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Biotechnology and Bioprocess Engineering

, Volume 23, Issue 6, pp 662–669 | Cite as

Efficient Over-expression and Application of High-performance Pectin Lyase by Screening Aspergillus niger Pectin Lyase Gene Family

  • Yulan He
  • Li Pan
  • Bin WangEmail author
Research Paper
  • 10 Downloads

Abstract

Acidic pectin lyase is of great application value in fruit-processing industry. In this study, five pectin lyase genes (pelA, pelB, pelC, pelD and pelF) from A. niger pectin lyase gene family were cloned and over-expressed in A. niger strain SH-2, respectively. The pelA recombinant strain (SH2-pelA) owns the highest acidic pectin lyase activity: 11069.2 U/mL in flask fermentation, and 65148.8 U/mL in submerged fermentation. The recombinant pectin lyase A (PelA) was purified by Ni-affinity chromatography, and specificity of the protein was confirmed by western blotting. The recombinant PelA exhibits maximum activity at pH 4.5 and 50°C. The recombineant PelA shows increasing specific activity as the degree of methyl-esterization of the substrate raises. The maximum activity (22423.3 U/mL) was obtined using citrus pectin (≥85% esterified) as the substrate. After the treatment by PelA, the light transmittance of orange, apple and grape juice was increased 19.2-fold, 7.3-fold and 3.8-fold, respectively, suggesting that the recombinant acidic PelA had noticeable clarifying effects on orange, apple and grape juice.

Keywords

acidic pectin lyase Aspergillus niger overexpression characterization juice clarification 

