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Applied Biochemistry and Biotechnology

, Volume 164, Issue 1, pp 68–76 | Cite as

Purification of ß-glucosidases from Pichia etchellsii Using CIM Monolith Columns

  • Roopa K. Gaonkar
  • Saroj MishraEmail author
  • M. A. Vijayalakshmi
Article

Abstract

β-Glucosidases (EC 3.2.1.21) are industrially important glycosyl hydrolases used for cellulose saccharification as well as for synthesis of glyco-conjugates. Crystal structure of only one β-glucosidase of family 3 of the glycosyl hydrolase families is available due to difficulty in purification of these closely related enzymes from a given source. Multiple steps used during purification result in low yield, making it difficult to study their properties. Conditions for purification of two closely related β-glucosidases (BGL I and BGL II) of family 3 from Pichia etchellsii were investigated in this study. Two weak anion exchange columns convective interaction media-diethyl amino ethyl (CIM-DEAE) and CIM-ethylenediamine (CIM-EDA) were used for this purpose. The results obtained at 0.34 ml disk (CIM-DEAE) level were scaled up to 8 ml CIM-DEAE tube column wherein BGL I and BGL II were separated from the major contaminants in the cell-free extract. The recovered enzymes were completely resolved in the second step using CIM-EDA. A final specific activity of 9,180 IU/mg and 2,345.3 IU/mg was achieved for BGL I and BGL II respectively with an overall yield of 33%. The system should be applicable to resolution of other closely related enzymes from this family.

Keywords

Pichia etchellsii Family 3 β-glucosidases CIM-DEAE CIM-EDA Chromatographic scale up 

Abbreviations

CIM-DEAE

Convective interaction media-diethylaminoethyl

CIM-EDA

Convective interaction media-ethylenediamine

pNPG

p-Nitrophenyl-β-d-glucopyranoside

Notes

Acknowledgements

This work was supported by a funding from Department of Science and Technology, (No.SR/SO/BB-21/2006 dated 10-08-2007) Government of India. We thank Dr. Ales Podgornik (BIA separations, Slovenia) for giving required technical details regarding CIM monoliths and Prof. Nadir T. Mrabet (Nancy University, School of Medicine, France) for his help in designing the optimization experiments on CIM-EDA chromatogram using different gradients.

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Roopa K. Gaonkar
    • 1
  • Saroj Mishra
    • 2
    Email author
  • M. A. Vijayalakshmi
    • 1
    • 3
  1. 1.Centre for Bioseparation TechnologyVIT UniversityVelloreIndia
  2. 2.Department of Biochemical Engineering and BiotechnologyIndian Institute of Technology DelhiNew DelhiIndia
  3. 3.Département Génie BiologiqueLIMTech.S, Centre de Recherches de Royallieu, UTCCompiegneFrance

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