Skip to main content
Log in

Preparation of a Calcium Alginate-Coated Polypyrrole/Silver Nanocomposite for Site-Specific Immobilization of Polygalacturonase with High Reusability and Enhanced Stability

  • Published:
Catalysis Letters Aims and scope Submit manuscript

Abstract

Polygalacturonase (PG) catalyses the hydrolysis of pectin substances and is commonly used in the textile and food industries. Herein, PG was purified from Arabian balsam using three techniques (ammonium sulfate precipitation, ion exchange chromatography, and gel filtration) with a recovery of 11.2% and tenfold purification. The molecular weight of the purified PG was estimated to be 75.5 kDa using a Sephadex G-150 column. To improve the stability and reusability of the purified enzyme, a novel method to immobilize PG through calcium alginate-coated polypyrrole/silver nanocomposite was described. The immobilized PG was characterized by FTIR, TGA, SEM, EDX, and Raman spectroscopy. The immobilization efficiency was 84.4%. Excellent long-term storage stability of the immobilized PG was demonstrated with 83% of the initial activity preserved after 60 days. The immobilized PG was highly reusable, showing high activity (91% and 68%) after five and ten cycles. The immobilized PG showed improved stability to temperature and pH relative to that of the free enzyme. The Km and Vmax were determined to be 0.368 mg/mL and 5.33 µmol/mL for the immobilized PG and 0.667 mg/mL and 7.38 µmol/mL for free PG, respectively. Improved storage stability, catalytic efficiency (Vmax/Km), and reusability of the immobilized PG make it ideal for biotechnological and industrial applications.

Graphic Abstract

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

Abbreviations

FTIR:

Fourier transform infrared spectroscopy

TGA:

Thermogravimetric analysis

SEM:

Scanning electron microscopy

EDX:

Energy-dispersive X-ray spectroscopy

References

  1. Gupta R, Beg Q, Khan S, Chauhan B (2002) An overview on fermentation, downstream processing and properties of microbial alkaline proteases. Appl Microbiol Biotechnol 60:381–395

    PubMed  CAS  Google Scholar 

  2. Mohamed SA, AL-Malki AL, Kumosani TA (2009) Characterization of a polygalacturonase from Trichoderma harzianum grown on citrus peel with application for apple juice. Aust J Basic Appl Sci 3:2770–2777

    CAS  Google Scholar 

  3. Tochi BN, Wang Z, Xu SY, Zhang W (2009) The influence of pectinase and pectinase/hemicellulases enzyme preparations on percentage pineapple juice recovery, particulates and sensory attributes. Pak J Nutr 8:1184–1189

    CAS  Google Scholar 

  4. Jayani RS, Saxena S, Gupta R (2005) Microbial pectinolytic enzymes: a review. Process Biochem 40:2931–2944

    CAS  Google Scholar 

  5. Kobayashi T, Higaki N, Yajima N, Suzumatsu A, Hagihara H, KAWAI S, ITo S (2001) Purification and properties of a galacturonic acid-releasing exopolygalacturonase from a strain of Bacillus. Biosci Biotechnol Biochem 65:842–847

    PubMed  CAS  Google Scholar 

  6. Fontana RC, da Silveira MM (2012) Production of polygalacturonases by Aspergillus oryzae in stirred tank and internal- and external-loop airlift reactors. Bioresour Technol 123:157–163

    PubMed  CAS  Google Scholar 

  7. Tepe O, Dursun AY (2014) Exo-pectinase production by Bacillus pumilus using different agricultural wastes and optimizing of medium components using response surface methodology. Environ Sci Pollut Res 21:9911–9920

    CAS  Google Scholar 

  8. Khan M, Nakkeeran E, Umesh-Kumar S (2013) Potential application of pectinase in developing functional foods. Annu Rev Food Sci Technol 4:21–34

    PubMed  CAS  Google Scholar 

  9. Caffall KH, Mohnen D (2009) The structure, function, and biosynthesis of plant cell wall pectic polysaccharides. Carbohydr Res 344:1879–1900

    PubMed  CAS  Google Scholar 

  10. Mohamed SA, Al-Malki AL, Khan JA, Kabli SA, Al-Garni SM (2013) Solid state production of polygalacturonase and xylanase by Trichoderma species using cantaloupe and watermelon rinds. J Microbiol 51:605–611

    PubMed  CAS  Google Scholar 

  11. Niture SK, Pant A (2004) Purification and biochemical characterization of polygalacturonase II produced in semi-solid medium by a strain of Fusarium moniliforme. Microbiol Res 159:305–314

    PubMed  CAS  Google Scholar 

  12. Kashyap DR, Soni SK, Tewari R (2003) Enhanced production of polygalacturonase by Bacillus sp.DT7 using solid state fermentation. Bioresour Technol 88:251–254

    Google Scholar 

  13. Srivastava S, Nighojkar A, Kumar A (1994) Multiple forms of pectin methylesterase from Cuscuta reflexa filaments. Phytochemistry 37:1233–1236

    CAS  Google Scholar 

  14. Sheldon RA, Schoevaart R, Van Langen LM (2005) Cross-linked enzyme aggregates (CLEAs): a novel and versatilemethod for enzyme immobilization (a review). Biocatal Biotransform 23:141–147

