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Column Chromatography Free Purification of Recombinant α-Amylase from Bacillus licheniformis by Tagging with Hydrophobic Elastin Like Polypeptide

  • Rekha C. Akhani
  • Arti T. Patel
  • Manisha J. Patel
  • Samir R. Dedania
  • Jagdish S. Patel
  • Darshan H. Patel
Research Article
  • 73 Downloads

Abstract

Thermal sensitive and highly hydrophobic elastin like polypeptide (ELP) tends to undergo inverse transition cycling (ITC) which can be used for non chromatographic purification. The present study reports non-chromatographic purification of industrially important α-amylase tagged with ELP and compared with IMAC (Immobilized metal affinity chromatography) purified α-amylase. amyL-Gene encoding α-amylase from Bacillus licheniformis was cloned and expressed in E. coli BL21. The expressed protein with His-tag was purified through IMAC using Ni–NTA matrix. Three ELP genes encoding repeats of pentapeptide (Val-Pro-Gly-Val-Gly)n with variable length (V = 20, 21, 22) were synthesized through PCR using overlapping primers. To generate ELP tagged α-amylase, amyL was placed at N-terminal of ELPs and transformed to E. coli BL21 for expression. After ITC, ELP22 at 30 °C showed maximum yield. α-Amylase purification through ITC and IMAC showed 2.9 and 1.72-fold purification, respectively. Furthermore, physical parameters of ELP tagged α-amylase have shown improvement with working temperature and thermal stability in comparison to His-tag α-amylase. The k cat /K m for ELP tag and His-tag α-amylase was found to be 61.4 and 23.7 mg−1 min−1, respectively, which shows that ELP-tag increased the enzyme efficiency. In conclusion, the ELP-tag purification strategy can be applied to industrially relevant enzyme for purification by the non-chromatography method.

Keywords

α-Amylase Elastin like polypeptide Inverse transition cycling Transition temperature Hydrophobic peptide 

Notes

Acknowledgements

The authors wish to thank the Department of Biotechnology (DBT) (Project No BT/PR9727/GBD/27/505/2013), New Delhi for financial support.

Compliance with Ethical Standards

Conflict of interest

The authors do not have any conflict of interest to declare.

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Copyright information

© The National Academy of Sciences, India 2017

Authors and Affiliations

  • Rekha C. Akhani
    • 1
  • Arti T. Patel
    • 1
  • Manisha J. Patel
    • 1
  • Samir R. Dedania
    • 1
  • Jagdish S. Patel
    • 1
  • Darshan H. Patel
    • 1
    • 2
  1. 1.Department of Biochemistry, P. D. Patel Institute of Applied SciencesCHARUSATChangaIndia
  2. 2.K. C. Patel Research and Development CentreCHARUSATChangaIndia

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