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

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

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

6. 6 Desalting proteins aa 2 Elution volume (ml) 0 4681012 Desalted sample salt Column: Sample: Buffer: Hi_Trap Desalting B.Subtilis FS2 20mM Sodium phosphate pH7.5

7. Desalting figure

8. 8 What is expected in cation exchange? Sample application and wash ElutionEquilibrationCleaning/ 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

11. Stepwise gradient versus Linear gradient Stepwise elution would be used afterwards

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

13. Results and Discussions !!! unstable protein binding capacities Comparison among linear gradient elution profiles

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

15. 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.

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

17. Thank YOU!