    CAS  Google Scholar 

  15. Bilal M, Asgher M, Iqbal HMN, Hu H, Zhang X (2016) Gelatin-immobilized manganese peroxidase with novelcatalytic characteristics and its industrial exploitation for fruitjuice clarification purposes. Catal Lett 146:2221–2228

    CAS  Google Scholar 

  16. Bilal M, Iqbal HMN, Hu H, Wang W, Zhang X (2017) Enhanced bio-catalytic performance and dye degradation potential of chitosan-encapsulated horseradish peroxidase in a packed bed reactor system. Sci Total Environ 575:1352–1360

    PubMed  CAS  Google Scholar 

  17. Abdulaal WH, Almulaiky YQ, El-Shishtawy RM (2020) Encapsulation of HRP enzyme onto a magnetic Fe3O4 Np–PMMA film via casting with sustainable biocatalytic activity. Catalysts 10:181

    CAS  Google Scholar 

  18. Almulaiky YQ, Aqlan FM, Aldhahri M, Baeshen M, Khan TJ, Khan KA, Alayafi AA (2018) α-Amylase immobilization on amidoximated acrylic microfibres activated by cyanuric chloride. R Soc Open Sci 5:172164

    PubMed  PubMed Central  CAS  Google Scholar 

  19. Al-Najada AR, Almulaiky YQ, Aldhahri M, El-Shishtawy RM, Mohamed SA, Baeshen M, Al-Harbi SA (2019) Immobilisation of α-amylase on activated amidrazone acrylic fabric: a new approach for the enhancement of enzyme stability and reusability. Sci Rep 9:1–9

    CAS  Google Scholar 

  20. Jia YJ, Feng BZ, Sun WX, Zhang XG (2009) Polygalacturonase, pectate lyase and pectin methylesteraseactivity in pathogenic strains of Phytophthora capsici incubatedunder different conditions. J Phytopathol 157:585–591

    CAS  Google Scholar 

  21. Gan J, Bagheri AR, Aramesh N, Gul I, Franco M, Almulaiky YQ, Bilal M (2020) Covalent organic frameworks as emerging host platforms for enzyme immobilization and robust biocatalysis–A review. Int J Biol Macromol 167:502–515

    PubMed  Google Scholar 

  22. Brayner R, Vaulay M-J, Fiévet F, Coradin T (2007) Alginate-mediated growth of Co, Ni, and CoNi nanoparticles: influence of the biopolymer structure. Chem Mater 19:1190–1198

    CAS  Google Scholar 

  23. Krueger KM, Al-Somali AM, Mejia M, Colvin VL (2007) The hydrodynamic size of polymer stabilized nanocrystals. Nanotechnology 18:475709

    Google Scholar 

  24. Otari SV, Patil RM, Waghmare SR, Ghosh SJ, Pawar SH (2013) A novel microbial synthesis of catalytically active Ag-alginate biohydrogel and its antimicrobial activity. Dalton Trans 42:9966–9975

    PubMed  CAS  Google Scholar 

  25. Sayo K, Deki S, Hayashi S (1999) A novel method of preparing nano-sized gold and palladium particles dispersed in composites that uses the thermal relaxation technique. Eur Phys J D 9:429–432

    CAS  Google Scholar 

  26. 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 72:248–254

    CAS  Google Scholar 

  27. Contreras-Esquivel JC, Hours RA, Voget CE, Mignone CF (1999) Aspergillus kawachii produces acidic pectin releasing enzyme activity. J Biosci Bioeng 88:48–52

    PubMed  CAS  Google Scholar 

  28. Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analyt chem 31:426–428

    CAS  Google Scholar 

  29. Omastová M, Mosnáčková K, Fedorko P, Trchová M, Stejskal J (2013) Polypyrrole/silver composites prepared by single-step synthesis. Synth Met 66:57–62

    Google Scholar 

  30. Al-Harbi SA, Almulaiky YQ (2020) Purification and biochemical characterization of Arabian balsam α-amylase and enhancing the retention and reusability via encapsulation onto calcium alginate/Fe2O3 nanocomposite beads. Int J Biol Macromol 160:944–952

    PubMed  CAS  Google Scholar 

  31. Almulaiky YQ, Al-Harbi SA (2019) A novel peroxidase from Arabian balsam (Commiphora gileadensis) stems: its purification, characterization and immobilization on a carboxymethylcellulose/Fe3O4 magnetic hybrid material. Int J Biol Macromol 133:767–774

    PubMed  CAS  Google Scholar 

  32. Köktepe T, Altın S, Tohma H, Gülçin I, Köksal E (2017) Purification, characterization and selected inhibition properties of peroxidase from haricot bean (Phaseolus vulgaris L.). Int J Food Prop 20:1944–1953

    Google Scholar 

  33. Mohamed SA, Abulnaja KO, Ads AS, Khan JA, Kumosani TA (2011) Characterisation of an anionic peroxidase from horseradish Cv. Balady Food Chem 128:725–730

