scholar
  50. 50.
    Soriano, M., Diaz, P., Javier, F. I., et al. (2006). Pectate lyase C from Bacillus subtilis: A novel endo-cleaving enzyme with activity on highly methylated pectin. Microbiology, 152, 617–625.CrossRefPubMedGoogle Scholar
  51. 51.
    Starr, M. P., & Moran, F. (1962). Eliminative split of pectic substances by phytopathogenic soft-rot bacteria. Science, 135, 920–921.CrossRefPubMedGoogle Scholar
  52. 52.
    Takao, M., Nakaniwa, T., Yoshikawa, K., et al. (2000). Purification and characterization of thermostable pectate lyase with protopectinase activity from thermophilic Bacillus sp. TS 47. Bioscience, Biotechnology, and Biochemistry, 64, 2360–2367.CrossRefPubMedGoogle Scholar
  53. 53.
    Van den Burg, B. (2003). Extremophiles as a source for novel enzymes. Current Opinion in Microbiology, 6, 213–218.CrossRefPubMedGoogle Scholar
  54. 54.
    Wang, X., Lu, Z., Xu, T., et al. (2018). Improving the specific activity and thermo-stability of alkaline pectate lyase from Bacillus subtilis 168 for bioscouring. Biochemical Engineering Journal, 129, 74–83.CrossRefGoogle Scholar
  55. 55.
    Yadav, S., Yadav, P. K., Yadav, D., et al. (2009). Pectin lyase: A review. Process Biochemistry, 44, 1–10.CrossRefGoogle Scholar
  56. 56.
    Yuan, P., Meng, K., Luo, H., et al. (2011). A novel low-temperature active alkaline pectate lyase from Klebsiella sp. Y1 with potential in textile industry. Process Biochemistry, 46, 1921–1926.CrossRefGoogle Scholar
  57. 57.
    Zhang, C., Yao, J., Zhou, C., et al. (2013). The alkaline pectate lyase PEL168 of Bacillus subtilis heterologously expressed in Pichia pastoris is more stable and efficient for degumming ramie fiber. BMC Biotechnology, 13, 1–9.CrossRefGoogle Scholar
  58. 58.
    Zhou, C., Xue, Y., & Ma, Y. (2017). Characterization and overproduction of a thermo-alkaline pectate lyase from alkaliphilic Bacillus licheniformis with potential in ramie degumming. Process Biochemistry, 54, 49–58.  https://doi.org/10.1016/j.procbio.2017.01.010.CrossRefGoogle Scholar
  59. 59.
    Zhou, M., Guo, P., Wang, T., et al. (2017). Metagenomic mining pectinolytic microbes and enzymes from an apple pomace-adapted compost microbial community. Biotechnology for Biofuels, 10, 198.CrossRefPubMedPubMedCentralGoogle Scholar
  60. 60.
    Zhou, Z., Liu, Y., Chang, Z., et al. (2017). Structure-based engineering of a pectate lyase with improved specific activity for ramie degumming. Applied Microbiology and Biotechnology, 101, 2919–2929.CrossRefPubMedGoogle Scholar

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Authors and Affiliations

  1. 1.Department of BiologyGazi UniversityAnkaraTurkey
  2. 2.Department of Molecular Biology and GeneticsBurdur Mehmet Akif Ersoy UniversityBurdurTurkey
  3. 3.Department of BiologyKaradeniz Technical UniversityTrabzonTurkey

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