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Purification of Lipase

Purification of Lipase. from Bacillus subtilis FS2. NONG Yuan Supervisor: Prof. Jan-Christer Janson Department of Surface Biotechnology Uppsala Biomedical Center Uppsala University 2005.12. Overview. Research Training in B7:3 Department of Surface Biotechnology ;

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Purification of Lipase

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  1. Purification of Lipase from Bacillus subtilis FS2 NONG Yuan Supervisor: Prof. Jan-Christer JansonDepartment of Surface Biotechnology Uppsala Biomedical Center Uppsala University 2005.12

  2. Overview Research Training in B7:3 • Department of Surface Biotechnology ; • Period: 1-Nov to 23-Nov; • Mainly work: Purification ; • Partially attend: Fermentation.

  3. Introduction Purification of Lipase from Bacillus subtilis FS2 • Purpose of this project • Research on Lipase from Bacillus Subtilis FS2; • First phase: purify lipase! Why interesting? • Glycerol ester hydrolases, catalyze hydrolysis of triacylglycerols free fatty acids + glycerol ; • High biotechnological potential; • Widely application in bioindustry.

  4. B. Subtilis FS2 -- lipase producing bacteria • novel strain, isolated from traditional fish source in Vietnam; • 97% homology with that of B. subtilis 168, which has become an attractive enzyme for industry; • Remarkable properties of Lipase • Molecular Weight: 19 kDa; • pI = 9.9 • High activity under alkaline conditions

  5. Material and Method Fermentation of Bacillus subtilis FS2 (from Vietnam) Collection of supernatant contained extra-cellular lipase Ammonium Sulphate 60% (w. v) Precipitation Purification ÄKTAdesign, UNICORN. Desalting • Cation Echange Chromatography

  6. a a Desalting proteins salt Desalted sample Column: Hi_Trap Desalting Sample: B.Subtilis FS2 Buffer: 20mM Sodium phosphate pH7.5 0 2 4 6 8 10 12 Elution volume (ml)

  7. Desalting figure

  8. + - + + - - + + + + + + + + - + + - + - + + - - + + - - + + + + + + + - + + + + + - - + + - - - - - + + - - - - - - - - - - - - + + + - - + - - - - - - + + + - - - - - + + What is expected in cation exchange? Equilibration Sample application and wash Elution Cleaning/ Regeneration + + + + + + + + + + - - - + Column: HiTrap SPFF 1ml, Sample: B. subtilis FS2, ÄKTAdesign, UNICORN. Start buffer: 20mM Sodium Phosphate pH 7.5. Elution buffer: 20mM Sodium Phosphate pH 7.5 with 1M NaCl.

  9. Result

  10. Stepwise gradient versus Linear gradient Stepwise elution would be usedafterwards

  11. Results and Discussions Linear elution profiles Theoretical VS Experimental !!!Bottom level shift

  12. Results and Discussions Comparison among linear gradient elution profiles !!! unstableprotein binding capacities

  13. Conclusion • No exactly expected outcome; • Experience accumulation to go further; System control Technology master Project design

  14. Future work Enzyme activity assay • Suitable method for lipase activity test; Purification work • Gel Fltration (Homology protein before application to ion exchanger column); • Hydrophobic Interaction Chromatography (recommended by previous researcher); Consider back to materials • Optimization of lipase producing----good sample .

  15. Acknowledges Special thanks to Prof. Janson and Ms Nguyet For the happy memory of 2005 Fall

  16. Thank YOU!

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