Environmental Science and Pollution Research

, Volume 24, Issue 8, pp 7035–7041 | Cite as

Bio-based degradation of emerging endocrine-disrupting and dye-based pollutants using cross-linked enzyme aggregates

  • Muhammad Bilal
  • Muhammad Asgher
  • Hafiz M. N. IqbalEmail author
  • Hongbo Hu
  • Xuehong Zhang
Research Article


In this study, manganese peroxidase (MnP) from an indigenous white-rot fungus Ganoderma lucidum IBL-05 was insolubilized in the form of cross-linked enzyme aggregates (CLEAs) using various aggregating agents, i.e., acetone, ammonium sulfate, ethanol, 2-propanol, and tert-butanol, followed by glutaraldehyde (GA) cross-linking. The precipitant type, MnP, and GA concentrations affected the CLEAs activity recovery and aggregation yield. Among precipitants used, acetone appeared to be the most efficient aggregation agent, providing the highest activity recovery and aggregation yield of 31.26 and 73.46%, respectively. Optimal cross-linking was noticed using 2.0% (v/v) GA and 8:1 (v/v) MnP to GA ratio at 3.0 h cross-linking time under continuous agitation at 4 °C. The highest recovered activity and aggregation yield were determined to be 47.57 and 81.26%, respectively. The MnP-CLEAs, thus synthesized, were tested to investigate their bio-catalytic capacity for removing two known endocrine-disrupting chemicals (EDCs), e.g., nonylphenol and triclosan in a packed bed reactor system. The insolubilized MnP efficiently catalyzed the biodegradation of both EDCs, transforming over 80% in the presence of MnP-based system. A maximal of 100% decolorization was recorded for Sitara textile (SIT-based) effluent, followed by 95.5% for Crescent textile (CRT-based) effluent, 88.0% for K&N textile (KIT-based) effluent, and 84.2% for Nishat textile (NIT-based) effluent.


Cross-linked enzyme aggregates Insolubilization Catalytic activity Endocrine disrupting chemicals Biodegradation 



