Cross-linked enzyme aggregates of arylamidase from Cupriavidus oxalaticus ICTDB921: process optimization, characterization, and application for mitigation of acrylamide in industrial wastewater

  • Nidhi H. Kulkarni
  • Abhijeet B. Muley
  • Dattatray K. Bedade
  • Rekha S. SinghalEmail author
Research Paper


Acrylamidase produced by Cupriavidus oxalaticus ICTDB921 was recovered directly from the fermentation broth by ammonium sulfate (40–50%) precipitation and then stabilized by cross-linking with glutaraldehyde. The optimum conditions for the preparation of cross-linked enzyme aggregates of acrylamidase (acrylamidase-CLEAs) were using 60 mM glutaraldehyde for 10 min at 35 °C and initial broth pH of 7.0. Acrylamidase-CLEAs were characterized by SDS-PAGE, FTIR, particle size analyzer and SEM. Cross-linking shifted the optimal temperature and pH from 70 to 50 °C and 5–7 to 6–8, respectively. It also altered the secondary structure fractions, pH and thermal stability along with the kinetic constants, Km and Vmax, respectively. A complete degradation of acrylamide ~ 1.75 g/L in industrial wastewater was achieved after 60 min in a batch process under optimum operating conditions, and the kinetics was best represented by Edward model (R2 = 0.70). Acrylamidase-CLEAs retained ~ 40% of its initial activity after three cycles for both pure acrylamide and industrial wastewater, and were stable for 15 days at 4 °C, retaining ~ 25% of its original activity.

Article highlights

  • Acrylamidase from broth was recovered as cross linked enzyme aggregates (CLEAs)

  • Acrylamidase-CLEAs retained good kinetic and pH/thermal stability

  • Acrylamidase-CLEAs possessed good storage stability and reusability

  • Acrylamidase-CLEAs efficiently degraded acrylamide from industrial waste water


Cupriavidus oxalaticus ICTDB921 acrylamidase CLEAs Acrylamide Industrial wastewater Kinetic modeling 



The authors would like to thank the Department of Biotechnology, Ministry of Science and Technology, Government of India, New Delhi, for providing the financial supports to carry out this research work.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest in this research.

Ethical approval

This article does not include any studies with human participants or animals performed by any authors.

Supplementary material

449_2019_2240_MOESM1_ESM.doc (204 kb)
Supplementary file1 (DOC 203 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Nidhi H. Kulkarni
    • 1
  • Abhijeet B. Muley
    • 1
  • Dattatray K. Bedade
    • 1
  • Rekha S. Singhal
    • 1
    Email author
  1. 1.Food Engineering and Technology DepartmentInstitute of Chemical TechnologyMumbaiIndia

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