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References

  1. 1.
    Latarullo, M. B., E. Q. Tavares, G. P. Maldonado, D. C. Leite, and M. S. Buckeridge (2016) Pectins, endopolygalacturonases, and bioenergy. Frontiers in Plant Science 7: 1401.CrossRefGoogle Scholar
  2. 2.
    Sharma, N., M. Rathore, and M. Sharma (2013) Microbial pectinase: sources, characterization and applications. Rev. Environ. Sci. Bio. 12: 45–60.CrossRefGoogle Scholar
  3. 3.
    Dubey, A. K., S. Y., M. Kumar, G. Anand, and D. Yadav (2016) Molecular biology of microbial pectate lyase: a review. British Biotechnology Journal 13: 1–26.CrossRefGoogle Scholar
  4. 4.
    Wu, M. C., C. M. Jiang, P. H. Huang, M. Y. Wu, and Y. T. Wang (2007) Separation and utilization of pectin lyase from commercial pectic enzyme via highly methoxylated cross–linked alcohol–insoluble solid chromatography for wine methanol reduction. J. Agr. Food Chem. 55: 1557–1562.CrossRefGoogle Scholar
  5. 5.
    Harmsen, J. A., M. A. Kusters–van Someren, and J. Visser (1990) Cloning and expression of a second Aspergillus niger pectin lyase gene (pelA): indications of a pectin lyase gene family in A. niger. Current Genetics 18: 161–166.CrossRefGoogle Scholar
  6. 6.
    Li, G., L. Rao, Y. Xue, C. Zhou, Y. Zhang, and Y. Ma (2010) Cloning, expression, and characterization of a highly active alkaline pectate lyase from alkaliphilic Bacillus sp. N16–5. Journal of Microbiology and Biotechnology 20: 670–677.CrossRefGoogle Scholar
  7. 7.
    Bartling, S., J. P. van den Hombergh, O. Olsen, D. von Wettstein, and J. Visser (1996) Expression of an Erwinia pectate lyase in three species of Aspergillus. Current Genetics 29: 474–481.Google Scholar
  8. 8.
    Templeton, M. D., K. R. Sharrock, J. K. Bowen, R. N. Crowhurst, and E. H. Rikkerink (1994) The pectin lyase–encoding gene (pnl) family from Glomerella cingulata: characterization of pnlA and its expression in yeast. Gene 142: 141–146.CrossRefGoogle Scholar
  9. 9.
    Martin, N., S. R. de Souza, R. da Silva, and E. Gomes (2004) Pectinase production by fungal strains in solid–state fermentation using agro–industrial bioproduct. Braz. Arch. Biol. Techn. 47: 813–819.CrossRefGoogle Scholar
  10. 10.
    Khan, M., E. Nakkeeran, and S. Umesh–Kumar (2013) Potential application of pectinase in developing functional foods. Annu. Rev. Food Sci. T. 4: 21–34.CrossRefGoogle Scholar
  11. 11.
    Hoondal, G. S., R. P. Tiwari, R. Tewari, N. Dahiya, and Q. K. Beg (2002) Microbial alkaline pectinases and their industrial applications: a review. Applied Microbiology and Biotechnology 59: 409–418.CrossRefGoogle Scholar
  12. 12.
    Yadav, S., P. K. Yadav, D. Yadav, and K. D. S. Yadav (2009) Pectin lyase: a review. Process Biochem. 44: 1–10.CrossRefGoogle Scholar
  13. 13.
    Patel, H., H. P. Sharma, and Sugandha (2017) Enzymatic added extraction and clarification of fruit juices–a review. Critical Reviews in Food Science and Nutrition 57: 1215–1227.CrossRefGoogle Scholar
  14. 14.
    Semenova, M. V., O. A. Sinitsyna, V. V. Morozova, E. A. Fedorova, A. V. Gusakov, O. N. Okunev, L. M. Sokolova, A. V. Koshelev, T. V. Bubnova, Y. P. Vinetskii, and A. P. Sinitsyn (2006) Use of a preparation from fungal pectin lyase in the food industry. Appl. Biochem. Micro+. 42: 598–602.CrossRefGoogle Scholar
  15. 15.
    Alana, A., A. Gabilondo, F. Hernando, M. D. Moragues, J. B. Dominguez, M. J. Llama, and J. L. Serra (1989) Pectin lyase production by a Penicillium italicum strain. Applied and Environmental Microbiology 55: 1612–1616.Google Scholar
  16. 16.
    Piccoli–Valle, R. H., F. M. L. Passos, F. J. V. Passos, and D. O. Silva (2001) Production of pectin lyase by Penicillium griseoroseum in bioreactors in the absence of inducer. Braz. J. Microbiol. 32: 135–140.Google Scholar
  17. 17.
    Kitamoto, N., S. Yoshino–Yasuda, K. Ohmiya, and N. Tsukagoshi (2001) A second pectin lyase gene (pel2) from Aspergillus oryzae KBN616: its sequence analysis and overexpression, and characterization of the gene products. J. Biosci. Bioeng. 91: 378–381.CrossRefGoogle Scholar
  18. 18.
    Perez–Fuentes, C., M. Cristina Ravanal, and J. Eyzaguirre (2014) Heterologous expression of a Penicillium purpurogenum pectin lyase in Pichia pastoris and its characterization. Fungal Biol–Uk. 118: 507–515.CrossRefGoogle Scholar
  19. 19.
    Kitamoto, N., S. Yoshino–Yasuda, K. Ohmiya, and N. Tsukagoshi (2001) Sequence analysis and overexpression of a pectin lyase gene (pel1) from Aspergillus oryzae KBN616. Bioscience, Biotechnology, and Biochemistry 65: 209–212.CrossRefGoogle Scholar
  20. 20.