The present study was a part of a research project focused on the development of ligninolytic enzymes for industrial applications. The financial support provided by the Higher Education Commission, Islamabad, Pakistan is thankfully acknowledged. The authors are also grateful to the State Key Laboratory of Microbial Metabolism, and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 200240 Shanghai, China for providing technical and analytical help.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Asgher M, Iqbal HMN (2013) Enhanced catalytic features of sol–gel immobilized MnP isolated from solid state culture of Pleurotus ostreatus IBL-02. Chin Chem Lett 24(4):344–346CrossRefGoogle Scholar
  2. Asgher M, Aslam B, Iqbal HMN (2013) Novel catalytic and effluent decolorization functionalities of sol-gel immobilized Pleurotus ostreatus IBL-02 manganese peroxidase produced from bio-processing of wheat straw. Chin J Catal 34(9):1756–1761CrossRefGoogle Scholar
  3. Asgher M, Kamal S, Iqbal HMN (2012) Improvement of catalytic efficiency, thermo-stability and dye decolorization capability of Pleurotus ostreatus IBL-02 laccase by hydrophobic sol gel entrapment. Chem Cent J 6(1):1CrossRefGoogle Scholar
  4. Asgher M, Ramzan M, Bilal M (2016) Purification and characterization of manganese peroxidases from native and mutant Trametes versicolor IBL-04. Chin J Catal 37(4):561–570CrossRefGoogle Scholar
  5. Auriol M, Filali-Meknassi Y, Tyagi RD, Adams CD, Surampalli RY (2006) Endocrine disrupting compounds removal from wastewater, a new challenge. Proc Biochem 41(3):525–539CrossRefGoogle Scholar
  6. Bilal M, Asgher M, Ramzan M (2015) Purification and biochemical characterization of extracellular manganese peroxidase from Ganoderma lucidum IBL-05 and its application. Sci Res Ess 10(14):456–464CrossRefGoogle Scholar
  7. Bilal M, Asgher M, Parra-Saldivar R, Hu H, Wang W, Zhang X, Iqbal HMN (2017a) Immobilized ligninolytic enzymes: an innovative and environmental responsive technology to tackle dye-based industrial pollutants—a review. Sci Total Environ 576:646–659CrossRefGoogle Scholar
  8. Bilal M, Iqbal HMN, Hu H, Wang W, Zhang X (2017b) Development of horseradish peroxidase-based cross-linked enzyme aggregates and their environmental exploitation for bioremediation purposes. J Environ Manag 188:137–143CrossRefGoogle Scholar
  9. Boyd GR, Palmeri JM, Zhang S, Grimm DA (2004) Pharmaceuticals and personal care products (PPCPs) and endocrine disrupting chemicals (EDCs) in stormwater canals and Bayou St. John in New Orleans, Louisiana, USA. Sci Total Environ 333(1):137–148CrossRefGoogle Scholar
  10. 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):248–254CrossRefGoogle Scholar
  11. Cabana H, Jiwan JLH, Rozenberg R, Elisashvili V, Penninckx M, Agathos SN, Jones JP (2007) Elimination of endocrine disrupting chemicals nonylphenol and bisphenol A and personal care product ingredient triclosan using enzyme preparation from the white rot fungus Coriolopsis polyzona. Chemosphere 67(4):770–778CrossRefGoogle Scholar
  12. Eibes G, López C, Moreira MT, Feijoo G, Lema JM (2007) Strategies for the design and operation of enzymatic reactors for the degradation of highly and poorly soluble recalcitrant compounds. Biocatal Biotransform 25(2–4):260–268CrossRefGoogle Scholar
  13. Iqbal HMN, Asgher M (2013) Characterization and decolorization applicability of xerogel matrix immobilized manganese peroxidase produced from Trametes versicolor IBL-04. Protein Pept Lett 20(5):591–600CrossRefGoogle Scholar
  14. Kolpin DW, Furlong ET, Meyer MT, Thurman EM, Zaugg SD, Barber LB, Buxton HT (2002) Pharmaceuticals, hormones, and other organic wastewater contaminants in US streams, 1999-2000: a national reconnaissance. Environ Sci Technol 36(6):1202–1211CrossRefGoogle Scholar
  15. Liu ZH, Kanjo Y, Mizutani S (2009) Removal mechanisms for endocrine disrupting compounds (EDCs) in wastewater treatment—physical means, biodegradation, and chemical advanced oxidation: a review. Sci Total Environ 407(2):731–748CrossRefGoogle Scholar
  16. Petrovic M, Solé M, López De Alda MJ, Barceló D (2002) Endocrine disruptors in sewage treatment plants, receiving river waters, and sediments: integration of chemical analysis and biological effects on feral carp. Environ Toxicol Chem 21(10):2146–2156CrossRefGoogle Scholar
  17. Rehman S, Bhatti HN, Bilal M, Asgher M (2016) Cross-linked enzyme aggregates (CLEAs) of Pencilluim notatum lipase enzyme with improved activity, stability and reusability characteristics. Int J Biol Macromol 91:1161–1169CrossRefGoogle Scholar
  18. Šekuljica NŽ, Prlainović NŽ, Jakovetić SM, Grbavčić SŽ, Ognjanović ND, Knežević-Jugović ZD, Mijin DŽ (2016) Removal of anthraquinone dye by cross-linked enzyme aggregates from fresh horseradish extract. CLEAN–Soil, Air, Water 44(9999):1–10Google Scholar
  19. Shah S, Sharma A, Gupta MN (2006) Preparation of cross-linked enzyme aggregates by using bovine serum albumin as a proteic feeder. Analytical Biochem 351(2):207–213CrossRefGoogle Scholar
  20. Sheldon RA (2011) Characteristic features and biotechnological applications of cross-linked enzyme aggregates (CLEAs). Appl Microbiol Biotechnol 92(3):467–477CrossRefGoogle Scholar
  21. Spahn C, Minteer SD (2008) Enzyme immobilization in biotechnology. Recent Pat Eng 2(3):195–200CrossRefGoogle Scholar
  22. Srinivasan A, Viraraghavan T (2010) Decolorization of dye wastewaters by biosorbents: a review. J Environ Manag 91:1915–1929CrossRefGoogle Scholar
  23. Šulek F, Fernández DP, Knez Ž, Habulin M, Sheldon RA (2011) Immobilization of horseradish peroxidase as crosslinked enzyme aggregates (CLEAs). Proc. Biochem. 46(3):765–769CrossRefGoogle Scholar
  24. Taboada-Puig R, Junghanns C, Demarche P, Moreira MT, Feijoo G, Lema JM, Agathos SN (2011) Combined cross-linked enzyme aggregates from versatile peroxidase and glucose oxidase: production, partial characterization and application for the elimination of endocrine disruptors. Bioresour Technol 102(11):6593–6599CrossRefGoogle Scholar
  25. Tandjaoui N, Tassist A, Abouseoud M, Couvert A, Amrane A (2015) Preparation and characterization of cross-linked enzyme aggregates (CLEAs) of Brassica rapa peroxidase. Biocatal Agric Biotechnol 4(2):208–213Google Scholar
  26. Yamak O, Kalkan NA, Aksoy S, Altinok H, Hasirci N (2009) Semi-interpenetrating polymer networks (semi-IPNs) for entrapment of laccase and their use in Acid Orange 52 decolorization. Proc. Biochem. 44(4):440–445CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Muhammad Bilal
    • 1
    • 2
  • Muhammad Asgher
    • 1
  • Hafiz M. N. Iqbal
    • 3
    Email author
  • Hongbo Hu
    • 2
  • Xuehong Zhang
    • 2
  1. 1.Industrial Biotechnology Laboratory, Department of BiochemistryUniversity of AgricultureFaisalabadPakistan
  2. 2.State Key Laboratory of Microbial Metabolism, and School of Life Sciences and BiotechnologyShanghai Jiao Tong UniversityShanghaiChina
  3. 3.ENCIT—Science, Engineering and Technology School, Tecnologico de MonterreyMonterreyMexico

Personalised recommendations