    CAS  Google Scholar 

  34. Wingfield PT (Ed.) (2016) Protein precipitation using ammonium sulfate. Curr Protoc Protein Sci, 84(1), A-3F.‏

  35. Welinder KG (1992) Superfamily of plant, fungal and bacterial peroxidases. Curr Opin Struc Biol 2:388–393

    CAS  Google Scholar 

  36. Pedrolli DB, Carmona EC (2010) Purification and characterization of the exopolygalacturonase produced by Aspergillus giganteus in submerged cultures. J Ind Microbiol Biot 37:567–573

    CAS  Google Scholar 

  37. Rha E, Park HJ, Kim MO, Chung YR, Lee CW, Kim JW (2001) Expression of exo-polygalacturonases in Botrytis cinerea. FEMS Microbiol Lett 201:105–109

    PubMed  CAS  Google Scholar 

  38. Gomes E, Leite RSR, Da Silva R (2009) Purification of an exopolygalacturonase from Penicillium viridicatum RFC3 produced in submerged fermentation. Int J Microbiol. https://doi.org/10.1155/2009/631942

    Article  PubMed  Google Scholar 

  39. Rahman M, Choi YS, Kim YK, Park C, Yoo JC (2019) Production of novel polygalacturonase from Bacillus paralicheniformis CBS32 and application to depolymerization of ramie fiber. Polymers 11:1525

    PubMed Central  CAS  Google Scholar 

  40. Ernest V, Gajalakshmi S, Mukherjee A (2014) Enhanced activity of lysozyme-AgNP conjugate with synergic antibacterial effect without damaging the catalytic site of lysozyme. Artif Cells Nanomed Biotechnol 42:336–343

    PubMed  CAS  Google Scholar 

  41. Mohamed SA, Al-Harbi MH, Almulaiky YQ, Ibrahim IH, Salah HA, El-Badry MO, El-Shishtawy RM (2018) Immobilization of Trichoderma harzianum α-amylase on PPyAgNp/Fe3O4-nanocomposite: chemical and physical properties. Artif Cells Nanomed Biotechnol 46:201–206

    PubMed  CAS  Google Scholar 

  42. Almulaiky YQ, El-Shishtawy RM, Aldhahri M, Mohamed SA, Afifi M, Abdulaal WH, Mahyoub JA (2019) Amidrazone modified acrylic fabric activated with cyanuric chloride: a novel and efficient support for horseradish peroxidase immobilization and 401 phenol removal. Int J Biol Macromol 140:949–958

    PubMed  CAS  Google Scholar 

  43. Lin SY, Li MJ, Cheng WT (2007) FT-IR and Raman vibrational microspectroscopies used for spectral biodiagnosis of human tissues. Spectroscopy 21:1–30

    Google Scholar 

  44. Liu YC, Hwang BJ (2000) Identification of oxidized polypyrrole on Raman spectrum. Synth Met 113:203–207

    CAS  Google Scholar 

  45. Buga ML, Ibrahim S, Nok AJ (2010) Physico-chemical characteristics of immobilized polygalacturonase from Aspergillus niger (SA6). Artif Cells Nanomed Biotechnol 9:8934–8943

    CAS  Google Scholar 

  46. Anand G, Yadav S, Yadav D (2017) Purification and biochemical characterization of an exo-polygalacturonase from Aspergillus flavus MTCC 7589. Biocatal Agric Biotechnol 10:264–269

    Google Scholar 

  47. Almulaiky YQ, Khalil NM, El-Shishtawy RM, Altalhi T, Algamal Y, Aldhahri M, Mohammed MM (2021) Hydroxyapatite-decorated ZrO2 for α-amylase immobilization: toward the enhancement of enzyme stability and reusability. Int J Biol Macromol 167:299–308

    PubMed  CAS  Google Scholar 

  48. Mazlan SZ, Hanifah SA (2017) Effects of temperature and pH on immobilized laccase activity in conjugated methacrylate-acrylate microspheres. Int J Polym Sci. https://doi.org/10.1155/2017/5657271

    Article  Google Scholar 

  49. Rehman HU, Aman A, Nawaz MA, Karim A, Ghani M, Baloch AH, Qader SA (2016) Immobilization of pectin depolymerising polygalacturonase using different polymers. Int J Boil Macromol 82:127–133

    CAS  Google Scholar 

  50. Aldhahri M, Almulaiky YQ, El-Shishtawy RM, Al-Shawafi WM, Salah N, Alshahrie A, Alzahrani HA (2020) Ultra-thin 2D CuO nanosheet for HRP immobilization supported by encapsulation in a polymer matrix: characterization and dye degradation. Lett Catal. https://doi.org/10.1007/s10562-020-03289-7

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Yaaser Q. Almulaiky.

Ethics declarations

Conflict of interest

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 16 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Almulaiky, Y.Q., Al-Harbi, S.A. Preparation of a Calcium Alginate-Coated Polypyrrole/Silver Nanocomposite for Site-Specific Immobilization of Polygalacturonase with High Reusability and Enhanced Stability. Catal Lett 152, 28–42 (2022). https://doi.org/10.1007/s10562-021-03631-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10562-021-03631-7

Keywords

Navigation