    Yin, C., B. Wang, P. He, Y. Lin, and L. Pan (2014) Genomic analysis of the aconidial and high–performance protein producer, industrially relevant Aspergillus niger SH2 strain. Gene 541: 107–114.CrossRefGoogle Scholar
  21. 21.
    van Hartingsveldt, W., I. E. M. M. J. Cora, and van Zeijl (1987) Development of a homologous transformation system for Aspergillus niger based on the pyrG gene. Molecular and General Genetics MGG. 206: 71–75.CrossRefGoogle Scholar
  22. 22.
    Albersheim, P. (1966) Pectin lyase from fungi. Methods in Enzymology 8: 628–631.CrossRefGoogle Scholar
  23. 23.
    Gruben, B. S., M. R. Makela, J. E. Kowalczyk, M. Zhou, I. Benoit–Gelber, and R. P. De Vries (2017) Expression–based clustering of CAZyme–encoding genes of Aspergillus niger. BMC Genomics. 18: 900.CrossRefGoogle Scholar
  24. 24.
    Martens–Uzunova, E. S., and P. J. Schaap (2009) Assessment of the pectin degrading enzyme network of Aspergillus niger by functional genomics. Fungal Genet. Biol. 46: S170–S179.CrossRefGoogle Scholar
  25. 25.
    Fleissner, A. and P. Dersch (2010) Expression and export: recombinant protein production systems for Aspergillus. Applied Microbiology and Biotechnology 87: 1255–1270.CrossRefGoogle Scholar
  26. 26.
    Ganzlin, M. and U. Rinas (2008) In–depth analysis of the Aspergillus niger glucoamylase (glaA) promoter performance using high–throughput screening and controlled bioreactor cultivation techniques. J. Biotechnol. 135: 266–271.CrossRefGoogle Scholar
  27. 27.
    Xu, S. X., X. Qin, B. Liu, D. Q. Zhang, W. Zhang, K. Wu, and Y. H. Zhang (2015) An acidic pectin lyase from Aspergillus niger with favourable efficiency in fruit juice clarification. Lett. Appl. Microbiol. 60: 181–187.CrossRefGoogle Scholar
  28. 28.
    Yadav, S., P. K. Yadav, D. Yadav, and K. D. S. Yadav (2008) Purification and characterisation of an acidic pectin lyase produced by Aspergillus ficuum strain MTCC 7591 suitable for clarification of fruit juices. Ann. Microbiol. 58: 61–65.CrossRefGoogle Scholar
  29. 29.
    Martial–Didier, A. K, and K. K. H (2017) Partial purification and characterization of two pectinases (polygalacturonase and pectin lyase) from Papaya Pericarp (Carica papaya cv. solo 8). Int. J. Curr. Microbiol. App. Sci. 6: 2729–2739.CrossRefGoogle Scholar
  30. 30.
    Poturcu, K., I. Ozmen, and H. H. Biyik (2017) Characterization of an alkaline thermostable pectin lyase from newly isolated Aspergillus niger_WHAK1 and its application on fruit juice clarification. Arab. J. Sci. Eng. 42: 19–29.CrossRefGoogle Scholar
  31. 31.
    Limberg, G., R. Korner, H. C. Buchholt, T. M. I. E. Christensen, P. Roepstorff, and J. D. Mikkelsen (2000) Analysis of pectin structure part 1–analysis of different de–esterification mechanisms for pectin by enzymatic fingerprinting using endopectin lyase and endopolygalacturonase II from A. niger. Carbohyd. Res. 327: 293–307.CrossRefGoogle Scholar
  32. 32.
    Yadav, S., P. K. Yadav, D. Yadav, and K. D. S. Yadav (2009) Purification and characterization of pectin lyase produced by Aspergillus terricola and its application in retting of natural fibers. Appl. Biochem. Biotech. 159: 270–283.CrossRefGoogle Scholar
  33. 33.
    Liu, M. Q., W. K. Huo, X. J. Dai, and Y. H. Dang (2018) Preparation of low–molecular–weight citrus pectin by recombinant Bacillus subtilis pectate lyase and promotion of growth of Bifidobacterium longum. Catal. Commun. 107: 39–42.CrossRefGoogle Scholar
  34. 34.
    Zhou, C., Y. F. Xue, and Y. H. Ma (2017) Cloning, evaluation, and high–level expression of a thermo–alkaline pectate lyase from alkaliphilic Bacillus clausii with potential in ramie degumming. Applied Microbiology and Biotechnology 101: 3663–3676.CrossRefGoogle Scholar
  35. 35.
    Lim JY, Y. F, and S. Ueda (1983) Purification and characterization of pectinesterase and pectin lyase from Aspergillus oryzae A–3. J. Appl. Biochem. 5: 1–8.Google Scholar
  36. 36.
    Yadav, S., P. K. Yadav, D. Yadav, and K. D. S. Yadav (2008) Purification and characterization of an alkaline pectin lyase from Aspergillus flavus. Process Biochem. 43: 547–552.CrossRefGoogle Scholar
  37. 37.
    Ishii S, Y. T. (1975) Purification and properties of pectin lyase from Aspergillus japonicus. Agric. Biol. Chem. 39: 13–21.Google Scholar

Copyright information

© The Korean Society for Biotechnology and Bioengineering and Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.School of Biology and Biological EngineeringSouth China University of TechnologyGuangzhouChina
  2. 2.Guangdong Provincial Key Laboratory of Fermentation and Enzyme EngineeringGuangzhouChina

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